F1000Research F1000Research 2046-1402 F1000 Research Limited London, UK 10.12688/f1000research.177148.2 Research Article Articles PEPTIDE IDR-1002 REGULATES THE ANTIOXIDANT AND ANTI-INFLAMMATORY RESPONSES BY ACTIVATING THE KEAP1-NRF2 SIGNALING PATHWAY

[version 2; peer review: 1 approved, 1 not approved]

 Romero-Durán Marco Antonio Conceptualization Formal Analysis Investigation Writing – Original Draft Preparation 1 Maldonado-Pichardo María Cristina Conceptualization Formal Analysis Investigation 2 Perez-Aguilar Jose Manuel Supervision Writing – Review & Editing 3 Baizabal Aguirre Victor Manuel Conceptualization Formal Analysis Funding Acquisition Methodology Project Administration Resources Supervision Validation Writing – Original Draft Preparation Writing – Review & Editing https://orcid.org/0000-0001-6816-0404 a 4 Centro Multidisciplinario de Estudios en Biotecnología, Facultad de Medicina Veterinaria y Zootecnia, Universidad Michoacana de San Nicolás de Hidalgo, Morelia, Michoacan, 58100, Mexico Centro Multidisciplinario de Estudios en Biotecnología, Facultad de Medicina Veterinaria y Zootecnia, Universidad Michoana de San Nicolás de Hidalgo, Morelia, Michoacan, 58100, Mexico School of Chemical Sciences, Meritorious Autonomous University of Puebla, Puebla, Puebla, 72570, Mexico Centro Multidisciplinario de Estudios en Biotecnología, Facultad de Medicina Veterinaria y Zootecnia, Universidad Michoacana de San Nicolás de Hidalgo, Morelia, Michoacan, 58100, Mexico victor.baizabal@umich.mx

No competing interests were disclosed.

 28 4 2026 2026 15 204 20 4 2026 Copyright: © 2026 Romero-Durán MA et al. 2026 This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background

Oxidative stress and inflammation are mutually reinforcing physiological processes. The transcription factor Nrf2 (Nuclear factor erythroid 2-related factor 2) acts as the master regulator of redox homeostasis and anti-inflammatory signaling. To overcome the off-target limitations of conventional electrophilic Nrf2 activators, this study investigates selective peptide-based modulators. We present data indicating that an innate defense regulator (IDR)-1002 peptide is able to activate the Nrf2-mediated antioxidant response and mitigate inflammation in an endothelial cell model.

 Methods

We first screened several IDR peptides IDR-1 (Lys-Ser-Arg-Ile-Val-Pro-Ala-Ile-Pro-Val-Ser-Leu-Leu), IDR-1018 (Val-Arg-Leu-Ile-Val-Ala-Val-Arg-Ile-Trp-Arg-Arg), HH2 (Val-Gln-Leu-Arg-Ile-Arg-Val-Ala-Val-Ile-Arg-Ala) and IDR-1002 (Val-Gln-Arg-Trp-Leu-Ile-Val-Trp-Arg-Ile-Arg-Lys) for their ability to induce Nrf2 nuclear translocation in HEK293 and bovine endothelial cells (BEC) via Western blot and ELISA. Transcriptional activity was assessed in HepG2 cells using an ARE (Antioxidant Response Element)-luciferase reporter assay to determine the EC 50. Downstream expression of antioxidant enzymes, including HO-1 (Heme Oxygenase-1), NQO1 (NAD(P)H:quinone oxidoreductase 1), and GCLM (Glutamate-Cysteine Ligase Modifier Subunit), was quantified by ELISA. GST (Glutathione S-Transferase) activity induced by IDR-1002 was measured using a CDNB-GSH conjugation assay. Finally, the functional capacity to reduce H 2O 2-induced ROS (Reactive Oxygen Species) and TNF-stimulated inflammation was measured in BECs.

 Results

Among the tested peptides, IDR-1002 induced the most potent concentration-dependent nuclear translocation of Nrf2 in both cell lines. IDR-1002 activated ARE-dependent transcription with an EC 50 of 18.57 μM. This activation led to a significant time-dependent increase in HO-1, NQO1, and GCLM protein levels, alongside enhanced GST activity. Functionally, IDR-1002 pre-treatment resulted in a dose-dependent reduction of intracellular ROS (up to 5.4-fold at 50 μM) and a significant decrease in TNF-α expression in stimulated BECs.

 Conclusions

IDR-1002 acts as a distinct dual-function regulator that simultaneously modulates the Nrf2 antioxidant response and inhibits NF-κB-mediated inflammation. These findings highlight the potential of IDR-1002 as a promising molecular template for the design of new therapeutic approaches against chronic diseases characterized by the interplay between oxidative stress and inflammation.

 IDR-1002 peptide Nrf2 oxidative stress inflammation TNF-α. Instituto de Ciencia Tecnología e Innovación PICIR-004-2022 Secretaría de Ciencia, Humanidades, Tecnología e Innovación (SECIHTI) MARDdoctoralscholarship814880 Coordinación de la Investigación Científica, Universidad Michoacana de San Nicolás de Hidalgo ResearchProgram2023-2025 Authors would like to thank the financial support from Coordinación de la Investigación Científica, Universidad Michoacana de San Nicolás de Hidalgo, Research Program 2023-2025, and Instituto de Ciencia Tecnología e Innovación (PICIR-004-2022) to VMBA. MARD is a recipient of the doctoral scholarship 814880 from Secretaría de Ciencia, Humanidades, Tecnología e Innovación (SECIHTI). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Amendments from Version 1

This new modified article version 2 has incorporated most of the critical comments made by the Reviewers. We have updated 1) the Conclusion in the abstract; 2) the Introduction section regarding the importance of HEK293 and Endothelial cells; 3) the Methods section by explaining in more detail the techniques used; 4) the Results section with respect to Lamin control data (Figure 1A), new beta-actin controls in Figures 2B and C. Also, the Y axis legend in Figure 4 was changed to "Relative intracellular redox state"; 5) the Discussion section has been thoroughly revised to better clarify our findings and incorporate the some comments from the Reviewers and 6) the References section was modified to include recent high impact reports (2021-2025).

 Introduction

Reactive oxygen species (ROS) are essential signaling molecules that, at low concentrations, contribute to physiological cellular functions, including cell proliferation, differentiation, and host defense. However, excessive accumulation of ROS or reactive nitrogen species (RNS) disrupts redox homeostasis and induces oxidative stress, leading to damage of lipids, proteins, and DNA that may cause numerous chronic and acute diseases, including neurodegenerative disorders, cancer, cardiovascular diseases, and infections. 1– 3

The transcription factor nuclear factor erythroid 2–related factor 2 (Nrf2) is a central regulator of the cellular antioxidant response that orchestrates a protective gene expression program against oxidative and electrophilic stress. 3– 5 Under basal conditions, Nrf2 is sequestered in the cytoplasm by the Kelch-like ECH-associated protein 1 (Keap1), which targets it for ubiquitination and proteasomal degradation. Upon exposure to various stress agents, Nrf2 dissociates from Keap1 and translocates to the nucleus, where it binds to the antioxidant response element (ARE) and induces the transcription of phase II detoxification enzymes such as heme oxygenase-1 (HO-1), NAD(P) H quinone oxidoreductase 1 (NQO1), and glutamate–cysteine ligase modifier subunit (GCLM), among others. 3, 6

Due to its central role in cytoprotection, Nrf2 is currently a promising therapeutic target for treating diseases associated with oxidative stress and chronic inflammation. Several small-molecule Nrf2 activators, including sulforaphane, chalcone, and fumaric acid derivatives such as dimethyl fumarate (DMF), have been investigated in preclinical and clinical settings. These compounds typically act as electrophiles that modify cysteine residues (e.g., Cys151, Cys273, Cys288) on Keap1 to disrupt its interaction with Nrf2. 3, 6, 7 Although these molecules appear to have appropriate characteristics, they have been observed to alter the activity of other proteins bearing Cys residues. This may non-selectively modify other cysteine-containing proteins, leading to off-target effects. Because of these limitations, there is increasing interest in the development of selective peptide-based modulators that can disrupt the Nrf2–Keap1 interaction with higher specificity and fewer side effects. 8 Nevertheless, the ability of peptides to simultaneously activate Nrf2 signaling while inhibiting a pro-inflammatory response mediated by nuclear factor kappa B (NF-κB) remains underexplored. 9– 12

Interestingly, some natural peptides have shown dual regulatory functions by activating Nrf2 and concurrently inhibiting NF-κB-mediated inflammation. For instance, the decapeptide YD1 (Ala-Pro-Lys-Gly-Val-Gln-Gly-Pro-Asn-Gly) induces an increase in Nrf2 activity and expression of downstream antioxidants such as HO-1 and NQO1, while suppressing pro-inflammatory mediators through TLR4/MyD88/NF-κB pathway inhibition. 11 Similarly, peptides such as K-8-K (Lys-Val-Leu-Pro-Val-Pro-Gly-Lys), S-10-S (Ser-Leu-Val-Asn-Asn-Asp-Asp-Arg-Asp-Ser), and LP-5 (Leu-Pro-Val-Thr-Lys), derived from milk, soy, and walnuts, respectively, 13, 14 show comparable dual activity by promoting antioxidant enzyme expression and reducing inflammasome activation. These findings highlight that modulation of both oxidative stress and inflammation is an emerging characteristic among certain natural peptides. 11

This dual regulatory capacity is particularly relevant for the vascular endothelium. Endothelial cells (ECs) are no longer viewed as passive vascular barriers, but as active immunological sentinels positioned at the critical interface between systemic circulation and tissues. As primary sensors of hemodynamic and chemical stimuli, ECs maintain vascular homeostasis through a unique metabolic profile that relies heavily on glycolysis to minimize mitochondrial ROS production. 15, 16 This strategic position allows them to function as early checkpoints in the transition from systemic inflammatory cues to localized tissue injury. 17 Upon encountering inflammatory stimuli, ECs rapidly respond by producing pro-inflammatory cytokines, most notably tumor necrosis factor-alpha (TNF-α). This production triggers a cascade that upregulates adhesion molecules (e.g., ICAM-1 and VCAM-1) and chemokines, orchestrating leukocyte recruitment and transmigration into the parenchyma. 18, 19 Beyond immune cell trafficking, endothelial-derived TNF-α directly disrupts intercellular tight junctions (e.g., ZO-1 and Occludin), leading to vascular ‘ leakiness’ and subsequent tissue damage. 20 Therefore, fortifying this “ vascular gateway” through the Keap1-Nrf2 signaling pathway represents a strategic approach to mitigating chronic inflammation and oxidative stress. In this study, we utilize Bovine Endothelial Cells (BECs) as a high-fidelity model. BECs provide a robust representation of large-mammal vascular responses and hold significant clinical relevance in bovine-specific inflammatory conditions, such as mastitis and respiratory disease. 21 Given the importance of the endothelium in the inflammatory response, synthetic peptides derived from natural host defense templates represent a valuable strategy for targeted intervention.

In this context, the innate defense regulator (IDR) anti-inflammatory peptides IDR-1 (Lys-Ser-Arg-Ile-Val-Pro-Ala-Ile-Pro-Val-Ser-Leu-Leu), 22, 23 IDR-1018 (Val-Arg-Leu-Ile-Val-Ala-Val- Arg-Ile-Trp-Arg-Arg), 17 HH2 (Val-Gln-Leu-Arg-Ile-Arg-Val-Ala-Val-Ile-Arg-Ala), 24 and IDR-1002 (Val- Gln-Arg-Trp-Leu-Ile-Val-Trp-Arg-Ile-Arg-Lys) 24– 26 emerge as good candidates to test their ability to modulate the activity of Nrf2.

In particular, IDR-1002 was identified from a library of bactenecin, an antimicrobial peptide found in bovine neutrophils, 25 as a peptide able to confer protection against invasive Staphylococcus aureus infection through chemokine induction. 26 Furthermore, IDR-1002 significantly reduced the production of reactive oxygen and nitrogen species (ROS/RNS) and attenuated inflammation and tissue damage in vivo in a mouse ear inflammation model. 27 Our group has also previously reported that IDR-1002 modulates inflammation in RAW 264.7 macrophages challenged with lipopolysaccharide (LPS), TNFα, or IL-1β via inhibition of IκBα phosphorylation and NF-κB p65 nuclear translocation. 28 Based on these findings, we hypothesized that IDR-1002 may also be able to upregulate an antioxidant response through Nrf2 signaling in addition to its known anti-inflammatory activity. Supporting this notion, another study using a chicken hepatocyte non-parenchymal cell co-culture showed that IDR-1002 reduces pro-inflammatory cytokine release while increasing Nrf2 production, highlighting its dual role in regulating inflammation and antioxidant responses; however, definitive proof of its direct effects on Nrf2 activation and subsequently the downstream antioxidant enzyme expression was not assessed. 12

Thus, in this study, we present experimental evidence regarding the potential of IDR-1002 to modulate Nrf2-mediated antioxidant responses in human embryonic kidney (HEK293) cells and bovine endothelial cells (BEC). Our data demonstrate that IDR-1002 induces Nrf2 nuclear translocation, enhances ARE-driven transcriptional activity, upregulates the production of key antioxidant enzymes (HO-1, NQO1 and GCLM), and promotes Glutathione S-Transferase (GST) activity. Furthermore, IDR-1002 reduced general oxidative stress in H 2O 2-stimulated cells and attenuated TNF-α production in TNFα-challenged BECs. Together, these results highlight the dual regulatory potential of IDR-1002 on redox and inflammatory pathways under the experimental conditions tested. These findings suggest that IDR-1002 could serve as a promising molecular scaffold for the development of peptide-based agents aimed at fine-tuning the Nrf2 and NF-κB signaling axes.

 Materials and methods Peptide synthesis

The immunomodulatory peptides IDR-1018 (VRLIVAVRIWRR-NH2), IDR-HH2 (VQLRIRVAVIRA-NH2), IDR-1 (KSRIVPAI-PVSLL-NH2), and IDR-1002, (VQRWLIVWRIRK-NH2) with the C-terminal amidated were synthesized by solid-phase Fmoc chemistry (CPC Scientific, USA). 29 The purity of the synthetic peptides was confirmed to be greater than 95% by high-performance liquid chromatography (HPLC) and mass spectrometry (MS). Lyophilized peptides were reconstituted in sterile water or PBS and stored at −80°C until use.

