A cytokine storm is a condition of uncontrolled systemic hyperinflammation caused by excess cytokines, leading to multiorgan failure. ¹ Cytokine storms may occur for many reasons, including malignancy, rheumatoid arthritis, and sepsis. Recently, cytokine storms were found to be related to mortality and morbidity in many cases of coronavirus disease 2019 (COVID-19). ² Since coronavirus disease is characterized by hyperinflammation and an excessive immune response, the need to develop anti-cytokine drugs has increased. ³ Lipopolysaccharide (LPS), a component of the outer membrane of gram-negative bacteria, signals toll-like receptor 4 (TLR 4) to activate macrophages, which stimulates several intracellular signaling pathways, including those for nuclear transcription factor kappa-B (NF-B) and mitogen-activated protein kinases (MAPKs). Interleukin IL-6, IL-1, and tumor necrosis factor (TNF-α) are proinflammatory cytokines activated macrophages release. ⁴

Fraxin, a coumarin derived from the plant Fraxinus and Cortex fraxin, is referred to as 7,8-Dihydroxy-6-methoxy coumarin, 8-D glucopyranoside. ⁵ Fraxin possesses different pharmacological activities, including anticancer, antiviral, anti-inflammatory, and antioxidant. ⁶ For this vast potential, fraxin is a target for further immunomodulating studies.

Quercetin is a bioflavonoid widely distributed in apples, berries, grapes, and onions. Quercetin reported in previous studies to have a wide range of biological actions, including anti-inflammatory properties due to the inhibition of inflammation-related enzymes, cyclooxygenase (COX), and lipoxygenase (LOX). ⁷ RAW 264.7, a standard monocyte/macrophage cell line, is mainly used to study the anti-inflammatory activity of plant-derived extracts and their active constituents by evaluating the reduction in the production of inflammatory mediators, cytokines, and chemokines in LPS-stimulated RAW 264.7 cells (RAW 264.7 a macrophage cell line that was established from a tumor in a male mouse induced with the Abelson murine leukemia virus). ⁸

Peroxisome proliferator-activated receptor γ (PPAR-γ) is a nuclear hormone receptor and a ligand-activated transcription factor family member. Increasing evidence indicates promising anti-inflammatory properties of cancer cells exerted by activating PPAR-γ with synthetic ligands. ⁹

PPAR-γ agonists have been thought to inhibit the production of monocyte inflammatory cytokines and the expression of inducible nitric oxide synthase (iNOS), which has been observed in response to synthetic anti-diabetic thiazolidinedione drugs (such as BRL 49653 and ciglitizone), and negatively regulates the expression of proinflammatory genes and suppresses tumor cell growth. ¹⁰ The anti-inflammatory effect derived from the activation of PPAR-γ is likely the result of inhibition of NF-kB, STAT signaling pathway, and MAPK signaling pathway. The inhibition of pro-inflammatory transcriptional factors and signaling pathways results in a reduction in the transcription of genes encoding pro-inflammatory cytokines (e.g., TNF-α, IL-1β, IL-6, IL-12), chemokines, and adhesion molecules. Additionally, it enhances the release of IL-10 and suppresses macrophage polarization toward the M1 phenotype (pro-inflammatory) while promoting the M2 phenotype (anti-inflammatory, tissue repair). ^{11 , 12}

Drug combinations have been previously used as a new approach for treating many diseases. Their beneficial effects appear to be enhancing pharmacological activity and minimizing the dose to avoid any unwanted side effects of drugs without compromising their efficacy. ¹³ Furthermore, previous literature mentioned that both fraxin and quercetin possess some antioxidant and anti-inflammatory activity in different disease models. This research was designed to investigate the cytokine release inhibitory activity of fraxin and quercetin from the LPS-induced murine macrophage RAW 264.7 cell line, the possible mechanism underlying it, through changes in expression of TLR4 and PPAR-γ signaling pathways, and whether exhibiting a potential synergy as combination using iso-bolographic analysis, based on the median effect principle. ¹⁴

