Anti-cytokine storm activity of fraxin and quercetin, alone and in combination, and their possible molecular mechanisms via TLR4 and PPAR-γ signaling pathways in lipopolysaccharide-induced RAW 264.7 cell line

Ethical consideration

The investigations followed the guidelines established by the Ethics Committee of Al-Nahrain University, College of Medicine (approval number Nah. Co). Pha.12 on 27 June 2022.

Chemicals and reagents

Quercetin hydrated 2-(3,4-dihydroxy phenyl)-3,5,7-trihydroxy-4Hchromenen-4-one dihydrate (purity ≥ 96%), fraxin (7,8-Dihydroxy-6-methoxy coumarin-8-beta-D-glucoside) (purity ≥ 98%), dexamethasone (purity ≥ 98%), and lipopolysaccharide (LPS) (Escherichia coli, 055: B5) were purchased from Hangzhou-Hyper Chem. Limited/China, dimethyl sulfoxide (DMSO) from Thomas Baker/India, Mouse Interleukin1β (IL-1β), (IL-6), and (TNF-α) enzyme-linked immunosorbent assay (ELISA) kits were purchased from MyBiosource, USA, RAW 264.7, (TIB-71) murine macrophage cell line (ATCC^® TIB-71™), and Dulbecco’s modified Eagle’s medium (DMEM) from American Type Culture Collection (ATCC, USA), fetal bovine serum (10% FBS), Trypsin- EDTA, penicillin/streptomycin solution from Capricorn Scientific/Germany, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) kit from MyBioSource, USA; TRIazol^® reagent (Invitrogen); RT-PCR primers: glyceraldehyde 3-phosphate dehydrogenase (GAPDH), PPAR-γ, and TLR-4 from OriGene/USA, LightCycler^® FastStart™ SYBR^® Green master kit/Roche, Germany, Revert AidTM first strand Complementary Deoxyribonucleic acid (cDNA) synthesis kit/Thermo Scientific, USA (Tables 1 and 2).

Cell culture

The RAW264.7 TIB-71 murine macrophage cell line was maintained in Dulbecco’s Modified Eagle Medium (Capricorn Scientific) with 10% fetal bovine serum (FBS) 1% penicillin-streptomycin and then kept in an incubator at 37°C with 5% carbon dioxide until confluent. Trypsin-EDTA was used to clean and harvest cells.¹³

Method of trypsinization, cell harvesting using trypsin-EDTA

The trypsinization procedure was performed in a laminar flow hood using proper aseptic technique. Trypsin, media, and phosphate buffer solution (PBS) were warmed in a 36°C water bath for at least 20 minutes prior to the procedure. All growth media was aspirated from the cell culture flask, washed once with PBS, and gently shaken with the PBS around the flask and aspirated. Half of the culture volume of trypsin was ejected directly onto cells, which was enough to cover the cells. The cells were incubated for 5 minutes at 37°C. cells were quite adherent to the flask. Cell lifter was needed at this point, trypsin was neutralized with serum containing medium (half of culture volume + 0.5 mL); warm media was directly ejected onto cells and a cell lifter was used gently to scrape cells off the flask. The cell suspension was collected from the flask and centrifuged for 4 minutes at 2500 rpm, after which the trypsin-containing supernatant was then discarded, and the cell pellet was resuspended with fresh medium, and counted or cultured as desired.¹³

Cell viability assay (MTT assay)