 Antibodies and reagents

Primary and secondary antibodies used: mouse β-actin (sc-47778), laminin (sc-7293), GAPDH-IgG (sc-32233), and anti-mouse IgG-BP-HRP (sc-525409) (Santa Cruz Biotechnology, USA); Histone H3 Rabbit (9717), rabbit NQO1 (62262S), and anti-rabbit IgG-HRP (7074S) (Cell Signaling Technology, USA); rabbit Nrf2 (ADI-KAP-TF125) and HO-1 (ADI-OSA-150-F) (Enzo Life Sciences, USA). Additional reagents included non-fat dry milk (Bio-Rad, USA), Luminol (Millipore, USA), protease and phosphatase inhibitor cocktail (cOmplete™, Roche, Switzerland), tert-butyl hydroperoxide (TBHP; Sigma-Aldrich, cat. 458139), ferrous sulfate (FeSO 4; cat. 7782-63-0), D, L-sulforaphane (SFN; cat. S4441), Tris-HCl, NaCl, 1-chloro-2,4-dinitrobenzene (CDNB), hydrogen peroxide (H 2O 2; cat. 7722-84-1), DCFDA (cat. 4091-99-0), Igepal CA-930, Na-pyrophosphate, NaF, Na-orthovanadate, RIPA buffer (cat. R0278), and chemiluminescent substrate (Millipore, USA). TNFα was purchased from (ACROBiosystem-Switzerland; cat. TNA-H4211). Cell culture media and supplements were obtained from BPS Bioscience (USA), including MEM, Thaw Medium 1, 1K nutrient medium, fetal bovine serum (FBS), non-essential amino acids, sodium pyruvate, penicillin/streptomycin, geneticin (cat. 79533), and Luciferase ONE-Step reagent (cat. 60690). Trypsin-EDTA 1X (cat. T2601), L-glutathione reduced (gsh, cat. G4251), Bradford Reagent (cat. B6916) and MTT (3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (cat. 475989) were purchased from Sigma-Aldrich.

 Cell culture

ARE-Hep-G2 cells (Hep-G2 cells) (BPS Bioscience, cat. 60513) which express luciferase under control of ARE sequences were cultured in 1K medium containing DMEM supplemented with 10% FBS, 1% non-essential amino acids, 1 mM sodium pyruvate, 1% penicillin/streptomycin, and 600 μg/mL geneticin at 37°C in a humidified atmosphere with 5% CO 2. Cells between passages 8–23 were used. BEC cells (bovine umbilical vein endothelial cells) were kindly provided by Dr. Carmen Clapp from the Institute of Neurobiology, National Autonomous University of Mexico (UNAM). These cells were immortalized by transfection with HPV16 E6E7 oncogenes, that extend their replicative lifespan of primary BEC from 40 to more than 120 passages without signs of senescence. Importantly, BEC cells retain key endothelial characteristics, including the uptake of acetylated low-density lipoproteins, von Willebrand factor expression, specific lectin binding, and proliferative responses to vascular endothelial growth factor. 21 HEK-293 cells were purchased from the American Type Culture Collection (ATCC; cat. CRL-1573). HEK-293 and BEC cells were cultured in DMEM supplemented with 10% FBS, 10,000 U/mL penicillin, and 1 mg/mL streptomycin, at 37°C in 5% CO 2. Cells between passages 8 and 20 were used.

 Luciferase reporter assay

Hep-G2 cells 30 were seeded at a density of 40,000 cells/well in 96-well plates using 45 μL of 1 K medium without geneticin. Cells were treated with 5 μL of IDR-1002 (final concentration: 0.5–300 μM) or FeSO 4 (100 μM) as a positive control. Following an 8–10 h incubation period at 37°C, 100 μL of Luciferase ONE-Step reagent was added, and plates were shaken for 15 min. Luminescence was measured using a Varioskan TM LUX multimode microplate reader (Thermo Fisher Scientific, USA). To ensure high signal-to-noise ratio and data accuracy background signal and cellular autofluorescence were subtracted from all treatment groups. Fold induction was calculated by normalizing the corrected luminescence values to the untreated controls. Tert-butyl hydroperoxide (TBHP), FeSO 4, and sulforaphane (SFN) were utilized as established Nrf2-pathway activators.

 MTT cell viability assay

Hep-G2 and BEC cells were seeded in 96-well plates at 5 × 10 4 cells/mL and incubated for 24 h at 37°C. Cells were then treated with IDR-1002 (1–100 μM) or TBHP (10 μM) for 8 h. Following treatment, 50 μL of MTT solution (5 mg/mL in PBS, for a final concentration of 10% v/v) was added to each well and incubated for 4 h at 37°C. The culture medium was then carefully aspirated, and the resulting formazan crystals were solubilized in 100 μL of 100% DMSO. Absorbance was read at 570 nm using a VarioskanTM LUX reader (Thermo Fisher Scientific, USA). Cell viability was calculated as a percentage relative to the untreated control cells (100% viability), and background absorbance from cell-free blank wells was subtracted from all readings to ensure data accuracy.

 Assessment of general intracellular oxidative stress

General intracellular oxidative stress levels were determined using 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA; Sigma-Aldrich, USA). 31– 33 BEC cells were seeded in black-walled, clear-bottom 96-well plates (Corning, USA) to prevent fluorescence cross-talk between adjacent wells. Cells were pretreated with IDR-1002 (1–50 μM) for 4 h and subsequently incubated with 30 μM DCFH-DA in PBS at 37°C for 30 min in the dark. During this period, the non-fluorescent DCFH-DA is internalized and hydrolyzed by intracellular esterases into DCFH. Following incubation, cells were washed with PBS to remove the extracellular probe and then stimulated with 50 μM H 2O 2 for 20 min. The oxidation-mediated conversion of DCFH to the highly fluorescent 2′,7′-dichlorofluorescein (DCF) was quantified using a Varioskan TM LUX multimode microplate reader (Thermo Fisher Scientific, USA) at excitation/emission wavelengths of 485/530 nm. To ensure data accuracy, background fluorescence was corrected by subtracting the signal from cell-free blank wells (to account for probe auto-oxidation) and untreated control cells (to account for cellular autofluorescence). Results were expressed as Relative Intracellular Redox State (Fold Change) normalized to control groups.

 Protein extraction and Western blotting

BEC cells were grown in 6-well plates to ~90% confluence, serum-starved for ≥4 h, and then treated with 1, 10, 25, or 50 μM IDR-1002 for 1 h or 4 h before lysis for subsequent Western blots of Nrf2, HO-1, and NQO1. Total protein (cytosolic plus nuclear from control and treated cells) was extracted by washing cells with cold PBS and lysing them with 80 μL of a cold buffer containing 20 mM Tris–HCl pH 7.5, 150 mM NaCl, 1% Igepal CA-930, 10 mM Na-pyrophosphate, and 50 mM NaF supplemented with 1 mM Na-orthovanadate and 1x protease inhibitors. Lysates were centrifuged at 13,000 × g (20 min, 4°C), and supernatants collected and transferred to ice-cold Eppendorf tubes. For subcellular distribution studies, cytoplasmic and nuclear fractions were isolated using the NE-PER Nuclear and Cytoplasmic Extraction Kit (Thermo Scientific) according to the manufacturer’s instructions. The resulting nuclear pellets were resuspended in 80 μL of RIPA buffer and sonicated with a Qsonica Q125 sonicator (Newtown, USA) at 25% amplitude for 15 s (3 × 5 s cycles, with 7 s rest intervals) to ensure complete protein solubilization. 28 Protein concentration was measured by the Bradford method using BSA as standard. 34 Samples (50-60 μg) were separated by 10% SDS-PAGE and transferred in a wet chamber to 0.22 μm nitrocellulose membranes for 1 h at 250–300 mA. Detection was performed using appropriate antibodies and Immobilon HRP chemiluminescent kit. Imaging was done with the LI-COR Odyssey system.

 Nrf2 and antioxidant enzymes quantification

Intracellular levels of Nrf2, HO-1, NQO1, and GCLM were quantified using competitive ELISA kits (MyBioSource, USA). Briefly, 100 μL of standards or cell lysates were added to pre-coated plates along with 10 μL of Balance Solution. Samples were then incubated with 50 μL of HRP-conjugate for 1 h at 37°C, where endogenous antigens competed for conjugate binding sites. After five washes, TMB substrate was added for color development and incubated in the dark at 37°C. The reaction was stopped with 50 μL of Stop Solution and the absorbance was measured at 450 nm. Results were inversely proportional to the target protein concentration and were normalized to total protein content for each sample.

 Glutathione S-transferase activity assay

BEC cells were treated with 50 μM IDR-1002 for 6, 12, 18, or 24 h. Following treatment, cell lysates were prepared in 50 mM Tris-HCl buffer (pH 7.4) containing 150 mM NaCl and protease inhibitors. Total GST activity was determined by monitoring the conjugation of 1-chloro-2,4-dinitrobezene (CDNB) with reduced glutathione (GSH). Briefly, cell lysates (containing 35 μg of total protein) were incubated with a reaction mixture consisting of 1 mM CDNB and 1 mM GSH (prepared from 100 mM concentrated stocks) in a phosphate-buffered saline (PBS) solution adjusted to pH 6.5. The enzymatic reaction was monitored at 340 nm using a microplate reader every 30 seconds for 5 minutes at [30°C] to determine the initial reaction velocity ( V 0). 35– 37

To account for non-enzymatic (spontaneous) conjugation of CDNB, a reagent blank (without cell lysate) was included in each run. This background absorbance was subtracted from all experimental readings to calculate the net enzymatic activity. Specific activity was determined using the molar extinction coefficient for the GSH-CDNB conjugate ( ε = 9.6 mM −1cm −1) and a light path of [0.6 cm for a 96-well plate or 1 cm for a cuvette]. Final values were calculated as units of activity per milligram of protein (U/mg). To account for variations in basal enzymatic levels across independent experiments and facilitate the comparison of biological induction, data were normalized and expressed as fold change relative to the unstimulated control (CTRL).

 TNFα quantification

Human TNFα levels in BEC supernatants were quantified by a sandwich enzyme-linked immunosorbent assay (ELISA) using the MBS267654 kit (MyBioSource, USA). Briefly, cell culture supernatants were centrifuged at 1000–3000 rpm for 10 min to remove debris. Standards (ranging from 15.6 to 1000 pg/mL) and 100 μL of samples were added to pre-coated wells and incubated at 37°C for 90 min. Following two washes with 350 μL of buffer, 100 μL of biotinylated detection antibody (1:100) was added and incubated at 37°C for 60 min. After three additional washes, the plate was incubated with 100 μL of HRP-avidin conjugate for 30 min at 37°C. The colorimetric reaction was developed by adding 100 μL of TMB substrate for up to 30 min in the dark at 37°C, then stopped with 100 μL of Color Reagent C (H 2SO 4). Absorbance was measured at 450 nm using a microplate reader within 10 minutes of stopping the reaction. Sample concentrations were determined by interpolation from the standard curve, with a minimum detectable sensitivity of 5 pg/mL.

 Statistical analysis

All data are presented as mean ± standard deviation (SD). Analyses were conducted using GraphPad Prism 8 (GraphPad Software, USA). Unpaired Student’s t test was used for two-group comparisons while one-way ANOVA with Tukey’s post hoc test was applied for multiple comparisons. Statistical significance is indicated by * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; ns = not significant and ND = not detected. We employed Tukey’s post-hoc test as it provides a rigorous framework for all-to-all comparisons while strictly controlling the family-wise error rate, ensuring that the observed dose-dependent effects of IDR-1002 are statistically robust across all experimental groups as established in the Honestly Significant Difference (HSD) methodology. 38

Results IDR peptides promote Nrf2 nuclear translocation in HEK293 cells

Several synthetic protein-protein interaction (PPI) inhibitors of Keap1-Nrf2 have been reported to activate the Nrf2 pathway; however, their use is limited by low selectivity and cytotoxicity effects. 39, 40 In this context, IDR peptides have emerged as promising alternatives due to their immunomodulatory properties and low cytotoxic potential. 24, 41– 43 Notably, among the IDR peptides group, IDR-1002 exhibits a potent inhibitory NF-κB activity. 1, 28 Thus, we initially explored the ability of IDR-1002 and three other IDR peptides, namely IDR-1018, IDR-HH2, and IDR-1, to activate Nrf2 nuclear translocation. HEK293 cells were selected as the initial screening model due to their robust metabolic machinery and validated resistance to oxidative stress, ensuring a reliable platform for studying signal transduction pathways. 44

HEK293 cells were treated with 10 μM of each peptide and Nrf2 abundance was assessed in whole-cell, cytoplasmic, and nuclear fractions by Western blot ( Figure 1A, B). Among all peptides tested, IDR-1002 induced the strongest nuclear translocation of Nrf2. IDR-HH2 also promoted Nrf2 nuclear accumulation, albeit to a lesser extent, while IDR-1 had minimal effect. To confirm the dose-dependency of IDR-1002, increasing concentrations (up to 50 μM) were tested ( Figure 1C, D). A 15-fold increase in nuclear Nrf2 abundance was observed at 50 μM compared to a 6-fold increase at 10 μM, supporting IDR-1002 as the most potent Nrf2 activator among the tested peptides. These data suggest that sequence variations between IDR peptides influence their ability to modulate the Nrf2 signaling pathway. Based on these results and those we previously reported on NF-κB inhibition, 28 IDR-1002 was selected for subsequent experiments.

Nrf2 nuclear translocation induced by IDR peptides in HEK293 cells and by IDR-1002 in bovine endothelial cells (BEC cells).

( A, C) Western blot detection showing the relative abundance of the Nrf2 protein (MW ~100 kDa) in whole (W), cytoplasmic (C), and nuclear (N) protein enriched extracts from HEK293 cells. Cells were treated for 1 h with 10 μM IDR-1002, IDR-1018, IDR-1, and IDR-HH2 or with 50 μM IDR-1002. β-Actin (MW ~42 kDa) and laminin (MW ~70 kDa) were used as loading controls for the cytoplasmic and nuclear extracts, respectively. (B, D) Nrf2 nuclear accumulation is expressed as fold changes normalized to the untreated control (CTRL). (E) Representative Western blot showing nuclear Nrf2 levels in BEC cells treated for 1 h with 1, 10, 25, and 50 μM IDR-1002 and a positive control, tert-butyl hydroperoxide (TBHP, 10 μM). The graph above the blot shows the densitometric analysis of the relative fold change in nuclear Nrf2 levels, normalized to the untreated control (CTRL). Histone H3 (H3, 17 kDa) was used as a nuclear loading control. (F) Quantification of nuclear Nrf2 levels by competitive ELISA in BEC cells treated under the same conditions as in (E). Mean values for IDR-1002 at 10, 25, and 50 μM, as well as TBHP, were significantly increased compared to CTRL. Comparisons not indicated by asterisks were not statistically significant (ns) or not detect (ND). These results are representative of three independent experiments (n = 3). Bars indicate the mean ± standard deviation (SD). Asterisks indicate a statistically significant difference, determined by two-way ANOVA, followed by Tukey's post hoc test with the following significance levels: *p < 0.05, **p < 0.01 and ***p < 0.001.