The devastating epidemic caused by SARS-CoV-2 in 2019 prompted researchers to make a considerable effort to search for a possible solution to limit infection. Following the demonstration of the pathological role of the “cytokine storm”, evidence for a cytokine release syndrome can be seen in increased proinflammatory cytokines in late-stage COVID-19. As seen in previous epidemics caused by SARS-CoV and MERS-CoV, dysregulated cytokine production and an influx of inflammatory myeloid cells can cause lung infiltration, septic shock, respiratory failure, acute respiratory distress syndrome (ARDS), multiorgan failure, and death. ^{20 , 21} Gram-negative bacteria’s outer membrane lipopolysaccharide (LPS) is used in inducing a cytokine storm model both in vivo and in vitro studies. The stimulation of macrophages with LPS can cause the excessive release of proinflammatory cytokines by activating the nuclear factor κB (NF- κB) and mitogen-activated protein kinase (MAPK) signaling pathways, increasing (COX-2) and (iNOS). ²² RAW 264.7 cells induced by LPS is the most widely used model for evaluating anti-cytokines in vitro. For centuries, plants have been used as a natural remedy for numerous illnesses. Hong et al. (2012) reported a dose-dependent reduction in cell spreading and pseudopodia production after treatment with an ethanol extract of Fraxinus rhynchophylla bark on LPS-stimulated macrophages. ²³ Whang et al. (2005) suggested in their study that fraxin and fraxin-related chemicals improved cell survival rate in human umbilical vein endothelial cells (HUVECs) when exposed to hydrogen peroxide (H ₂O ₂) mediated oxidative stress; other previous studies discussed the effect of quercetin on cell migration, which plays a vital role in the development of cancer. ²⁴

Quercetin strongly inhibited LPS-induced macrophage adhesion and migration in a dose-dependent manner. ²⁵ Previous research has highlighted the various biological activities of fraxin and quercetin, including anti-inflammatory and antioxidant effects, raising the need for additional investigation into their role in cytokine storms. Our study observed that fraxin, quercetin, and fraxin + quercetin exerted low cytotoxic activity on RAW 264.7, and only when cells were exposed to higher concentrations of fraxin and quercetin, which was in agreement with a study by Cui et al. that suggested that only the highest concentration of quercetin reduced macrophage viability when administered together with LPS (1 μg/mL). ²⁵ Other previous studies compatible with our study support that the presence or absence of LPS did not compromise the cell viability of RAW 264.7. ^{23 , 26}

Li et al. (2019) concluded that fraxin confers protection against LPS-induced lung injury and the inflammatory response in A549 cells. ²⁶ In an approach to modulate virus hyperinflammation such as chronic systemic symptoms, the anti-inflammatory effects of quercetin were investigated in mouse macrophage cells exposed to polyinosinic-polycytidylic acid (poly (I:C) as an experimental model for viral inflammation by Kim YJ and Park W. (2016). They found that quercetin might suppress poly (I:C)-induced inflammation by reducing the levels of inflammatory mediators. ²⁷

Our findings support previous research that found that both fraxin and quercetin were effective at suppressing the release of proinflammatory mediators from LPS-induced RAW 264.7, the mechanism underlying which may be related to interference with various inflammatory signaling pathways, including the TLR signaling system. Following their activation, proinflammatory molecules (such as IL-1, IL-6, and TNF-α) are abundantly generated, and NF-κB phosphorylation, nuclear translocation, and upregulated transcription of proinflammatory factors are all results of TLR-4 activation. ^{22 , 28}