Cell viability in RAW264.7 was evaluated using the MTT assay after treatment with quercetin, fraxin, and fraxin + quercetin (FQ). RAW264.7 cells were plated at a density of 1 × 10⁴ cells/well in a 96-well plate and allowed to grow for 24 h. before the medium was removed. Then the cells were treated with fraxin, quercetin, and FQ in a ratio of 1:1 in serial dilutions (200, 100, 50, 25, 12.5, and 6.25 μg/ml), each concentration in tri-replicate, for 2 h. from the treatment. First, LPS (1 μg/ml) was added to each well and incubated for 24 h. Next, 20 μL of MTT (5 mg/mL) was added to each well, followed by incubation for 4 h at 37°C with 5% CO₂. After removing the media, 150 μL of dimethyl sulfoxide (DMSO) was added to each well to dissolve the insoluble formazan crystals in viable cells. The ELISA plate reader read an absorbance of 540 nm. Cell viability was calculated relative to the untreated control cells, which were considered to have 100% viability. The following formula was used to determine cell viability:

Experiments were performed in triplicate, and the data are presented as mean ± standard error of the mean.

Sample preparation

Quercetin hydrated 2-(3,4-dihydroxy phenyl)-3,5,7-trihydroxy-4Hchromenen-4-one dihydrate (purity ˃ 96%), Fraxin (7,8-Dihydroxy-6-methoxy coumarin-8-beta-D-glucoside) (purity ˃ 98%), and LPS (lipopolysaccharide) (Escherichia coli, 055: B5) were all purchased from Hangzhou-Hyper Chem. Limited/China. Both quercetin and fraxin were dissolved using DMSO (Dimethyl sulfoxide, from Thomas Baker/India), and LPS was dissolved and diluted in PBS for preparation of 1 μg/ml solution. Each agent (quercetin and fraxin) was dissolved in DMSO and then diluted to final volume with (Mg²⁺, Ca²⁺)-free PBS buffers at pH 7.4 to prepare a stock solution of 1 mg/ml for each fraxin and quercetin. Serial dilutions were freshly prepared at the same day of the experiment from the stock solution. Agents were tested at concentrations of 200, 100, 50, 25, 12.5, and 6.25 μg/ml and for FQ (half the concentration was tested for each agent in the same well), cells were supplemented with 200 μl of fresh medium along with the tested agent. The concentration of DMSO used (<0.1%) did not influence the performed assays.

Calculation of combination index (CI)

The results of the MTT assay were analyzed using the isobologram equation for the median effect/combination index (CI) by Chou (2006) and Chou and Talalay (1984). A dose-response curve can be generated by experimenting with different concentrations of each drug and its combination. For example, D is the dose, and Dm is the dose for a 50% effect; in this case, it is equal to IC50%. The parameters of the dose-effect curves were calculated with the help of a computer program (Compusyn), which then determines the CI values using the general combination index equation, from which we can infer that synergism, additive effects, and antagonism are present if the CI value is less than one, equal to one, or more than one, respectively.¹²

Cytokine levels measurement

In comparison with dexamethasone as a positive control drug, the anti-cytokine storm activity of fraxin, quercetin, and FQ was evaluated by measuring levels of proinflammatory cytokines IL-1β, IL-6, and TNF-α in RAW 264.7 cells induced with (1 μg/ml) LPS. A 96-well plate was seeded with RAW264.7 cells at a density of 1 × 10⁴ cells/well and incubated for 24 h. Cells were pretreated with fraxin (concentration 25 μg/ml), quercetin (concentration 12.5 μg/ml), and FQ (concentration 6.25 μg/ml). These concentrations were selected according to the MTT assay results, and dexamethasone (5 μg/ml) was used as a positive control drug. All treatments were performed in triplicate; untreated cells were considered a negative control. After 2 hours adding treatment, LPS was added to all wells in the plate and incubated for 24 h at 37 °C in a humidified CO₂ 5% incubator. After that the medium was collected and centrifuged at 2000xg for 10 min. Culture supernatants were collected to quantify proinflammatory cytokines by enzyme-linked immunosorbent assay (ELISA) kits for the targeted cytokines IL-1β, IL-6, and TNF-α. All reagents, and dilutions were prepared on instructions provided by the manufacturer (My BioSource, USA). From each, 100 μl of dilution of standards, blank, and the collected samples were added to each well in a 96-well plate then covered, gently shaken, and incubated at 37°C for 60 minutes. The liquid was removed from each well, without washing. A volume of. 100 μl of detection reagent A was added to each well and incubated at 37°C for 60 minutes. The washing was repeated three times, 100 μl detection reagent B was added to each well, and was incubated for 30 minutes at 37°C. The washing was repeated five times, 90 μl substrate Solution was added to each well and incubated for 10–20 minutes at 37°C. Away from light, the liquid turned blue, 50 μl stop Solution was added and the liquid turned yellow. Under 450 nm wavelength, the optical density (OD) was calculated. The linear regression equation of the standard curve was computed based on concentrations of standards and related OD values. Then the concentration of the corresponding sample was calculated, and the levels of IL-1β, IL-6, and TNF-α in cell culture supernatants were expressed as pg/ml. The OD was calculated at a wavelength of 450 nm. The linear regression equation of the standard curve was computed based on the concentrations of the standards and corresponding OD values. Then the attention of the corresponding sample was calculated, and the levels of IL-1β, IL-6, and TNF-α in cell culture supernatants were expressed as pg/ml.