 IDR-1002 activates Nrf2 nuclear translocation in BEC cells

After identifying IDR-1002 as a lead candidate for Nrf2 modulation in the robust HEK293 signaling model, 44 our objective was to determine whether this effect was conserved in a physiologically specialized context. Given the central role of endothelial cells in redox signaling and inflammation, and the antagonistic interplay between NF-κB and Nrf2 in vascular homeostasis, 26 , 45 – 48 we chose BEC cells as a relevant model for evaluating peptide-driven cytoprotection. As shown in ( Figure 1E), treatment with increasing concentrations of IDR-1002 induced a pronounced, concentration-dependent increase in Nrf2 nuclear translocation in BECs. These findings were further corroborated by ELISA quantification of Nrf2 levels in BEC lysates, which showed a significant increase following treatment with the peptide from 10 to 50 μM ( Figure 1F). Collectively, these data indicate that IDR-1002 effectively activates the Nrf2 nuclear translocation in both HEK293 and BEC cells, reinforcing the peptide’s potential to fortify the vascular gateway against oxidative and inflammatory challenges.

 IDR-1002 induces Nrf2 transcriptional activity

Under basal conditions, Nrf2 is retained in the cytoplasm by the kelch domain of Keap1. Upon activation, Nrf2 dissociates from Keap1, translocates to the nucleus and binds to ARE in target genes promoters, driving the expression of antioxidant genes. 49– 51

To further explore the cytoprotective potential of IDR-1002 in a tissue condition characterized by high oxidative load and detoxification demands, we evaluated Nrf2 transcriptional activity in HepG2 liver cells stably expressing an ARE-luciferase reporter. This cell line is a well-established gold standard for quantifying the potency of Nrf2-ARE signaling, as it acts as a master regulatory sensor that coordinates redox homeostasis. 52 Following stimulation of HepG2 cells with IDR-1002 for 8 h, a concentration-dependent increase in ARE-luciferase activity was observed with an EC 50 of 18.57 μM ( Figure 2A). Importantly, cell viability assays confirmed that IDR-1002 did not induce cytotoxicity neither in Hep-G2 or BEC cells at 1, 10, 25, 50, or 100 μM ( Extended data Figure E1).

IDR-1002 activates the Nrf2 transcriptional pathway and downstream phase II antioxidant enzymes in BEC cells.

(A) Measurement of Nrf2-mediated transcriptional activity in HepG2-ARE-luciferase reporter cells treated with IDR-1002 (0.5 to 300 μM) for 8 h. Activity is measured as a fold change in luminescence relative to the untreated control, resulting in an EC 50 of 18.57 μM. (B, C) Western blot analysis of the protein levels of the antioxidant enzymes HO-1 (28 kDa) and NQO1 (29 kDa) in BEC cells treated for 4 h with 1, 10, 25, and 50 μM of IDR-1002. β-Actin (45 kDa) served as a loading control for both blots. Iron (II) sulfate (FeSO 4, 150  μM) was used as a positive control. Iron (II) sulfate (FeSO 4, 150 μM) was used as a positive control. The graph above the blot represents the densitometric analysis of the relative fold change in HO-1 expression, normalized to the unstimulated control. Mean values for HO-1 at 10, 25, and 50 μM IDR-1002, and those for NQO1 at 25 and 50 μM IDR-1002 were significantly different from the control group. (D, E, F) ELISA quantification of the protein levels of the antioxidant enzymes, HO-1 (D), NQO1 (E), and GCLM (F) in BEC cells treated with 50 μM of IDR-1002 for 2 or 4 h. Protein levels were normalized to total protein content and compared to the respective time-matched untreated control (CTRL 2 h or CTRL 4 h). Treatment with IDR-1002 significantly increased the production of these antioxidant enzymes in a sustained manner across the evaluated time points. Comparisons not indicated by asterisks were not statistically significant (ns) or not detect (ND). These results are representative of three independent experiments (n = 3). Bars indicate the mean ± standard deviation (SD). Asterisks indicate a statistically significant difference, determined by two-way ANOVA, followed by Tukey’s post hoc test with the following significance levels: *p < 0.05, **p < 0.01 and *** p < 0.001.

 IDR-1002 induces the production of antioxidant enzymes

The Nrf2-dependent phase II antioxidant enzymes HO-1, NQO-1 and GCLM mitigate oxidative stress and can suppress NF-κB activity. 7 , 51 , 53 , 54 Western blot analysis confirmed the increased production of HO-1 and NQO1 following IDR-1002 treatment ( Figure 2B, C), supporting its role in Nrf2 activation and downstream antioxidant induction. This was further corroborated by ELISA-based quantification of HO-1, NQO1, and GCLM at 2 and 4 h post-treatment with 50 μM IDR-1002. Treatment with IDR-1002 triggered a significant and sustained upregulation of these antioxidant enzymes. Although the production levels remained elevated from 2 to 4 hours post-treatment, no statistically significant difference was observed between these two time points ( Figure 2D, E, F). This indicates that the induction reaches a plateau or remains stable following the initial response, maintaining the antioxidant defense throughout the evaluated period.

 IDR-1002 induces glutathione S-transferase activity

Glutathione S-transferases (GSTs) are a family of phase II detoxifying enzymes that catalyze the conjugation of glutathione (GSH) to reactive intermediates, thereby protecting the cell against oxidative damage. 53 , 55 To evaluate whether IDR-1002 modulates GST activity, BEC cells were treated with 50 μM of the peptide for 18 and 24 h. GST activity was significantly increased at 18 h ( Figure 3A). At 24 h ( Figure 3B), activity remained elevated relative to the untreated control, although a slight reduction compared to 18 h was observed, without a statistically significant difference between these two time points. No detectable GST activity was observed at earlier time points (6 and 12 h; data not shown). These findings suggest that GST activation occurs at later time points relative to the early induction of HO-1 and NQO1, supporting a temporally distinct response in which Phase II detoxification capacity develops after the initial activation of antioxidant signaling pathways.

Induction of GST activity by IDR-1002 in BEC cells.

BEC cells were treated with 50 μM IDR-1002 for 18 ( A) and 24 h ( B). Sulforaphane (SFN, 10 μM) was used as a positive control. Glutathione S-transferase (GST) activity was quantified after each treatment using the 1-chloro-2,4-dinitrobenzene (CDNB) colorimetric assay. IDR-1002-stimulated cells showed a significant increase in GST activity compared to the untreated control. Data are representative of three independent experiments (n = 3) and are presented as the mean ± standard deviation (SD). Statistical significance was determined by the two-way ANOVA with Tukey’s post hoc test. Asterisks indicate significance levels as follows: *p < 0.05, **p < 0.01, and ***p < 0.001.

 IDR-1002 modulates the intracellular redox state and mitigates general oxidative stress in H <sub>2</sub>O <sub>2</sub>-stimulated BEC cells

To assess the antioxidant capacity of IDR-1002, general intracellular oxidative stress was measured in BEC cells challenged with H 2O 2 following peptide pretreatment. BEC cells were pre-incubated with the indicated concentrations of IDR-1002 for 4 h and subsequently exposed to 50 μM H 2O 2 for 15 min. Basal redox levels remained unchanged in both control and 50 μM IDR-1002-treated cells, indicating that the peptide does not exert pro-oxidant effects under resting conditions ( Figure 4). In H 2O 2-stimulated cells, IDR-1002 pre-treatment resulted in a dose-dependent reduction of oxidative stress levels. At 25 and 50 μM, IDR-1002 decreased the fluorescent signal by approximately 2.4- and 5.4-fold, respectively, compared to the H 2O 2-only group. These findings are consistent with previous reports in which 18 μM IDR-1002 reduced H 2O 2-induced oxidative stress by 1.6-fold in different models. 12 These results demonstrate that IDR-1002 effectively modulates the intracellular redox environment most likely through the activation of the Nrf2-mediated antioxidant response.

IDR-1002 modulates the intracellular redox state and mitigates general oxidative stress in H <sub>2</sub>O <sub>2</sub>-stimulated BEC cells.

BEC cells were pretreated with 1, 10, 25, and 50 μM IDR-1002 for 4 h and subsequently incubated with 50 μM H 2O 2 for 15 min to induce oxidative stress. Intracellular ROS production was then quantified using the 2',7'-dichlorofluorescin diacetate (DCFH-DA) fluorescence assay. Data are representative of three independent experiments (n = 3) and are presented as the mean ± standard deviation (SD). Asterisks indicate a statistical difference compared to the H 2O 2-treated control, with a significance level of ***p < 0.001.

 IDR-1002 reduces TNF-α production

Chronic oxidative stress is tightly linked to inflammatory processes, with TNF-α acting as a central mediator in multiple redox-associated pathologies, including cancer, neurodegenerative disorders, metabolic diseases, and cardiovascular conditions. 56 – 60 A bidirectional relationship exists between ROS and TNF-α, where oxidative stress can stimulate TNF-α production, which in turn further amplifies intracellular ROS levels, reinforcing the inflammatory response. To evaluate the effect of IDR-1002 on TNF-α production, BEC cells were pre-incubated with 1, 10, 25, and 50 μM of the peptide for 1 h, followed by stimulation with 10 ng/mL TNF-α for an additional 1 h. Supernatants were then collected, and TNF-α levels were quantified by ELISA. IDR-1002 significantly reduced TNF-α production at concentrations ≥10 μM ( Figure 5), supporting its anti-inflammatory activity. These findings are consistent with previous reports describing IDR-1002 as a modulator of NF-κB signaling in macrophages. 28

IDR-1002 decreases TNF-α production in BEC cells stimulated with TNFα.

BEC cells were pretreated with 1, 10, 25, and 50 μM IDR-1002 for 1 h and subsequently incubated with 10 ng/mL TNFα for an additional 1 h. Supernatants were collected and TNFα levels were quantified by ELISA. These results are representative of three independent experiments (n = 3). Bars indicate the mean ± SD, and asterisks indicate statistical difference, ** p <0.01 ; ***P ˂0.001, n = 3, by the 2-way ANOVA method post hoc Tukey.

 Discussion

This study demonstrates that the immunomodulatory 12-residue cationic peptide IDR-1002 effectively activates the Keap1–Nrf2 signaling pathway, thereby promoting both antioxidant and anti-inflammatory responses in both human and bovine cell models. The choice of HEK293 cells for the initial characterization of IDR-1002 is supported by recent metabolic profiling, which identifies this lineage as a robust model for studying oxidative stress resistance. According to Sapeta-Nowińska et al. (2025), 44 HEK293 cells exhibit a highly efficient adaptive metabolic response, characterized by the precise regulation of key metabolites that support cellular defense mechanisms. This innate resilience makes them an ideal ‘baseline’ to evaluate the efficacy of Nrf2 activators, ensuring that the observed nuclear translocation and subsequent enzymatic induction are mediated by specific signaling modulations rather than generalized cellular distress. Building upon this mechanistic foundation, the inclusion of HepG2 cells in our study strengthens the rationale for IDR-1002 as a systemic modulator of the antioxidant response. While HEK293 cells served as a robust initial model for signaling fidelity, 44 and BECs offered a context to link Nrf2 activation with its theoretical role in preserving vascular integrity, 16 the results in HepG2 cells highlight its potential in high-demand metabolic tissues. The liver is the primary site for neutralizing reactive species and maintaining systemic redox balance, 52 consequently, the ability of IDR-1002 to upregulate the Nrf2-driven program in this model suggests a broad therapeutic utility. By activating Nrf2 in HepG2, IDR-1002 demonstrates a capacity to upregulate cytoprotective pathways where oxidative load is prevalent, which suggests a role in mitigating localized vascular damage and systemic oxidative stress through Nrf2-mediated defenses. The inclusion of HEK-293 and HepG2 models alongside BECs provides a comprehensive assessment of IDR-1002’s versatility. By demonstrating consistent activation of the Keap1-Nrf2 pathway across vascular, renal, and hepatic lineages, we establish that the peptide’s anti-inflammatory and antioxidant regulatory effects are part of a conserved, robust pharmacological mechanism and not a cell-specific artifact.

In addition, our data demonstrate that IDR-1002 promotes the induction of Nrf2-dependent proteins, which play a central role in maintaining cellular redox homeostasis and mitigating oxidative damage. The increased production of HO-1, NQO1, and GCLM was significant compared to control groups at both 2 and 4 hours, reflecting a durable activation of the antioxidant response rather than a linear time-dependent increase within the studied temporal window. This sustained profile suggests that IDR-1002 effectively primes the cellular defense mechanism potentially enhancing the cell’s readiness to withstand subsequent oxidative insults. Furthermore, GST activity evaluation suggests that IDR-1002 induces a robust antioxidant response that eventually stabilizes over prolonged periods. Since GSTs mediate the conjugation of glutathione to electrophilic compounds promoting their clearance and reducing cumulative damage from oxidative stress and lipid peroxidation-derived products, 61 their significant activity at 24 h strengthens the cellular defense against sustained oxidative challenges. This temporal profile, following the earlier production of HO-1 and NQO1, highlights a coordinated and sequential enzymatic response triggered by the peptide.

Nuclear translocation of Nrf2 induced by IDR-1002 in both HEK293 and BEC cells was dose-dependent and correlated well with a significant increase in ARE-luciferase reporter activity in HepG2 cells, with an EC 50 under 20  μM. These results are comparable in terms of efficacy to those reported for established Nrf2 activators such as TBHP and SFN. 62 While SFN exhibits higher potency achieving pathway activation at lower concentrations, IDR-1002 demonstrates a similar maximal induction of the Nrf2-ARE signaling.

Consistent with these antioxidant effects, IDR-1002 significantly reduced intracellular oxidative stress following H 2O 2 stimulation. This attenuation of cellular redox imbalance contributes to its anti-inflammatory activity, as evidenced by a marked reduction in TNF- α protein levels in TNF-α-stimulated BECs. The observed decrease in endothelial TNF-α highlights a key anti-inflammatory mechanism operating at the vascular interface. Given that endothelial-derived TNF-α amplifies inflammatory signaling through both autocrine and paracrine pathways, its suppression suggests that IDR-1002 interferes with early events required for leukocyte recruitment and endothelial activation. 15 , 19 Of note, TNF-α is also a major driver of endothelial barrier dysfunction, promoting cytoskeletal rearrangement and disruption of tight junction proteins, which leads to increased vascular permeability and tissue injury. 20 Elevated endothelial TNF-α is a hallmark of multiple inflammatory pathologies and contributes directly to vascular hyperpermeability and tissue damage. 17 Therefore, the ability of IDR-1002 to attenuate TNF-α expression may preserve vascular integrity, limiting both leukocyte recruitment and the propagation of inflammation from the circulation into target tissues. Mechanistically, this effect is in agreement with the established crosstalk between the Nrf2 and NF-κB pathways, whereby activation of Nrf2 negatively regulates NF-κB-dependent transcription of pro-inflammatory mediators, including TNF-α. 63 , 64 Hence, the inhibition of TNF-α serves as a functional validation of Nrf2 activation by IDR-1002 that leads to a biologically meaningful anti-inflammatory outcome in a primary endothelial model.

