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

Background: Cytokine storm syndrome (CSS) is a leading cause of morbidity and mortality in patients with late-stage coronavirus disease 2019 (COVID-19), causing multiple organ failure and death. According to prior research, fraxin, and quercetin have anti-inflammatory, antioxidant, antimicrobial, and antiviral properties. Therefore, this study aimed to investigate the anti-cytokine storm activity of fraxin and quercetin, their combination, and the molecular mechanism behind this activity in Lipopolysaccharide (LPS)-induced RAW 264.7 cells. Methods: : LPS-induced macrophage cells were treated with fraxin, quercetin, or their combinations at various doses for cytotoxicity, anti-cytokine assays, and gene expression analyses. An enzyme-linked immunosorbent assay was used to quantify the levels of proinflammatory cytokines, interleukin 1 beta (IL1β), interleukin 6 (IL-6), and tumor necrosis factor-α (TNF-α), and RT-PCR was used to measure the gene expression of PPAR-γ and Toll-like receptor 4 (TLR-4) concerning GAPDH as a reference gene. Results: : The results revealed a slight decrease in cell viability only when higher concentrations were applied to the cells. Fraxin, quercetin, and their combination reduced the generation of proinflammatory cytokines. The combination (fraxin + quercetin (FQ)) reduced the levels of IL-1β, IL-6, and TNF-α by 56.2%, 58.5%, and 70.6% respectively, compared to the LPS-only control; pretreatment of cells with farxin, quercetin, and their combination resulted in significant inhibition of TLR-4 gene expression by 89%, 82%, and 93% respectively, compared to the control (P˂ 0.05); FQ upregulated PPAR-γ expression up to 60-fold compared to the control, while fraxin and quercetin increased PPAR-γ by 17.6 and 8.6-folds, respectively. Conclusions: : Based on these findings, fraxin, quercetin and their combination might be considered anti-cytokine storm agents, which could lay the groundwork for future research into other combinations of fraxin and quercetin, as well as the pathways involved in the molecular mechanisms underlying the synergistic anti-cytokine-storm-activity.