Gene expression analysis

Each well of a 6-well plate was inoculated with 1 × 10⁶ cells and incubated for 24 hours. Fraxin (25 μg/ml), quercetin (12.5 μg/ml), and FQ (6.25 μg/ml) were applied to cells in triplicate (1 μg/ml). After Two hours,cells were treated with fraxin, quercetin, and FQ, LPS was added. The cells were then incubated at 37°C for 24 hours in a humidified CO₂ 5% incubator. Prior to harvesting, the cells were rinsed three times with PBS. The cells were pre-treated with fraxin (25 μg/ml), quercetin (12.5 μg/ml), and FQ (6.25 μg/ml) all in triplicate, (1μg/ml) LPS was added after 2 h. Subsequently, cells were incubated for 24 h at 37 °C in a humidified CO2 5% incubator. The cells were washed three times with PBS before being harvested. The growth media was then removed. To lyse the cells, 1 mL of TRIzol™ Reagent was added directly. The lysate was pipetted up and down several times to homogenize, then incubated for 5 minutes at room temperature before 0.2 mL of chloroform was added, then thoroughly mixed by shaking and incubated for 2–3 minutes at room temperature. The sample was centrifuged at 12,000 × g at 4°C for 15 minutes. The mixture separated into a lower red phenol-chloroform, an interphase, and a colorless upper aqueous phase. By angling the tube at 45° and pipetting the solution out, the aqueous phase containing the RNA was transferred to a new tube. After 0.5 mL of isopropanol was added to the aqueous phase, it was incubated for 10 minutes at 4°C before being centrifuged for 10 minutes at 12,000 × g at 4°C. The total RNA precipitated formed a white gel-like pellet at the bottom of the tube, and the supernatant was discarded with a micro pipettor. The pellet was resuspended in 1 mL of 75% ethanol, briefly vortexed then centrifuged for 5 minutes at 7500 × g at 4°C. The supernatant was discarded with a micro pipettor, and the RNA pellet was vacuumed or air dried for 5–10 minutes. By pipetting up and down, the pellet was resuspended in 20–50 μL of RNase-free water, 0.1 mM EDTA, or 0.5% SDS solution, incubated in a water bath at 55–60°C for 10–15 minutes, then proceeded to downstream applications, or stored the RNA at -70°C. Total RNA was extracted from stimulated cells using TRIzol^® reagent, and the first strand of cDNA was produced using a commercial kit according to the manufacturer’s instructions, the detailed steps were as the following: after thawing, the kit’s components were mixed and briefly centrifuged. The following reagents were added to an ice-cold sterile, nuclease-free tube in the following order:

- Total volume 12 μL

This was gently mixed, briefly centrifuged and incubated at 65°C for 5 min then placed on ice for 1 min. The following components were added in the indicated order:

Total volume 20 μL

This was gently mixed, and centrifuged briefly, incubated for 5 min at 25°C followed by 60 min at 42°C. The reaction was terminated by heating at 70°C for 5 min.