In light of the evidence presented in our previous work, 28 regarding the inhibitory effect of IDR-1002 on the IKK/IκBα axis and the activation of p38/ERK1/2–MSK1-dependent CREB phosphorylation, a direct modulation of NF-κB signaling was initially considered. However, the strong association observed here between Nrf2 activation and the induction of Phase II enzymes (HO-1, NQO1, GCLM) supports a more integrated mechanism. Accordingly, we have refined our interpretation to propose the Nrf2 signaling as the primary driver of the observed immunomodulatory phenotype. This model moves beyond a strictly p65-centered explanation and instead supports a mechanism of “ redox interference,” which means IDR-1002 promotes a redox-stable intracellular environment through Nrf2 that indirectly antagonizes the pro-inflammatory signaling. 65 , 66 By simultaneously preventing IκBα degradation and enhancing antioxidant defenses, IDR-1002 orchestrates a comprehensive cytoprotective response, positioning it as a sophisticated candidate for modulating inflammatory diseases through dual signaling pathways.

Based on its reported capacity to inhibit the NF-κB pathway and activate Nrf2, IDR-1002 shares functional similarities with several naturally occurring peptides such as YD1 (a decapeptide from kimchi), 11 K-8-K (an octapeptide from milk), and S-10-S (a decapeptide from soy). 13 These peptides promote Nrf2 nuclear translocation and suppress inflammation, at least in part, by preserving IκB. Another peptide in this category is LP-5, a pentapeptide derived from walnut protein, that mitigates oxidative stress and inflammation through Nrf2 activation, increasing the activity of superoxide dismutase and catalase, and reducing the activation of the NLRP3 inflammasome. 14 While these natural peptides offer promising biological profiles, IDR-1002 provides distinct advantages, including synthetic accessibility, the modularity of its amino acid sequence for optimization, and a well-characterized immunomodulatory profile across diverse models. 12 , 26 , 28 , 42 Recent evidence underscores the growing importance of bioactive peptides as strategic tools to target Nrf2 for alleviating inflammation. 67 Specifically, in endothelial cell physiology, these peptides are now recognized as pivotal regulators at the mechanistic and pharmacological crossroads of vascular health. 68

The broader immunomodulatory activities previously described for IDR peptides help contextualize the differential Nrf2 responses observed in this study. Evidence from other innate defense regulator peptides supports the dual antioxidant and anti-inflammatory profile observed for IDR-1002. Studies in human neutrophils have shown that IDR-1018 and HH2 reduce ROS production, suppress TNF-α release, and promote LL-37 secretion, demonstrating that IDRs can simultaneously modulate oxidative and inflammatory pathways. 17 Additionally, IDR-1 has been reported to interact with the ZZ domain of p62/SQSTM1, a key regulator of the Keap1–Nrf2 axis, providing a mechanistic explanation on how certain IDRs may facilitate Nrf2 stabilization through p62-dependent sequestration of Keap1. 69

Despite that IDR-1002 induces a more pronounced Nrf2 nuclear accumulation than IDR-1 in the HEK293 cell model, the specific contribution of p62-dependent pathways or other upstream regulatory kinases for this particular peptide remains to be fully elucidated. In our model, the calculated EC 50 for Nrf2-driven transcriptional activity was 18.57 μM, reflecting a nuanced potency in the reporter assay; however, the pronounced nuclear presence suggests that IDR-1002 effectively facilitates the initial stages of the antioxidant response. This Nrf2 nuclear accumulation, coupled with its previously reported NF-κB inhibition, supports the idea that IDR-1002 integrates both antioxidant and anti-inflammatory activities more effectively than other related synthetic peptides. These results indicate that while the peptide’s potency for gene induction is moderate, its capacity to modulate cellular redox sensors is notable. Although the precise molecular target of IDR-1002 that bridges these two nodes is currently under investigation, our functional data clearly position this peptide as a versatile dual-function regulator within the innate defense regulator family.

Although these findings are promising, further experimental validation is necessary to confirm direct binding between IDR-1002 and its biological target in order to determine if this interaction modulates Keap1-mediated degradation. Based on our previous finding that lactoferricin B-derived peptide (FKC) targets TNFR1, 70 and our recent observations that FKC also activates Nrf2, we investigated if IDR-1002 shared this mechanism. However, FRET-based monitoring of TNFR1 conformational dynamics showed no inter-monomeric changes, indicating that, unlike FKC, IDR-1002 is not a direct receptor antagonist or allosteric modulator. Consequently, its dual activity must involve an alternative receptor or signaling node, narrowing the search for its primary cellular target. A leading candidate for such a role is the Keap1-Nrf2 axis, which remains a premier target for therapy due to its central role in preventing chronic inflammatory and oxidative damage. 71 , 72 The recent development of high-affinity, selective inhibitors of the Keap1–Nrf2 protein-protein interaction (PPI) by Lin et al. (2025) provides a compelling structural precedent for such a possibility. 73

Given its specific size and cationic nature, it is plausible to suggest that IDR-1002 might act as a competitive or allosteric inhibitor of this PPI, potentially favored by the electrostatic affinity between the peptide’s positive residues and the anionic motifs of the Nrf2 Neh2 domain (specifically the DLG and ETGE). Under this hypothetical framework, IDR-1002 could potentially offer a more targeted and cytocompatible alternative for Nrf2 activation compared to classical electrophilic agents; by potentially avoiding the covalent modification of cellular thiols, the peptide is expected to minimize the risk of non-specific off-target effects. Nevertheless, further computational or biophysical validation remains essential to definitively characterize the binding kinetics of this interaction. Future studies utilizing Molecular Dynamics (MD), Surface Plasmon Resonance (SPR) or Isothermal Titration Calorimetry (ITC) will be required to confirm the physical binding constants and thermodynamic profile between the peptide and the specific Neh2 motifs, bridging the gap between our functional observations and the underlying molecular recognition. Although, the consistent correlation between Nrf2 nuclear translocation and the specific induction of downstream enzymes (HO-1, NQO1, GCLM) provides compelling evidence of the pathway’s activation by IDR-1002, we acknowledge that absolute Nrf2-dependence for the observed antioxidant protection remains to be definitively confirmed. Specifically, while IDR-1002 treatment resulted in a significant improvement in the intracellular redox state demonstrated by an enhanced capacity to neutralize H 2O 2 challenges, we recognize that proving this protection is exclusively Nrf2-dependent requires genetic silencing or chemical inhibition.

As an alternative we decided to determine a robust functional validation approach due to the significant biological confounding factors associated with Nrf2-deficient models. First, Nrf2-deficient cells exhibit profound mitochondrial instability and NADPH deficits, which in endothelial models like BECs, leads to hypersensitivity to routine handling and ‘handling-induced death’. 74 Second, current pharmacological tools present significant specificity risks; ML385 has been recently highlighted for its cross-reactivity with structurally homologous factors like Nrf1 and CREB, 75 , 76 while Brusatol acts as a global translation inhibitor affecting multiple essential regulators like c-Myc, 77 making it difficult to discriminate Nrf2-specific effects. Moreover, stable knockdown via CRISPR or shRNA often triggers deep redox reprogramming and compensatory mechanisms (e.g., Nrf1 stabilization), 78 , 79 potentially deviating from the original physiological state of BEC cells. Future studies using transient, high-specificity interference strategies will be essential to conclusively link the molecular activation of Nrf2 to the global redox fortification observed in our BEC model.

Finally, despite the encouraging evidence presented here, a deep insight on the pharmacokinetic properties, bioavailability, and potential off-target effects requires further investigation. Additionally, validation of these findings in animal models of oxidative and inflammatory diseases are mandatory. In spite of these limitations, IDR-1002 emerges as a non-cytotoxic, dual-function synthetic peptide that simultaneously modulates Nrf2 and NF-κB signaling. Its capacity to co-regulate these pathways provides a valuable model for studying the interplay between cellular redox homeostasis and inflammatory signaling, potentially informing the future development of more targeted immunomodulatory strategies.

 Data availability Underlying data

Figshare: Files that contain data on Nrf2 activation by the cationic peptide IDR-1002. https://doi.org/10.6084/m9.figshare.31093330. 80

WB peptides IDR, densitometry data.xlsx (Data used to generate Figures 1A-D).

WB Nrf2 data analysis HEK293 cells; peptides IDR.pzfx (Data used for statistical analysis of Figures 1A and D).

Densitometry data WB in BEC cells with (IDR-1002).xlsx (Data used to generate Figures 1E and F; Figures 2B and C).

WB Nrf2 data analysis BEC cells; IDR-1002.pzfx (Data used for statistical analysis of Figures 1E and F).

HepG2 EC50 assay IDR-1002; raw and normalized data.pzfx (Data used to generate Figure 2A).

HO-1_NQO1_GCLM ELISA normalized data 2h and 4h.pzfx (Data used to generate Figures 2E-F).

GST activity assay 6,12,18,24 h; raw and normalized data.pzfx (Data used to generate Figure 3).

General intracellular oxidative stress assay; raw data and normalized.pzfx (Data used to generate Figure 4).

TNF alpha assay; raw and normalized data.pzfx (Data used to generate Figure 5).

Quantitative Assessment of Subcellular Fractionation Purity and Signal Correction data.xlsx

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).

 Extended data

Figshare: Files that contain the supplementary Figure S1 and data on IDR-1002 cytotoxicity. https://doi.org/10.6084/m9.figshare.31116373.v1. 81

MTT assay in Hep G2 cells 8 h; raw and normalized data.pzfx (Data used to generate supplementary Figure 1A).

MTT assay in bovine endothelial cells (BEC) 8 h; raw and normalized data.pzfx (Data used to generate supplementary Figure 1B).

Supplementary FIGURE S1_Romero-Duran-2026.tif

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).

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Figshare. 2026. https://doi.org/10.6084/m9.figshare.31116373.v1 10.5256/f1000research.198845.r479955 Reviewer response for version 2 O'Rourke Sinead 1 Referee Trinity College Dublin, Dublin, Ireland

Competing interests: No competing interests were disclosed.

 12 6 2026 Copyright: © 2026 O'Rourke S 2026 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation reject

The authors have failed to provide the uncropped blots for the revised figure 1A. There are no such files included in the supplemental data. Furthermore, the authors fail to explain in their rebuttal or methods section how the representative blots are cropped from replicates. As it currently is presented, the lamin control blot still appears to contain cropped sections. 

This data is unsatisfactory for indexing and cannot be endorsed without the authors providing all uncropped blots for any and all blots included in the manuscript in the supplemental data. Moreover, this supplemental data should be presented as a supplemental figure that is clearly referenced in the text.

Is the work clearly and accurately presented and does it cite the current literature?

Yes

If applicable, is the statistical analysis and its interpretation appropriate?

Yes

Are all the source data underlying the results available to ensure full reproducibility?

Partly

Is the study design appropriate and is the work technically sound?

Yes

Are the conclusions drawn adequately supported by the results?

Partly

Are sufficient details of methods and analysis provided to allow replication by others?

Yes

Reviewer Expertise:

Immunology, Inflammation, Oxidative Stress, Regenerative Medicine, Innate Immunity

I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.

Baizabal Aguirre Victor Manuel CMEB-FMVZ, Universidad Michoacana de San Nicolás de Hidalgo, Morelia, Michoacán, Mexico

Competing interests: No competing interests.

 12 6 2026

Dear Dr. Synead O'Rourke,

I understand perfectly your decision because in the version 2 of our article does not appear listed the new results on Lamin western blots, in which we demonstrate that Lamin signal is the only observable one. You can access this and other results at https://doi.org/10.6084/m9.figshare.32002761. As you can see, we performed several repetitions and all of them show discrete and well-defined bands corresponding to Lamin and no other signal. We chose to crop the Lamin signal from the same blots we obtained the Nrf2 signals. Please revise our Figshare uploaded data. We will be happy to answer any question you may have. In the next few days, we will be working on the manuscript to cite these blots properly as you sensibly suggest.

 10.5256/f1000research.198845.r479956 Reviewer response for version 2 Zhao Qinjian 1 Referee Chongqing Medical University, Chongqing, China

Competing interests: No competing interests were disclosed.

 7 5 2026 Copyright: © 2026 Zhao Q 2026 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve

Authors revised the submission according to my last comments as much as possible, its seem to accept or consider for indexing in this form.

Thanks

Is the work clearly and accurately presented and does it cite the current literature?

Partly

If applicable, is the statistical analysis and its interpretation appropriate?

Yes

Are all the source data underlying the results available to ensure full reproducibility?

Partly

Is the study design appropriate and is the work technically sound?

Partly

Are the conclusions drawn adequately supported by the results?

Partly

Are sufficient details of methods and analysis provided to allow replication by others?

Partly

Reviewer Expertise:

Immunogenetics

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Baizabal Aguirre Victor Manuel CMEB-FMVZ, Universidad Michoacana de San Nicolás de Hidalgo, Morelia, Michoacán, Mexico

Competing interests: No competing interests were disclosed.

 7 5 2026

We truly appreciate your recommendation for its acceptance and indexing.

 10.5256/f1000research.195321.r459182 Reviewer response for version 1 O'Rourke Sinead 1 Referee Trinity College Dublin, Dublin, Ireland

Competing interests: No competing interests were disclosed.

 14 3 2026 Copyright: © 2026 O'Rourke S 2026 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve-with-reservations

In this research article, the authors aimed to investigate the capacity of innate defence regulator peptides to activate transcription factor, Nrf2. Specifically, they provide compelling findings for peptide, IDR-1002, and its ability to activate Nrf2, which in turn promotes both antioxidant and anti-inflammatory effects in bovine endothelial cells. While the results of this article can be considered a significant contribution to investigations of novel Nrf2 inducers, it is recommended that the authors add further detail to both the introduction and discussion of the article to strengthen the rationale of their chosen experimental model (HEK293 and BEC cells) to further underscore the significance of these findings. There are also several comments below which the authors should address. This article can be recommended for indexing following these revisions.

 

As mentioned above, the introduction and discussion section would benefit from discussing the role of endothelial cells in oxidative stress/damage, to strengthen rationale of BEC cells as an experimental model.

Similarly, results section should explain rationale for HEK cells and Hep-G2 cells being selected as experimental model in addition to BEC cells.

I commend the authors for transparently sharing their densitometry data, but there are concerns that the blot in Figure 1A has been photo edited at the control sites for laminin as the image appears distorted. Can the authors please provide all the uncropped blots for their western results and clarify this question.

Furthermore, regarding the blot presented in Figure 2 B-C, GAPDH is an inappropriate housekeeping control, given that Nrf2 induction has been observed to affect cell metabolism. Can authors provide an alternative house-keeping control, for example b-actin as used in Figure 1?