In numerous macrophage models, PPAR-γ has been demonstrated to exert anti-inflammatory effects by suppressing the expression of multiple pro-inflammatory genes, including IL-6, TNF-α, and IL-12. ²⁹ From earlier studies, fraxin, isolated from the roots of Ulmus macrocarpa Hance, significantly suppressed the expression of iNOS and COX-2, increased PPAR-γ expression, activated the nuclear factor erythroid 2-related factor 2/heme oxygenase-1 (HO-1) (Nrf2/HO-1) pathway, and inhibited NF-κB and ERK1/2 in a dose-dependent manner The neuroprotective and anti-inflammatory effects of fraxin were also diminished by treatment with GW9662 which is a PPAR-γ antagonist. ^{30 , 31}

In LPS-induced ARDS in mice, fraxin reduced the production of TNF-α, IL-1β, IL-6, Reactive oxygen species (ROS), and Malondialdehyde (MDA), increased superoxide dismutase (SOD), and suppressed NF-κB and MAPK signaling. ³²

Li et al., 2019 discovered that pretreatment with fraxin decreased protein expressions of NF-κB and nucleotide-binding domain, leucine-rich–containing family, pyrin domain–containing-3 (NLRP3) activated in response to lipopolysaccharide (LPS). ³³ Aesculin, a hydroxycoumarin, is the 6-O-beta-D-glucoside of esculetin, another organic compound isolated from Cortex fraxini that is structurally related to fraxin. Furthermore, studies in the peritoneum and macrophages demonstrated that aesculin inhibits the production of inflammatory mediators such as iNOS, IL-1, and TNF-α via the PPAR-γ/NF-κB pathway. ³⁴

While quercetin inhibits liver inflammation mainly through NF-κB/TLR/NLRP3, it also inhibits LPS-stimulated NO increase by suppressing iNOS. ^{35 , 36} In addition, in the differentiated human acute monocyte leukemia cell line (THP-1), quercetin might lower cholesterol levels in macrophages with elevated PPAR-γ expression. Quercetin metabolites, such as quercetin-3-glucuronide (Q3G) and quercetin-3′-sulfate, also upregulated PPAR-γ in A549 lung cancer cells. ^{37 , 38}

Flavonoids, such as quercetin and kaempferol, increase PPAR-γ-mediated gene expression through a mechanism distinct from conventional PPAR-γ agonists. ³⁹

Dihydroquercetin activates AMPK/Nrf2/HO-1 signaling in macrophages, which mediates its anti-inflammatory effects. ⁴⁰

Our results were consistent with previous studies supporting that fraxin and quercetin upregulated PPAR-γ expression and downregulated TLR-4, stimulated by LPS treatment in macrophage RAW 264.7 cells.

Fraxin + quercetin showed synergistic activity when combined, which may be due to multiple targets involved when coming to their anti-inflammatory mechanism, resulting in suppression of proinflammatory mediators IL-1, IL-6, TNF-α, and suppression of other pathways like iNOS, COX-2, Nrf2/HO-1, NF-κB, NLRP3, TLR-4, and upregulation of PPAR-γ.

A study described the synergistic combination of two bioflavonoids: quercetin and catechin; this combination caused inhibition of the LPS-activated upregulation of iNOS and COX-2. ²⁶ Previous studies have shown that drug combinations, especially in phytopharmaceuticals, may activate entirely different sets of genes than those started by each drug alone. ⁴¹ This may provide another theoretical explanation for the synergistic activity between fraxin and quercetin, despite the differences in their chemical structures.

Our study showed that fraxin, quercetin, and their combination exert anti-cytokine storm activity on LPS-induced RAW246.7 cells by targeting multiple signaling pathways and suppressing TLR-mediated NF-κB. Upregulation of PPAR-γ mediated gene expression ( Figure 4A and B) may serve as a foundation for future research into other combinations of fraxin and quercetin and pathways involved in their molecular mechanisms explaining the synergistic cytokine release inhibitory activity. As the current study demonstrates an effect against cytokine release with potential synergism, these outcomes need further exploration using in vivo models of LPS to validate the synergism through pharmacokinetic studies and explore other signaling pathways to explain and relate to the current outcomes.