RNA yield determination

Total nucleic acid content is determined by 260 nm absorbance, while sample purity is determined by 280 nm absorbance. Because free nucleotides, RNA, ssDNA, and dsDNS absorb at 260 nm, they all contribute to the sample’s total absorbance. Using the NanoDropTM spectrophotometer, RNA samples can be quantified by absorbance without prior dilution. In the reaction tube, 18 μl of SYBR Green PCR mix containing nuclease-free water, reverse and forward primers, SYBR Green I dye, 1U Taq DNA polymerase, 1.25 mM MgCl₂, PCR buffer, and 100 μM Deoxynucleotide triphosphate (dNTP) was added to 2 μl cDNA template to accomplish PCR in 20 μl of the reaction mixture. Primers were used for amplification, and their sequences (5′–3′) are listed in Table 3.

The amplification conditions were as follows: the RT-PCR reaction began with one cycle at 95°C for 3 min, followed by one process at 95°C for 25 s, 55°C for 25 s, and 40 cycles at 95 °C for 25 s each to prevent the amplification of non-specific products. A melting curve assay was performed from 60 to 94°C at a transition rate of 1°C/s. The relative expression ratio of each target gene in the experimental group relative to the control group was computed using the 2^−△△Ct method and normalized against GAPDH, which was used as an internal reference gene. The results are expressed as fold-changes compared to the control.

Statistical analysis

All tests were performed in triplicate, and the results are presented as mean ± standard error of the mean (SEM), analyzed by one-way ANOVA-Tukey post hoc test for multiple comparisons. SPSS (RRID: SCR_013726) version 25 was used for statistical analysis; P < 0.05, was considered significant.

Cell viability assay

The MTT assay determined the cell viability of RAW 264.7 cells³⁵; Figure 1 illustrates the effects of fraxin, quercetin, and FQ on cell viability (expressed as a percentage compared to the control-untreated cells-considered 100% cell viability) in the presence of LPS. A reduction in cell viability was noticeable with higher concentrations of both fraxin and quercetin. While FQ exhibited the highest cytotoxicity among all three treatment groups, the viability of cells was decreased in a dose-dependent manner.

Figure 1. Effects of fraxin, quercetin, and fraxin + quercetin on RAW 264.7 cell viability.

Cell viability was determined using an MTT assay. The lower red line indicates that cells were subjected to different concentrations of all treatment groups with the presence of lipopolysaccharide (LPS) (1 μg/ml), and the upper red line represents 70% cell viability in all treatments. Cell viability was expressed as a percentage compared with the control, which was considered 100% cell viability, and data are presented as mean ± SEM.

Half-maximal inhibitory concentration (IC₅₀) values were 248, 54, and 26.5 for fraxin, quercetin, and FQ, respectively. Based on these results, the threshold for cell viability was set at 70% or higher for the anti-cytokine storm assay,⁸ and the concentrations (25, 12.5, 6.25 μg/ml) for fraxin, quercetin, and FQ were selected, respectively, for the cytokine storm assay following assay.

Calculation of combination index (CI)

The combination index (CI) for FQ in a 1:1 ratio was calculated using CompuSyn based on the MTT results. Dose-effect and median-effect curves were plotted for each drug and its combination. The Results showed that FQ in a 1:1 ratio exhibited synergism, with CI values of 0.297 at IC₅₀ and CI values of 0.409, 0.333, 0.332, 0.267, 0.265, and 0.276 at the following concentrations FQ (200, 100, 50, 25, 12,5, 6.25 g/ml).