Based on graphs presented in Figure 2, the authors cannot state that increased expression of HO-1, NQO1, and GCLM is time-dependent, as it appears there is no statistical significance between IDR-1002 2 hr and 4 hr treatment. Only significance between Control and Treated cells. It is recommended that the authors rephrase this.

Can the authors provide more detail/explanation of their chosen time point for GST assays when 2 and 4 hrs were chosen for western blot analysis.

The authors state that “Altogether, these results indicate that IDR-1002 exerts its antioxidant effects through the activation of the Nrf2 pathway.” While it is very likely that IDR-1002 promotes antioxidant effects through Nrf2 induction, authors should acknowledge this cannot be confirmed without inhibitor experiments. This should be included in the discussion section.

The authors mention that IDR-1002 may offer advantages in specificity compared to previously established inducers of Nrf2. How are the authors assessing specificity to Nrf2? The authors should clarify whether this was assessed in their experiments, or else in the discussion describe future experiments which can do so.

It is unclear the rationale for assessing TNF expression specifically in endothelial cells, what role do they have in incidence of infection/inflammation? Again, this would be strengthened by further details in the introduction/discussion section regarding the role of endothelial cells in oxidative stress/inflammation, as well as the significance of showing reduced TNF expression in endothelial cells. For example, is there clinical significance to this effect? How does this help?

Can the authors provide more detail/explanation of their chosen 1 hr pre-treatment for Figure 5, as opposed to 4 hrs previously used in other experiments?

It is recommended that the authors rephrase Figure 5 caption, as expression can be misinterpreted as gene expression. The authors should change this to production for better clarity.

The authors state in their discussion that GST “remains” elevated, however, there was no significant changes at earlier timepoints, therefore authors should rephrase to say GST activity was most significant at 24 hrs suggesting prolonged cytotoxic effect compared to observed earlier expression of HO-1 etc…

The statement regarding “well-characterised immunomodulatory profile” in the discussion requires references.

Is the work clearly and accurately presented and does it cite the current literature?

Yes

If applicable, is the statistical analysis and its interpretation appropriate?

Yes

Are all the source data underlying the results available to ensure full reproducibility?

Partly

Is the study design appropriate and is the work technically sound?

Yes

Are the conclusions drawn adequately supported by the results?

Partly

Are sufficient details of methods and analysis provided to allow replication by others?

Yes

Reviewer Expertise:

Immunology, Inflammation, Oxidative Stress, Regenerative Medicine, Innate Immunity

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

Baizabal Aguirre Victor Manuel CMEB-FMVZ, Universidad Michoacana de San Nicolás de Hidalgo, Morelia, Michoacán, Mexico

Competing interests: No competing interests have been construed.

 6 5 2026

These are the responses to the Reviewer 2.

1. We appreciate the reviewer’s suggestion to further elaborate on the role of endothelial cells in oxidative damage to strengthen the rationale for our experimental model. We have expanded the Introduction and Discussion to highlight that Bovine Endothelial Cells (BECs) are not merely a structural barrier, but a primary metabolic and immunological interface highly susceptible to oxidative stress.

In the context of IDR-1002 and the Keap1-Nrf2 pathway, we present the following arguments to strengthen the rationale of our experimental model, maintaining a clear distinction between observed data and theoretical inferences:

Endothelial cells (ECs) function as the primary sensors of hemodynamic and chemical stimuli. By utilizing BECs, we demonstrate how IDR-1002 interacts with the interface responsible for regulating systemic-to-local inflammatory flux. The high metabolic activity of ECs essential for active transport and maintaining vascular tone—makes them an ideal model for studying antioxidant interventions. Based on contemporary signaling models, the robust activation of Nrf2 in this lineage is expected to support cellular defense mechanisms against the oxidative load inherent to vascular functions (Sapeta-Nowińska et al., 2025).

Unlike other cell types, oxidative stress in ECs has been linked to direct structural consequences, specifically the degradation of tight junction proteins. The antioxidant response triggered by IDR-1002 in our study suggests a potential mechanism to assist in preventing the vascular permeability associated with chronic inflammation. Existing literature indicates that Nrf2 activation in the endothelium plays a vital role in vascular protection, which could, theoretically, prevent the degradation of structural proteins under oxidative challenge (Citrin et al., 2025; He et al., 2011).

Since our study involves bovine cells, it is pertinent to emphasize that BECs are a gold-standard model for studying bovine-specific inflammatory conditions (e.g., mastitis or respiratory disease). This model provides a high-fidelity representation of the bovine vascular response (Cajero-Juárez et al., 2002), ensuring that our observations regarding IDR-1002 are theoretically translatable to veterinary applications and large-mammal vascular biology.

Evaluating Nrf2 activation in this specific cell type is justified as it represents a critical site where antioxidant regulation could contribute to avoiding vascular hyperpermeability. Furthermore, the functional activation of the ARE-luciferase reporter observed in this study aligns with established master regulatory patterns of redox homeostasis and systemic detoxification hubs described in the literature (Bogen, 2017).

 References:

Cajero-Juárez M, Avila B, Ochoa A, et al.: Immortalization of bovine umbilical vein endothelial cells: a model for the study of vascular endothelium. Eur. J. Cell Biol. 2002; 81: 1–8.

Sapeta-Nowińska M, Sołtys K, Gębczak K, et al.: Resistance of HEK-293 and COS-7 cell lines to oxidative stress as a model of metabolic response. Acta Biochim. Pol. 2025; 72: 14164.

Citrin KM, Chaube B, Fernández-Hernando C, et al.: Intracellular endothelial cell metabolism in vascular function and dysfunction. Trends Endocrinol. Metab. 2025; 36: 744–755.

He M, Siow RC, Sugden D, et al.: Induction of HO-1 and redox signaling in endothelial cells by advanced glycation end products: A role for Nrf2 in vascular protection in diabetes. Nutr. Metab. Cardiovasc. Dis. 2011; 21: 277–285.

Bogen KT: Low-dose dose–response for in vitro Nrf2-ARE activation in human HepG2 cells. Dose-Response. 2017; 15: 1559325817737687.

2. We appreciate the reviewer's comment regarding the rationale for using multiple cell lines. To address this, we have clarified our selection criteria in the Introduction, Results and Discussion sections.

While Bovine Endothelial Cells (BECs) represent the primary vascular interface of this study, HEK-293 cells were chosen as a gold-standard model to rigorously validate the molecular signaling of the Keap1-Nrf2 axis. This lineage has been recently characterized by its robust metabolic machinery and its reliability as a model for studying adaptive metabolic responses to oxidative stress (Sapeta-Nowińska et al., 2025). Their consistent phenotypic stability allows for a clear characterization of IDR-1002’s ability to trigger Nrf2 nuclear translocation, minimizing potential interference from cell-specific metabolic complexities.

Furthermore, HepG2 cells were included to evaluate the peptide’s efficacy in a high-metabolic-demand environment. As the liver acts as a master regulatory hub for systemic detoxification and coordinates redox homeostasis through the Nrf2-ARE signaling pathway (Bogen, 2017), evaluating Nrf2 activation in this model suggests a potential for the peptide to modulate systemic cytoprotection.

Together, the inclusion of HEK-293, HepG2, and BECs provides a comprehensive assessment of IDR-1002’s versatility. By demonstrating consistent activation of the Keap1-Nrf2 pathway across renal, hepatic, and vascular lineages, we propose that the peptide’s antioxidant and anti-inflammatory regulatory effects are part of a conserved, robust pharmacological mechanism rather than a cell-specific artifact. This cross-tissue validation strengthens the theoretical potential of IDR-1002 as a candidate for modulating oxidative stress-related conditions that could extend beyond the vascular system.

 References:

Sapeta-Nowińska M, Sołtys K, Gębczak K, et al.: Resistance of HEK-293 and COS-7 cell lines to oxidative stress as a model of metabolic response. Acta Biochim. Pol. 2025; 72: 14164.

Bogen KT: Low-dose dose–response for in vitro Nrf2-ARE activation in human HepG2 cells. Dose-Response. 2017; 15: 1559325817737687.

3. We sincerely thank the reviewer for their careful inspection of Figure 1A and for the opportunity to address these concerns. Data integrity is a cornerstone of our research, and we take this observation very seriously.

Regarding the original uncropped blots, we offer our most sincere apologies. Due to a hardware failure on the primary workstation where the high-resolution raw files were stored, we were unable to retrieve the original uncropped source images for the specific experiment shown in Figure 1A. To address your concern regarding the perceived distortion and to validate our findings, we performed new independent experiments using the same protein extracts from the original study. The following points clarify the situation:

New Western blots for Lamin A/C (Laminin) were conducted in triplicate. Each replicate was processed on separate membranes to ensure consistency. The new densitometric analyses strictly confirm the original data, with no significant deviations in the observed trends.

We have updated Figure 1A with a new, high-quality representative blot from these recent validation assays. We are now providing the uncropped, full-length blots for these new experiments as supplementary material to ensure total transparency.

Regarding the original image, we categorically state that no intentional manipulation occurred. Lamin A/C is a high-molecular-weight protein (~70 kDa for Lamin A/C), and artifacts such as uneven transfer or background noise are common. The perceived " distortion" was likely a combination of mechanical membrane noise and global brightness/contrast adjustments applied to the entire blot to improve visibility.

 We believe that providing these new, validated, and uncropped blots demonstrates our commitment to transparency and confirms that the numerical data shared is a faithful reflection of the biological phenomenon.

4. We completely agree with the reviewer’s concern regarding the potential metabolic interference of Nrf2 induction on GAPDH levels. To ensure the highest standards of normalization, we have replaced GAPDH with β-Actin as the loading control for Figure 2B–C. Detection of β-Actin was performed using the same protein extracts and, where possible, on the same membranes used for the target enzymes, by stripping or parallel blotting, to ensure a direct and accurate normalization. New densitometric analyses were performed for each enzyme, and the updated figures now reflect these more robust data, which fully confirm our initial findings with only minor variations.

5. We agree with the Reviewer’s observation that the absence of statistical significance between the 2-hour and 4-hour treatments precludes a definitive claim of a "time-dependent" progression. Accordingly, we have revised the manuscript to describe the effect of IDR-1002 as a "significant and sustained increase" in antioxidant enzyme production. While minor increments may be observed between 2 and 4 hours, the activation is consistently maintained at a stable plateau relative to the control across the evaluated timeframe. All references to time-dependence in this context have been removed for better clarity.

Results section:

Treatment with IDR-1002 triggered a significant and sustained upregulation of the antioxidant genes HO-1, NQO1, and GCLM. Although the expression levels remained elevated from 2 to 4 hours post-treatment, no statistically significant difference was observed between these two time points, indicating that the induction reaches a plateau or remains stable following the initial response.

Discussion section:

Our data demonstrates that IDR-1002 promotes the induction of Nrf2-dependent proteins. The increased production of HO-1, NQO1, and GCLM at both 2 and 4 hours suggests that, rather than eliciting a transient response, the peptide establishes a relatively stable antioxidant program within this time frame. This profile is consistent with a rapid activation of Nrf2 signaling followed by maintenance of downstream gene expression, indicating that IDR-1002 may effectively prime cellular defense mechanisms against oxidative stress. This pattern is particularly relevant in inflammatory contexts, where continuous antioxidant support is required to counterbalance persistent ROS production.

6. We thank the Reviewer for the opportunity to clarify the temporal design of our experiments. The temporal discrepancy between Western blot (2–4 h) and GST assays (18–24 h) reflects the biological transition from signaling initiation to functional metabolic reinforcement.

1. Early Phase (2–4 h): Protein Expression of HO-1 and NQO1

Our objective at these time points was to capture the first wave of protein translation following Nrf2 nuclear translocation. As established by Kobayashi et al. (2006) and further supported by Senger et al. (2016), Nrf2 stabilizes rapidly after stimulation (such as with IDR-1002) due to the inhibition of its ubiquitination. Heme Oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase 1 (NQO1) are characterized as primary, rapid-response targets of Nrf2. Gu et al. (2011) demonstrated that while Nrf2 nuclear translocation is an immediate event, the subsequent protein upregulation of these early effectors is robustly detectable by Western blot within 2 to 4 hours. This early window allowed us to confirm that IDR-1002 successfully "switches on" the protective signaling machinery to mitigate immediate oxidative or inflammatory bursts (Huante-Mendoza et al., 2016; Madera & Hancock, 2012; Tonelli et al., 2018).

2. Late Phase (18–24 h): Functional GST Enzymatic Activity

The rationale for extending the evaluation of Glutathione S-transferase (GST) (Hayes et al., 2005) activity to 18–24 h is based on the requirement for a significant expansion of the total cellular enzymatic pool. Unlike Western blot, which can detect the presence of a few protein molecules, functional assays measure the cumulative catalytic capacity of the cell (e.g., the conjugation of CDNB with glutathione). As demonstrated by (Leoncini et al., 2007; Angeloni et al., 2009) there is an intrinsic temporal " delay" in this process:

The de novo synthesis of Phase II enzymes requires adequate time for ribosomes to process newly transcribed mRNA.

GST proteins often accumulate more slowly due to their specific stability and metabolic turnover rates compared to HO-1.

A higher threshold of protein accumulation is necessary to achieve a statistically significant increase in enzymatic activity.

 While HO-1 and NQO1 act as the "first line of defense," the full expansion of the GST-mediated detoxification pool is a slower, cumulative process that typically requires 18–24 h to reach its maximal functional capacity.

References:

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Gu J, Cheng Y, Wu H, et al.: Nrf2 nuclear translocation to upregulate phase II detoxifying enzyme expression coupled with the ERK, Akt and JNK signaling pathways. Mol. Cell. Biochem. 2011; 353: 101–109. doi:10.1007/s11010-011-0778-0.

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Madera L, Hancock REW: Synthetic immunomodulatory peptide IDR-1002 enhances monocyte migration and adhesion on fibronectin. J. Innate Immun. 2012; 4: 553–568. doi:10.1159/000336619.

Hayes JD, Flanagan JU, Jowsey IR: Glutathione transferases. Annu. Rev. Pharmacol. Toxicol. 2005; 45: 51–88. doi:10.1146/annurev.pharmtox.45.120403.095857.

Huante-Mendoza A, Silva-García O, Oviedo-Boyso J, et al.: Peptide IDR-1002 inhibits NF-κB nuclear translocation by inhibition of IκBα degradation and activates p38/ERK1/2–MSK1-dependent CREB phosphorylation in macrophages. Front. Immunol. 2016; 7: 533. doi:10.3389/fimmu.2016.00533.

Angeloni C, Leoncini E, Malaguti M, et al.: Modulation of phase II enzymes by sulforaphane: implications for its cardioprotective potential. J. Agric. Food Chem. 2009; 57: 5615–5622. doi:10.1021/jf900549c.

Leoncini E, Angeloni C, Malaguti M, et al.: Sulforaphane modulates phase 2 enzyme and Nrf2 expression in cultured rat cardiomyocytes. Ital. J. Biochem. 2007; 56: 101.

7. We thank the Reviewer for this critical observation. We agree that genetic silencing or chemical inhibition is a common approach to demonstrate pathway dependence. However, we have revised our manuscript to acknowledge this limitation and to clarify the rationale behind our functional validation strategy.

While the consistent correlation between Nrf2 nuclear translocation and the specific induction of downstream enzymes (HO-1, NQO1, GCLM) provides marked evidence of the pathway's activation by IDR-1002, we recognize that absolute Nrf2-dependence for the observed antioxidant protection specifically the enhanced capacity to neutralize H 2O 2 challenges remains to be definitively confirmed.

We emphasize that such experiments are necessary to confirm this molecular requirement; however, we opted for a robust functional approach due to significant biological confounding factors associated with current Nrf2-deficient models:

In endothelial models like BECs, Nrf2-deficiency leads to profound mitochondrial instability and NADPH deficits, resulting in "handling-induced death" that can obscure specific peptide-induced effects (Holmström et al., 2013).

Recent evidence highlights that ML385 carries risks of cross-reactivity with structurally homologous factors like Nrf1/CREB (Yan et al., 2024; Juszczak et al., 2024), while Brusatol acts as a global translation inhibitor (Harder et al., 2017).

Stable knockdown (CRISPR/shRNA) often triggers deep redox reprogramming and compensatory mechanisms (e.g., Nrf1 stabilization), potentially deviating from the original physiological state of the BECs (Peretz et al., 2018; Deng et al., 2024).

 In light of these challenges, we have updated the Discussion (Lines 655-664) to explicitly acknowledge that, while our data strongly support the involvement of Nrf2, additional studies employing transient, high-specificity approaches (such as inducible degron systems or biophysical binding assays including SPR and ITC) will be valuable to more directly link molecular Nrf2 activation with the broader redox fortification observed in our model.

References:

Holmström KM, Baird L, Zhang Y, et al.: Nrf2 impacts cellular bioenergetics by controlling substrate availability for mitochondrial respiration. Biol. Open. 2013; 2(8): 761–770. DOI: 10.1242/bio.20134853.

Harder B, Tian W, La Clair JJ, et al.: Brusatol overcomes chemoresistance through inhibition of protein translation. Mol. Carcinog. 2017; 56(5): 1493-1500. DOI: 10.1002/mc.22609.

Yan L, Hu H, Feng L, et al.: ML385 promotes ferroptosis and radiotherapy sensitivity by inhibiting the NRF2-SLC7A11 pathway in esophageal squamous cell carcinoma. Med. Oncol. 2024; 41(12): 309. DOI: 10.1007/s12032-024-02483-6.

Juszczak M, Tokarz P, Woźniak K: Potential of NRF2 Inhibitors-Retinoic Acid, K67, and ML-385-In Overcoming Doxorubicin Resistance in Promyelocytic Leukemia Cells. Int. J. Mol. Sci. 2024; 25(19): 10257. DOI: 10.3390/ijms251910257.

Peretz L, Besser E, Hajbi R, et al.: Combined shRNA over CRISPR/cas9 as a methodology to detect off-target effects and a potential compensatory mechanism. Sci. Rep. 2018; 8(1): 93. DOI: 10.1038/s41598-017-18551-z.

Deng R, Zheng Z, Hu S, et al.: Loss of Nrf1 rather than Nrf2 leads to inflammatory accumulation of lipids and reactive oxygen species in human hepatoma cells, which is alleviated by 2-bromopalmitate. Biochim. Biophys. Acta Mol. Cell Res. 2024; 1871(2): 119644. DOI: 10.1016/j.bbamcr.2023.119644.

8. We appreciate the Reviewer’s request for clarification on the term 'specificity.' In our study, this refers to the hypothesis that IDR-1002 may act as a Protein-Protein Interaction (PPI) modulator of the Keap1-Nrf2 axis, distinguishing it from classical electrophilic inducers (e.g., sulforaphane) that rely on the non-specific covalent modification of Keap1 cysteine residues.

Our suggestion of specificity is derived from the inherent physicochemical properties of IDR-1002. Given its cationic nature and specific 12-residue sequence, it is plausible that the peptide exhibits an electrostatic affinity for the anionic motifs within the Nrf2 Neh2 domain (such as the DLG and ETGE motifs). This potential interaction suggests a targeted displacement mechanism similar to recently developed non-electrophilic PPI inhibitors (Lin et al., 2025) which would avoid the systemic thiol-reactivity and collateral oxidative stress associated with traditional electrophilic agents.

The following molecular coupling calculations may help to clarify our statement on specificity. These results, were not included in the manuscript, suggest IDR-1002 may interact with the Neh2 domain of Nrf2, blocking the natural interaction between the kelch domain of Keap1 and Nrf2.

Peptide Docking calculations were carried out using the PIPER-FlexPepDock program, an ab-initio protocol designed to create high-resolution models of complexes between flexible peptides and globular proteins. PIPER-FlexPepDock is part of the well-known Rosetta Commons software. The results suggest a potential binding mode of the IDR-1002 at the low-affinity DLG motif within the Neh2 domain (Figure 1a). Additionally, we utilized AlphaFold-multimer to predict peptide-protein interactions at the Neh2 domain. In this case, the interaction also occurs at the low-affinity (DLG) Neh2 domain (Figure 1b).

Note: Please find this Figure in the Figshare repository under Extended data.

Taking together, both the PIPER-FlexPepDock protocol and AlphaFold-multimer predictions suggest a consistent peptide-protein interaction localized at the low-affinity DLG motif within the Nrf2 Neh2 domain. These findings provide structural support for the direct interaction between IDR-1002 and its target site.

While our current experiments characterize the downstream activation of canonical Nrf2-target genes (HO-1, NQO1, GCLM), we acknowledge that absolute specificity against all other signaling pathways was not definitively quantified in this study. We have revised the Discussion to clarify that our focus on this pathway is based on these structural observations and the consistent activation of the Nrf2-ARE signaling (Lines 624-644).

References:

Lin C, Narayanan D, Barreca M, et al.: Fragment-Based Drug Discovery of Novel High-affinity, Selective, and Anti-inflammatory Inhibitors of the Keap1-Nrf2 Protein-Protein Interaction. Angew. Chem. Int. Ed. Engl. 2025; 64: e202508121. doi:10.1002/anie.202508121.

9. We thank the Reviewer for this insightful comment regarding the rationale for assessing TNF-α expression in endothelial cells. In response, we have expanded the Introduction and Discussion sections (Lines 109-120 and 539-558) to better clarify the physiological and clinical relevance of this observation.

Endothelial cells are now widely recognized as active participants in innate immunity, functioning as immunological sentinels at the interface between systemic circulation and tissues (Pober & Sessa, 2007). Upon stimulation, they produce pro-inflammatory cytokines such as TNF-α, which act in both autocrine and paracrine manners to induce the expression of adhesion molecules (e.g., ICAM-1 and VCAM-1) and chemokines that are essential for leukocyte recruitment and extravasation (Ley et al., 2007; Bradley, 2008). Thus, endothelial TNF-α represents a critical upstream regulator of inflammatory cell trafficking.

Importantly, TNF-α also plays a central role in the regulation of endothelial barrier function. Elevated levels of this cytokine promote the disruption of intercellular junctions, leading to increased vascular permeability and facilitating the transition of inflammatory signals from the bloodstream into surrounding tissues (Mehta & Malik, 2006). This process is a hallmark of multiple inflammatory pathologies, including sepsis and chronic inflammatory diseases (Aird, 2007). Therefore, the observed reduction of TNF-α expression in endothelial cells following IDR-1002 treatment suggests a protective effect at the vascular level, limiting both leukocyte recruitment and vascular leakiness.

Finally, the suppression of TNF-α provides functional support for the proposed crosstalk between the Nrf2 and NF-κB pathways. Activation of Nrf2 is known to negatively regulate NF-κB signaling, thereby reducing the transcription of pro-inflammatory mediators such as TNF-α (Wardyn et al., 2015; Ahmed et al., 2017). In this context, our findings indicate that IDR-1002-mediated activation of Nrf2 translates into a biologically relevant anti-inflammatory outcome in endothelial cells.

 References:

Pober JS, Sessa WC: Evolving functions of endothelial cells in inflammation. Nat. Rev. Immunol. 2007; 7: 803–815. doi:10.1038/nri2137.

Ley K, Laudanna C, Cybulsky MI, et al.: Getting to the site of inflammation: the leukocyte adhesion cascade updated. Nat. Rev. Immunol. 2007; 7: 678–689. doi:10.1038/nri2156.

Bradley JR: TNF-mediated inflammatory disease. J. Pathol. 2008; 214: 149–160. doi:10.1002/path.2287.

Mehta D, Malik AB: Signaling mechanisms regulating endothelial permeability. Physiol. Rev. 2006; 86: 279–367. doi:10.1152/physrev.00012.2005.

Aird WC: Phenotypic heterogeneity of the endothelium: II. Representative vascular beds. Circ. Res. 2007; 100: 174–190. doi:10.1161/01.RES.0000255690.03436.d3.

Wardyn JD, Ponsford AH, Sanderson CM: Dissecting molecular cross-talk between Nrf2 and NF-κB response pathways. Biochem. Soc. Trans. 2015; 43: 621–626. doi:10.1042/BST20150014.

Ahmed SM, Luo L, Namani A, et al.: Nrf2 signaling pathway: Pivotal roles in inflammation. Biochim. Biophys. Acta Mol. Basis Dis. 2017; 1863: 585–597. doi:10.1016/j.bbadis.2016.11.005.

10. We thank the Reviewer for the opportunity to clarify the experimental design. We would like to emphasize that the 1-h pre-treatment used in Figure 5 was strategically selected to evaluate the early priming and interference phase of the signaling response, whereas the 4-h periods in other experiments were intended to assess the late-stage accumulation of antioxidant enzymes.

This 1-h incubation time is supported by three integrated molecular arguments:

As established by Kobayashi et al. (2006), Nrf2 activation occurs via the rapid inhibition of its ubiquitination. This biochemical "switch" happens shortly after the initial peptide stimulation, allowing Nrf2 to accumulate in the nucleus within 60 minutes, placing the cell in a "pro-antioxidant state" prior to any further challenge.

The work by Huante-Mendoza et al. (2016) demonstrates that IDR-1002 effectively inhibits NF-κB nuclear translocation by preventing IκBα degradation. Given the known crosstalk between NF-κB and Nrf2 where NF-κB-driven inflammation can mutually antagonize Nrf2 signaling a 1-h pre-treatment is critical. This ensures that the peptide blocks the pro-inflammatory machinery and promotes a cytoprotective environment before the secondary challenge (e.g., TNF-α) can trigger the NF-κB cascade.

Consistent with Madera & Hancock (2012), IDR-1002 is a rapid immunomodulator that triggers signaling flux (such as PI3K-Akt and MAPK) within minutes. Choosing a 1-h window allows the peptide's signaling to reach its peak potency exactly when the secondary inflammatory challenge (10 ng/mL TNF-α) is introduced.

 This is clearly evidenced in Figure 5, where the 1-h pre-treatment was sufficient to significantly decrease subsequent TNF-α production. This demonstrates that the peptide successfully "primed" the cells to interrupt the inflammatory feedback loop, a result that might be confounded by long-term metabolic adaptations if a 4-h window were used for this specific functional assay.

References:

Kobayashi M, Yamamoto M: Nrf2–Keap1 regulation of cellular defense mechanisms against electrophiles and reactive oxygen species. Adv. Enzyme Regul. 2006; 46: 113–140. doi:10.1016/j.advenzreg.2006.01.007.

Dinkova-Kostova AT, Kostov RV, Canning P: Keap1, the cysteine-based mammalian intracellular sensor for electrophiles and oxidants. Arch. Biochem. Biophys. 2017; 617: 84–93. doi:10.1016/j.abb.2016.08.005.

Huante-Mendoza A, Silva-García O, Oviedo-Boyso J, et al.: Peptide IDR-1002 inhibits NF-κB nuclear translocation by inhibition of IκBα degradation and activates p38/ERK1/2–MSK1-dependent CREB phosphorylation in macrophages. Front. Immunol. 2016; 7: 533. doi:10.3389/fimmu.2016.00533.

Wardyn JD, Ponsford AH, Sanderson CM: Dissecting molecular cross-talk between Nrf2 and NF-κB response pathways. Biochem. Soc. Trans. 2015; 43: 621–626. doi:10.1042/BST20150014.

Ahmed SM, Luo L, Namani A, et al.: Nrf2 signaling pathway: pivotal roles in inflammation. Biochim. Biophys. Acta Mol. Basis Dis. 2017; 1863: 585–597. doi:10.1016/j.bbadis.2016.11.005.

Madera L, Hancock REW: Synthetic immunomodulatory peptide IDR-1002 enhances monocyte migration and adhesion on fibronectin. J. Innate Immun. 2012; 4: 553–568. doi:10.1159/000336619.

Niyonsaba F, Madera L, Afacan N, et al.: The innate defense regulator peptides IDR-HH2, IDR-1002, and IDR-1018 modulate human neutrophil functions. J. Leukoc. Biol. 2013; 94: 159–170. doi:10.1189/jlb.0213062.

11. We agree with the reviewer’s observation. To avoid any potential confusion with gene expression (mRNA levels), we have replaced the term "expression" with " production" throughout the Figure 5 caption and the corresponding text in the Results section. This term more accurately reflects the protein quantification performed by ELISA.

12. We appreciate the reviewer's precision regarding the enzymatic kinetics. We have rephrased the Discussion section to clarify that GST activity reached its peak significance at 24 h. This distinction highlights a sequential response, where the maximal induction of GST occurs later than the earlier production of HO-1 and NQO1, suggesting a prolonged protective or physiological effect.

13. We thank the Reviewer for pointing this out. We have updated the Discussion (Lines 583-584) to include the following key references that support the statement regarding the "well-characterised immunomodulatory profile" of IDR-1002 across different biological contexts:

Nijnik A, Madera L, Ma S, et al.: Synthetic cationic peptide IDR-1002 provides protection against bacterial infections through chemokine induction and recruitment of neutrophils. J. Immunol. 2010; 184: 2539–2550. doi:10.4049/jimmunol.0901813. (Established the peptide’s role in providing protection against bacterial infections by modulating chemokine responses)

Madera L, Hancock REW: Synthetic immunomodulatory peptide IDR-1002 enhances monocyte migration and adhesion on fibronectin. J. Innate Immun. 2012; 4: 553–568. doi:10.1159/000339324. (Characterized its effects on monocyte migration and adhesión)

Huante-Mendoza A, Silva-García O, Oviedo-Boyso J, et al.: Peptide IDR-1002 inhibits NF-κB nuclear translocation by inhibition of IκBα degradation and activates p38/ERK1/2-MSK1-dependent CREB phosphorylation in macrophages. Front. Immunol. 2016; 7: 533. (Demonstrated that IDR-1002 inhibits NF-κB nuclear translocation, a central mechanism in its anti-inflammatory profile)

Sebők C, Pelyhe C, Giay L, et al.: Modulation of the immune response by the host defense peptide IDR-1002 in chicken hepatic cell culture. Sci. Rep. 2023; 13: 14530. doi:10.1038/s41598-023-41710-5. (Recently validated its immunomodulatory activity in hepatic cell models, supporting its relevance in metabolic tissues)

 10.5256/f1000research.195321.r459174 Reviewer response for version 1 Zhao Qinjian 1 Referee Chongqing Medical University, Chongqing, China

Competing interests: No competing interests were disclosed.