Anti-cytokine storm assay

Fraxin, quercetin, and FQ in concentrations of 25, 12.5, 6.25 μg/ml, respectively, significantly suppressed the production of IL-1β, IL-6, and TNF-α (P ˂ 0.01) in a dose-dependent manner when compared to control (cells treated with LPS only). LPS significantly upregulated production of proinflammatory cytokines compared to the control group (P ˂ 0.05). The highest inhibition activity was recorded with dexamethasone (5 μg/ml) (positive, treated group) which significantly (P ˂ 0.05) suppressed IL-1β, IL-6, and TNF-α by 75.7%, 69%, and 79% respectively compared with the LPS treated control. In the case of the combination, FQ, the levels of IL-1β, IL-6, and TNF-α reduced by 56.2%, 58.5%, and 70.6% respectively, suggesting it is more effective in inactivating cytokine production than each drug alone. The results are shown in Figure 2A, B, and C).

Figure 2. A: Effects of fraxin, quercetin, and fraxin + quercetin on cytokine levels (IL-1) in RAW 264.7 cells at varied doses (25, 12.5,6.25 g/ml), 2 hours apart. Subsequently, all the cells were exposed to lipopolysaccharide (LPS) (1 μg/ml) for 24 h. All data are presented as mean ± SEM. * P ˂ 0.01 vs. LPS group, ** P ˂ 0.05 vs. control. B: Effects of fraxin, quercetin, and fraxin + quercetin on cytokine levels (IL-6) in RAW 264.7 cells at various doses (25, 12.5,6.25 g/ml) after 2 hours. Subsequently, all cells were exposed to lipopolysaccharide (LPS) (1 μg/ml) for 24 h. *P ˂ 0.01 vs. LPS group, **P ˂ 0.05 vs. control. C: Effects of fraxin, quercetin, and fraxin + quercetin on cytokines levels (TNF-α) in RAW 264.7 cells using different concentrations (25, 12.5,6.25 μg/ml), after 2 h. Later all cells were exposed to LPS (1 μg/ml) for 24 h. All data are presented as mean ± SEM. *P ˂ 0.01 vs. LPS group, **P ˂0.05 vs. control.

Gene expression analysis

When compared to the control, pretreatment of RAW 264.7 cells with farxin (25 μg/ml), quercetin (12.5 μg/ml), and FQ (6.25 μg/ml) for 2 hours before LPS (1 μg/ml) resulted in significant (P ˂ 0.05) suppression of TLR-4 gene upregulation by (89%, 82%, and 93%, respectively). Treatment with LPS activated the TLR-4 pathway, as shown in Figure 3A, and treatment with fraxin, quercetin, and FQ successfully counteracted the stimulatory impact of LPS on RAW 264.7 cells. Furthermore, compared to either treatment alone, the combination synergistically reversed the impact of LPS on cells.

Figure 3. A: The effects of treatment on the gene expression of Toll-like receptor 4. Cells were pretreated with fraxin (25 μg/ml), quercetin (12.5 μg/ml), and quercetin + fraxin (6.25 μg/ml) for 2 h. Cells were then treated with lipopolysaccharide (LPS) (1 μg/ml) and incubated for 24 h. **P < 0.001; compared with the control, each graph has been represented as Mean. B: The effects of treatment on gene expression of PPAR-γ, RAW 264.7 Cells were pretreated with fraxin (25 μg/m), quercetin (12.5 μg/m), and quercetin + fraxin (6.25 μg/m) for 2 h. Cells were treated with lipopolysaccharide (LPS) (1 μg/ml) and incubated for 24 h. **P < 0.001; compared with the control, each graph has been represented as Mean.

While Figure 3B reveals that there is a considerable stimulatory impact on the PPAR-γ pathway, this effect is shown as enhanced gene expression. FQ increased up to 60-fold relative to the control, whereas fraxin and quercetin (17.6, 8.6-folds, respectively) decreased proinflammatory cytokines (Figure 3B), indicating a mechanism by which fraxin, quercetin, and their combination reduce proinflammatory cytokines.