 2 3 2026 Copyright: © 2026 Zhao Q 2026 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve-with-reservations

The title overstates the mechanism by KEAP1–NRF2 pathway activation without direct evidence of Keap1 binding or disruption via molecular docking or another tools; the claim should be moderated.

Absent of Nrf2 knockdown or inhibition experiments were conducted, so Nrf2-dependence of the effects is not conclusively demonstrated.

NF-κB involvement is suggested, but no direct assays for activation of NF-κB (e.g., reporter assays or p65 translocation) are included.

Nuclear translocation data lack strong validation of fraction purity, weakening confidence in Nrf2 localization results.

ELISA measurement of Nrf2 does not validate the transcriptional activity or functional DNA binding.

The moderate potency was suggested according to reported EC50, yet the discussion presents IDR-1002 as a strong activator.

ROS assessment relies only on DCFDA, which is non-specific and prone to artifacts.

GST activity slightly declines at later time points, but the discussion frames it as sustained activation.

Mechanistic interpretations (e.g., p62 or upstream kinases) are speculative and not experimentally addressed.

Translational claims in conclusion are bit strong, but no in vivo experimental validation is provided.

The novelty is somewhat overstated given prior reports of IDR-1002’s anti-inflammatory effects.

Figures quality is not up to the mark.

References need to be updated.

Methodology needs to be cited where needed.

Overall current study offers compelling evidence that IDR-1002 activates Nrf2 signaling and reduces oxidative stress and inflammation in vitro. However, the manuscript lacks mechanistic insight into how IDR-1002 modulates the Keap1–Nrf2 axis, and causality has not been established through Nrf2 loss-of-function experiments. The claims regarding direct activation of the Keap1–Nrf2 pathway should therefore be moderated. Additional mechanistic studies would substantially strengthen the manuscript.

Is the work clearly and accurately presented and does it cite the current literature?

Partly

If applicable, is the statistical analysis and its interpretation appropriate?

Yes

Are all the source data underlying the results available to ensure full reproducibility?

Partly

Is the study design appropriate and is the work technically sound?

Partly

Are the conclusions drawn adequately supported by the results?

Partly

Are sufficient details of methods and analysis provided to allow replication by others?

Partly

Reviewer Expertise:

Immunogenetics

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

Baizabal Aguirre Victor Manuel CMEB-FMVZ, Universidad Michoacana de San Nicolás de Hidalgo, Morelia, Michoacán, Mexico

Competing interests: No competing interests have been construed.

 6 5 2026

These are the responses to the Reviewer 1. 

1. We thank the reviewer for this comment and constructive suggestion and agree that that a more moderate title is appropriate for the current findings. Even though is it outside of the scope of this manuscript and prompted by your comment, we utilized computational techniques to assess the possibility of a direct interaction between the Neh2 domain of Nrf2 and the twelve-residue peptide IDR-1002.

Peptide Docking calculations were carried out using the PIPER-FlexPepDock program, an ab-initio protocol designed to create high-resolution models of complexes between flexible peptides and globular proteins. PIPER-FlexPepDock is part of the well-known Rosetta Commons software. The results suggest a potential binding mode of the IDR-1002 at the low-affinity DLG motif within the Neh2 domain (Figure 1a). Additionally, we utilized AlphaFold-multimer to predict peptide-protein interactions at the Neh2 domain. In this case, the interaction also occurs at the low-affinity (DLG) Neh2 domain (Figure 1b).

Note: Please find this Figure in the Figshare repository under Extended data.

Taking together, both the PIPER-FlexPepDock protocol and AlphaFold-multimer predictions suggest a consistent peptide-protein interaction localized at the low-affinity DLG motif within the Nrf2 Neh2 domain. These findings provide structural support for the direct interaction between IDR-1002 and its target site.

2. We thank you for this critical observation. We acknowledge that Nrf2 silencing is a standard approach; however, we deliberately prioritized robust functional validation over knockdown (KD) or chemical inhibition due to significant technical and biological confounding factors such as:

Metabolic Artifacts (KD): Nrf2-deficient cells exhibit profound mitochondrial instability, NADPH deficits, and hypersensitivity to routine handling (trypsinization), which can lead to "handling-induced death" creating technical artifacts that obscure the specific effects of IDR-1002 (Holmström et al., 2013).

Use of inhibitor ML385: Recent studies (Yan et al., 2024; Juszczak et al., 2024) highlight its risk of cross-reactivity with structurally homologous factors (Nrf1/CREB). Furthermore, its poor solubility requires high DMSO concentrations, which independently alter membrane fluidity and basal ROS.

Use of inhibitor Brusatol: Acts as a global translation inhibitor, affecting essential regulators like c-Myc, making it impossible to attribute effects solely to Nrf2 (Harder et al., 2017).

Stable knockdown with shRNA or CRISPR: These approaches often lead to the selection of "robust" clones that have activated compensatory mechanisms (e.g., Nrf1 stabilization or HSF1 activation) (Peretz et al., 2018; Rongzhen et al., 2024). The problem is that such genetically modified cells no longer accurately represent the original model, as they have undergone deep redox reprogramming to survive the loss of Nrf2.

Comprehensive functional validation: To ensure data accuracy without these artifacts, we confirmed pathway activation through the following a alternative approaches:

ARE-Luciferase Reporter Assay: Real-time monitoring of transcriptional activity without compromising the basal metabolic viability of the cells.

Downstream Effectors: Dose-dependent induction of high-fidelity targets (HO-1, NQO1, GCLM).

Enzymatic Activity: Confirmed a significant correlation between Nrf2 activation and a measurable induction of GST activity, which aligns with a general improvement in the intracellular redox state by showing an approximately 5.4-fold enhancement in the cellular antioxidant capacity.

The consistent correlation between Nrf2 nuclear translocation, specific enzyme induction, and the resulting antioxidant effect provides a reliable representation of the Keap1-Nrf2-ARE axis activation by IDR-1002, avoiding the compromised data of inhibited or knockdown models.

References

Holmström KM, Baird L, Zhang Y, et al.: Nrf2 impacts cellular bioenergetics by controlling substrate availability for mitochondrial respiration. Biol. Open. 2013; 2(8): 761–770. DOI: 10.1242/bio.20134853.

Harder B, Tian W, La Clair JJ, et al.: Brusatol overcomes chemoresistance through inhibition of protein translation. Mol. Carcinog. 2017; 56(5): 1493-1500. DOI: 10.1002/mc.22609.

Yan L, Hu H, Feng L, et al.: ML385 promotes ferroptosis and radiotherapy sensitivity by inhibiting the NRF2-SLC7A11 pathway in esophageal squamous cell carcinoma. Med. Oncol. 2024; 41(12): 309. DOI: 10.1007/s12032-024-02483-6.

Juszczak M, Tokarz P, Woźniak K: Potential of NRF2 Inhibitors-Retinoic Acid, K67, and ML-385-In Overcoming Doxorubicin Resistance in Promyelocytic Leukemia Cells. Int. J. Mol. Sci. 2024; 25(19): 10257. DOI: 10.3390/ijms251910257.

Peretz L, Besser E, Hajbi R, et al.: Combined shRNA over CRISPR/cas9 as a methodology to detect off-target effects and a potential compensatory mechanism. Sci. Rep. 2018; 8(1): 93. DOI: 10.1038/s41598-017-18551-z.

Deng R, Zheng Z, Hu S, et al.: Loss of Nrf1 rather than Nrf2 leads to inflammatory accumulation of lipids and reactive oxygen species in human hepatoma cells, which is alleviated by 2-bromopalmitate. Biochim. Biophys. Acta Mol. Cell Res. 2024; 1871(2): 119644. DOI: 10.1016/j.bbamcr.2023.119644.

3. We appreciate this good observation. While NF-kB is a central mediator of inflammation, we prioritized Nrf2 signaling in this study based on the following mechanistic considerations:

Previous experimental evidence reported: Our previous research established that IDR-1002 exerts its anti-inflammatory effects by inhibiting the activity of the IKK complex, preventing the phosphorylation and subsequent degradation of IκBα. By stabilizing IκBα, the peptide effectively sequesters NF-κB in the cytoplasm, blocking its nuclear translocation and DNA binding (Huante-Mendoza et al., 2016). This report demonstrated that reduction of pro-inflammatory cytokines, such as TNF-α, is mediated by the inhibition of upstream activation signals rather than direct antagonism of p65. Furthermore, IDR-1002 modulated the inflammatory response in similar contexts, primarily through the p38/ERK1/2-MSK1 signaling pathway, which triggered the phosphorylation of CREB. This activation promoted an immunomodulatory profile that worked in conjunction with the cytoplasmic sequestration of NF-κB. These findings support a mechanism where the reduction of pro-inflammatory cytokines, such as TNF-α, is a result of upstream signal interference and alternative transcriptional regulation, bypassing the need for direct intervention at the level of p65 nuclear translocation.

Nrf2-NF-κB indirect crosstalk: Nrf2 activation exerts a potent indirect inhibitory effect on inflammation. By inducing antioxidant enzymes like HO-1, Nrf2 prevents the degradation of IkBα, thereby sequestering NF-kB in the cytoplasm (Huante-Mendoza et al., 2016; Gao et al., 2022). In this model, the reduction of TNFα is a downstream consequence of Nrf2-mediated redox stabilization rather than a direct antagonism of the NF-kB p65 subunit.

In endothelial cells, the TNF promoter is not exclusively dependent on NF-κB (Pober, 2002). The transcriptional activation of TNF involves a coordinated enhanceosome complex that includes:

AP-1 (c-Jun/c-Fos): Highly sensitive to the MAPK signaling that we have previously linked to IDR-1002.

Ets-1 and Elk-1: Crucial for vascular inflammation and TNF-α induction.

Redox Interference: Our results show a robust correlation between Nrf2 activation, Phase II enzyme induction (HO-1, GCLM, NQO1), and TNF-α reduction. This suggests that IDR-1002 acts via "redox-interference" where Nrf2 activation antagonizes the pro-inflammatory milieu.

We have revised the Discussion to clarify that the observed reduction in TNF-α is a consequence of Nrf2-mediated antioxidant induction (Lines 539-558 and 559-571). In light of the evidence from our previous work (Huante-Mendoza et al., 2016) and the robust correlation between Nrf2 activation and Phase II gene expression (HO-1, NQO1, GCLM) shown in this study, we have prioritized Nrf2 signaling as the primary driver of the observed immunomodulatory phenotype. This interpretation replaces the previously suggested direct p65-mediated pathway. We now propose that the peptide promotes a redox-stable environment that indirectly antagonizes pro-inflammatory signaling, a mechanistic link that is more consistent with our current experimental data.

References

Huante-Mendoza A, Silva-García O, Oviedo-Boyso J, et al.: Peptide IDR-1002 inhibits NF-κB nuclear translocation by inhibition of IκBα degradation and activates p38/ERK1/2-MSK1-dependent CREB phosphorylation in macrophages stimulated with lipopolysaccharide. Front. Immunol. 2016; 7: 533. DOI: 10.3389/fimmu.2016.00533.

Gao W, Guo L, Yang Y, et al.: Dissecting the crosstalk between Nrf2 and NF-κB response pathways in drug-induced toxicity. Front. Cell Dev. Biol. 2022; 9: 809952. DOI: 10.3389/fcell.2021.809952.

Pober, JS. Activación endotelial: vías de señalización intracelular. Arthritis Res Ther 4, 2002 S109. https://doi.org/10.1186/ar576.

4. We appreciate this important comment. We agree that demonstrating fraction purity is essential for an accurate interpretation of Nrf2 translocation data. To ensure the integrity of our results, we followed a rigorous and optimized protocol:

Fractionation protocol: BEC cells were grown to ~90% confluence and serum-starved (≥ 4 h) prior to IDR-1002 treatment. To separate the compartments, we used the NE-PER Nuclear and Cytoplasmic Extraction Kit (Thermo Scientific), strictly following the manufacturer’s instructions to obtain high-purity fractions. Nuclear pellets were then lysed with 80 μL of RIPA buffer and sonicated using a Qsonica Q125 sonicator at 25% amplitude (3 cycles of 5 s) to ensure complete extraction of chromatin-bound proteins. This fractionation methodology is consistent with our previous work (Huante et al., 2016), where we successfully isolated highly pure cellular compartments to study MAPK-mediated signaling. In those studies, the same validation criteria were applied to confirm that peptide-induced changes were strictly compartment-specific, further supporting the reliability of our current findings.

Purity monitoring and normalization: Fraction purity was monitored using high-fidelity loading controls: β-Actin (cytoplasm) and Lamin (nucleus). The absence of significant cross-reactivity confirmed the efficacy of the NE-PER kit in our cellular model. To prevent artifacts, nuclear Nrf2 levels were specifically normalized against Lamin, while cytoplasmic Nrf2 was normalized against β-Actin.

Densitometric analysis and correction in Figure 1A: To ensure the highest precision in our findings, we performed a cross-contamination analysis for all lanes. Specifically for cases like IDR-1 and HH2, where minor traces of markers were detected in the opposite fraction, the Nrf2 signal was corrected by subtracting the proportional intensity attributed to technical contamination (see quantitative assessment of subcellular fractionation purity and signal correction). This mathematical normalization procedure ensures that Nuclear/Cytoplasmic ratio reported is a faithful reflection of the net compartmental distribution.

A correction for compartmental mass transfer was applied. For each sample, a contamination coefficient (Fc) was calculated based on the nuclear marker (Lamin) signal in the cytoplasmic fraction. The net cytoplasmic signal of Nrf2 was obtained by subtracting the technical noise proportional to the nuclear pool:

(Nrf2C_corr = Nrf2C - [Nrf2N * Fc])

This approach ensures that the reported levels are biologically accurate and strictly represent the protein's localization. Regardless of technical variations in fractionation (marker carryover), the corrected densitometric analysis demonstrates that the peptides IDR-1002, 1018, IDR-1, and HH2 induce translocation of Nrf2 to the nuclear compartment. Furthermore, the mathematical cancellation of the residual cytoplasmic signal confirms that the pool of stabilized Nrf2 is predominantly located in the nucleus, thereby validating the activation of the Keap1-Nrf2-ARE antioxidant response pathway. Finally, the reliability of our fractionation procedure is the direct correlation between nuclear Nrf2 accumulation and the expression of these enzymes, which confirms that the protein detected in the nuclear fraction is functionally active.

Reference

Huante-Mendoza A, Silva-García O, Oviedo-Boyso J, et al.: Peptide IDR-1002 inhibits NF-κB nuclear translocation by inhibition of IκBα degradation and activates p38/ERK1/2-MSK1-dependent CREB phosphorylation in macrophages stimulated with lipopolysaccharide. Front. Immunol. 2016; 7: 533. DOI: 10.3389/fimmu.2016.00533.

5. We appreciate your critical assessment regarding the functional implications of Nrf2 nuclear levels. While we agree that ELISA primarily quantifies protein abundance, we maintain that in our experimental model, the increase in nuclear Nrf2 is a high-fidelity surrogate for its transcriptional activation, based on the following integrated evidence:

The gold standard for Nrf2 activity is the induction of its downstream effectors. Our data show a dose-dependent upregulation of HO-1, NQO1, and GCLM. Since these genes are strictly regulated by ARE-promoters, their induction confirms that the translocated Nrf2 (measured by ELISA) is functionally binding to DNA (Zhang et al., 2017). Our results mirror the activation kinetics of ARE-luciferase systems, where nuclear Nrf2 enrichment correlates linearly with enhanced promoter activity.

The Keap1-Nrf2 signaling axis is primarily regulated at the level of protein stability and subcellular localization. Under the stimulus of IDR-1002, the disruption of the Keap1-Nrf2 interaction leads to the saturation of the nuclear compartment. Extensive literature confirms that once Nrf2 escapes cytoplasmic degradation and translocates to the nucleus, its heterodimerization with small Maf proteins and subsequent binding to the ARE sequence is the inevitable functional outcome.

In summary, the ELISA-based detection of nuclear Nrf2 in our study does not stand in isolation; it is mechanistically linked to the robust induction of HO-1, GCLM, and NQO1. This 'triangulation' of data nuclear enrichment, canonical gene induction, and consistency with ARE-reporter models provides conclusive evidence of the transcriptional activity of Nrf2 induced by IDR-1002.

Reference

Zhang QY, Chu XY, Jiang LH, et al.: Identification of non-electrophilic Nrf2 activators from approved drugs. Molecules. 2017; 22(6): 883. DOI: 10.3390/molecules22060883.

6. We thank the reviewer for this critical observation. While our results show that IDR-1002 induces a robust nuclear translocation of Nrf2, we agree that the term 'strong activator' should be adjusted to better align with the reported EC50 values, which suggest a more moderate potency. Consequently, we have revised the terminology to describe IDR-1002 simply as an activator, ensuring a more precise and objective representation of our findings while maintaining that the observed Nrf2 stabilization is a direct result of peptide-induced signaling.

7. We thank you for this insightful comment regarding the inherent limitations of the DCFDA (2',7'-dichlorofluorescein diacetate) assay. We acknowledge that DCFDA serves as a general indicator of the cellular redox state rather than a specific sensor for individual oxygen radicals, such as hydrogen peroxide or superoxide (Deng et al., 2015).

To address your concern and ensure the correct conclusion of our findings, we have refined our interpretation and methodology as follows:

Terminological precision: We have revised the manuscript (see section: Assessment of General Intracellular Oxidative Stress in Materials and Methods) to refer to the observed changes as an increase in "general intracellular oxidative stress" or "overall redox disequilibrium" rather than attributing the signal to specific Reactive Oxygen Species (ROS).

Control of artifacts: To minimize the risk of photo-oxidation and artificial redox cycling, all measurements were conducted under the following strictly controlled conditions.

Background correction: As detailed in our methodology, fluorescence was corrected by subtracting signals from cell-free blank wells (to account for probe auto-oxidation) and untreated control cells (to account for cellular autofluorescence).

Standardized protocol: Cells were incubated in the dark with 30 μM DCFH-DA for a consistent period (30 min) in phenol red-free conditions to reduce background interference.

Data normalization: Results are now expressed as Relative Intracellular Redox State (Fold Change), ensuring that the signal reflects chemical oxidation rather than changes in probe concentration or cell density.

Reference

Deng R, Hua X, Li J, et al.: Oxidative stress markers induced by hyperosmolarity in primary human corneal epithelial cells. PLoS One. 2015; 10(5): e0126561. DOI: 10.1371/journal.pone.0126561.

8. We thank the reviewer for this observation. We agree that the term 'sustained activation' should be refined to better reflect the experimental data. Regarding GST, while its activity remained elevated for much of the study, we observed a slight decrease in GST activity by the end of the 24-h incubation period. This suggests that while the peptide induces an initial antioxidant response, the enzymatic activation may undergo a gradual attenuation over prolonged periods. Since GSTs mediate the binding of glutathione to electrophilic compounds, promoting their clearance and reducing the cumulative damage caused by ROS and lipid peroxidation-derived products (Strazielle et al., 2025), their overall induction could provide a global defense against recurrent or prolonged oxidative stress episodes.

Reference

Strazielle N, Silva K, Rault E, et al.: The glutathione-dependent neuroprotective activity of the blood-CSF barrier is inducible through the Nrf2 signaling pathway during postnatal development. Fluids Barriers CNS. 2025; 22: 19.

9. We sincerely thank the Reviewer for this insightful and critical observation. We fully agree that establishing the precise molecular interface between IDR-1002 and the Keap1–Nrf2 axis is a fundamental step for the complete characterization of this peptide. In accordance with your suggestion, we have moderated our mechanistic claims and expanded the Discussion to explicitly acknowledge these current limitations (Lines 597–609).

Regarding the mechanistic interpretations, we have carefully rephrased our discussion of p62 and upstream regulatory kinases. Rather than presenting them as confirmed pathways for IDR-1002, we now describe them as potential regulatory nodes that warrant future experimental validation (Lines 597-599). This ensures a clear distinction between our solid functional observations and the theoretical signaling pathways that may be involved.

10. We thank the Reviewer for this constructive critique regarding the translational language in our conclusions. We agree that the term "translational" should be used with caution, as our study is primarily focused on the molecular and cellular functional responses elicited by IDR-1002 in a bovine endothelial cell model.

To ensure a balanced and scientifically rigorous interpretation of our findings, we have implemented the following changes:

We have revised the concluding remarks (Lines 129-143, 559-571, and 662-667) to remove any definitive claims regarding clinical outcomes. Instead, we now emphasize that IDR-1002 acts as a "potential lead candidate" and that its ability to trigger the Keap1-Nrf2 axis provides a "functional rationale" for exploring its efficacy in more complex systems. While our current study is in vitro, the therapeutic interest in IDR-1002 is grounded in in vivo models of infection and inflammation (Turner-Brannen et al., 2011). Specifically, Nijnik et al. (2010) demonstrated that IDR-1002 significantly enhanced host protection in a mouse model of Staphylococcus aureus infection. In their study, the peptide reduced bacterial load and modulated the systemic inflammatory response without direct antimicrobial activity, highlighting its role as a potent Innate Defense Regulator (IDR). Our discovery that IDR-1002 promotes the induction of HO-1, GCLM, and NQO1 identifies Nrf2-mediated signaling as a candidate pathway that may contribute to the host protection observed in such studies, highlighting its role in neutralizing the pro-inflammatory milieu through redox stabilization. This evidence reinforces the potential of IDR-1002 as a promising translational candidate for the therapeutic management of complex inflammatory and oxidative disorders.

References

Nijnik A, Madera L, Ma S, et al.: Synthetic cationic peptide IDR-1002 provides protection against bacterial infections through chemokine induction and enhanced leukocyte recruitment. J. Immunol. 2010; 184(5): 2539–2550. DOI: 10.4049/jimmunol.0901813.

Turner-Brannen E, Choi KY, Lippert DN, et al.: Modulation of interleukin-1β-induced inflammatory responses by a synthetic cationic innate defence regulator peptide, IDR-1002, in synovial fibroblasts. Arthritis Res. Ther. 2011; 13(4): R129. DOI: 10.1186/ar3440.

11. We thank the Reviewer for this comment, which allows us to further clarify the unique contributions of our study. While it is true that the anti-inflammatory properties of IDR-1002 have been documented in previous literature, we have now demonstrated that this effect is driven by a dual molecular mechanism. IDR-1002 prevents the nuclear translocation of NF-κB by inhibiting IκBα phosphorylation and MAPK signaling (Huante-Mendoza et al, 2016), and simultaneously activates the Nrf2 signaling pathway.

Of note, this study identifies Nrf2 as a critical regulatory node that complements the anti-inflammatory action of the peptide by reprogramming the cellular redox state. By demonstrating the subsequent induction of Phase II enzymes such as HO-1, NQO1, and GCLM, we define a critical molecular pathway through which this innate defense regulator (IDR) exerts its cytoprotective effects. Using a combination of nuclear translocation assays and functional assessments of the intracellular redox state (DCFH-DA), we prove that the anti-inflammatory and pro-antioxidant effects of IDR-1002 are intrinsically linked. This mechanistic detail provides a more holistic view of how the peptide manages cellular homeostasis under stress, effectively bridging the gap between active redox regulation and the modulation of the inflammatory response.

In summary, while the observed outcome of reduced inflammation aligns with prior reports, the identification of the Keap1-Nrf2 signaling axis as a functional requirement is entirely novel. We have revised the Discussion sections (Lines 495-518) to more clearly articulate that this study identifies a new molecular 'node' in the peptide's signaling network. This finding effectively shifts the focus from simple cytokine inhibition to active redox regulation, providing a mechanistic link between the induction of antioxidant defenses and the modulation of inflammatory pathways.

Reference

Huante-Mendoza A, Silva-García O, Oviedo-Boyso J, et al.: Peptide IDR-1002 inhibits NF-κB nuclear translocation by inhibition of IκBα degradation and activates p38/ERK1/2-MSK1-dependent CREB phosphorylation in macrophages stimulated with lipopolysaccharide. Front. Immunol. 2016; 7: 533. DOI: 10.3389/fimmu.2016.00533.

12. We appreciate your feedback regarding the visual presentation of our data. We take the clarity and quality of our scientific illustrations seriously to ensure the accurate communication of our findings.

Importantly, all figures submitted with the manuscript were prepared according to the specific technical requirements of F1000Research, including resolution (minimum 600 DPI), and file format (TIFF/EPS). The Editorial Office conducted a preliminary technical screening and confirmed that the files met the journal's quality benchmarks for publication and peer review. Anyway, we decided to increase the resolution up to 1000 dpi for Figures 1 to 5 and 600 dpi for Figure S1.

13. We appreciate the suggestion to update our bibliography. We agree that the field of IDR peptides and Nrf2-mediated redox signaling is rapidly evolving. Consequently, we have revised the manuscript to incorporate recent high-impact studies (2021–2025) that strengthen the conceptual framework of our study. In accordance with your suggestion, we have updated the references to include recent evidence that reinforces the mechanistic link between host defense peptides (HDPs) and Nrf2-mediated redox regulation:

Notably, we included the study by Garg et al. (2024), which provides specific insights into how HDPs modulate endothelial cell physiology, and the review by Chu et al. (2025), which confirms the expanding role of bioactive peptides as potent Nrf2 activators for alleviating inflammation. To strengthen the mechanistic framework, we incorporated the reports by Garstkiewicz et al. (2017) and Mohan & Gupta (2018), which elucidate the non-transcriptional inhibition of the NLRP3 inflammasome and the essential crosstalk between TLR signaling and Nrf2, respectively.

Furthermore, the clinical and pharmacological relevance of targeting the Keap1-Nrf2 axis is supported by the recent works of Adinolfi et al. (2023) and Chen et al. (2024). Finally, the inclusion of Lin et al. (2025) regarding high-affinity protein-protein interaction inhibitors provides a contemporary perspective on the potential of IDR-1002 as a non-electrophilic Nrf2 inducer. Collectively, these references position our findings within a modern pharmacological paradigm where Nrf2-mediated redox stabilization is recognized as a primary driver of immunomodulation.

References

Garstkiewicz M, Strittmatter GE, Grossi S, et al.: Opposing effects of Nrf2 and Nrf2-activating compounds on the NLRP3 inflammasome independent of Nrf2-mediated gene expression. Eur. J. Immunol. 2017; 47(5): 806–817. DOI: 10.1002/eji.201646665.

Mohan S, Gupta D: Crosstalk of toll-like receptors signaling and Nrf2 pathway for regulation of inflammation. Biomed. Pharmacother. 2018; 108: 1866–1878. DOI: 10.1016/j.biopha.2018.10.019.

Adinolfi S, Patinen T, Jawahar Deen A, et al.: The KEAP1-NRF2 pathway: Targets for therapy and role in cancer. Redox Biol. 2023; 63: 102726. DOI: 10.1016/j.redox.2023.102726.

Chu Z, Zhu L, Zhou Y, et al.: Targeting Nrf2 by bioactive peptides alleviate inflammation: expanding the role of gut microbiota and metabolites. Crit. Rev. Food Sci. Nutr. 2025; 65(17): 3314–3333. DOI: 10.1080/10408398.2024.2367570.

Garg VK, Joshi H, Sharma AK, et al.: Host defense peptides at the crossroad of endothelial cell physiology: Insight into mechanistic and pharmacological implications. Peptides. 2024; 182: 171320. DOI: 10.1016/j.peptides.2024.171320.

Chen F, Xiao M, Hu S, et al.: Keap1-Nrf2 pathway: a key mechanism in the occurrence and development of cancer. Front. Oncol. 2024; 14: 1381467. DOI: 10.3389/fonc.2024.1381467.

Lin C, Narayanan D, Barreca M, et al.: Fragment-based drug discovery of novel high-affinity, selective, and anti-inflammatory inhibitors of the Keap1-Nrf2 protein-protein interaction. Angew. Chem. Int. Ed. Engl. 2025; 64(39): e202508121. DOI: 10.1002/anie.202508121.

14. We thank the Reviewer for the suggestion to enhance the technical transparency of our methodology. In the revised manuscript, we have integrated authoritative citations across all critical experimental stages to support the reproducibility of our findings. This includes the formal sourcing of cell lines and peptide synthesis standards, as well as the foundational protocols for subcellular fractionation and protein quantification. Furthermore, we have explicitly cited the mechanistic basis and known considerations of the DCFH-DA redox assay and the ARE-luciferase reporter validation, ensuring that our functional assays are anchored in established scientific literature. These references (detailed in the Materials and Methods section) provide the necessary detail to support the robust multi-tiered approach used to validate the Nrf2 signaling axis.