Quercetin Attenuates Cytokine Storm in Viral Pneumonia via Inhibition of the TLR3‐NF‐κB‐IL‐6 Signaling Axis

Cytokine storm is a critical determinant of mortality in viral pneumonia. The TLR3-NF-κB signaling pathway mediates excessive inflammatory responses, with IL-6 as a key effector molecule. This study aimed to identify natural compounds capable of attenuating cytokine storm through modulation of this signaling axis. Network pharmacology was employed to screen active components from Houttuynia cordata and identify therapeutic targets for viral pneumonia. Molecular docking evaluated binding affinities between candidate compounds and core targets. A Poly(I:C)-induced inflammatory model in human bronchial epithelial BEAS-2B cells was established to validate protective effects and molecular mechanisms. Post-treatment experiments were conducted to evaluate therapeutic (vs. preventive) efficacy of quercetin. siRNA-mediated knockdown of TLR3 and TRIF was performed to verify pathway dependence. An epithelial-macrophage (BEAS-2B/THP-1) co-culture model and primary normal human bronchial epithelial (NHBE) cells were employed to enhance physiological relevance. Cellular thermal shift assay (CETSA) was used to confirm direct target engagement of quercetin with NF-κB p65 and TLR3. Comparative experiments with other Houttuynia cordata flavonoids (isorhamnetin and kaempferol) were conducted to validate the unique contribution of quercetin. Among nine active components of Houttuynia cordata, quercetin exhibited the highest number of targets (142) with 13 intersection genes shared with viral pneumonia disease targets. Molecular docking demonstrated favorable binding of quercetin to NF-κB p65 (-7.7 kcal/mol) and TLR3 (-6.3 kcal/mol). Quercetin dose-dependently restored cell viability, maintained epithelial barrier integrity, inhibited apoptosis, and suppressed IL-6, TNF-α, and IFN-β expression. Mechanistically, quercetin inhibited TLR3 and TRIF expression, reduced IκBα phosphorylation, blocked p65 nuclear translocation, and decreased p65 binding to the IL6 promoter κB site. Rescue experiments confirmed NF-κB p65 as a key therapeutic target. Post-treatment experiments demonstrated that quercetin administered after Poly(I:C) stimulation (Post-0h, Post-2h, Post-6h) retained significant anti-inflammatory and barrier-protective effects in a time-dependent manner, supporting therapeutic rather than solely preventive efficacy. siRNA knockdown of TLR3 or TRIF abolished Poly(I:C)-induced inflammatory responses and eliminated the additional inhibitory effect of quercetin, genetically confirming pathway dependence. In the BEAS-2B/THP-1 co-culture system, epithelial-macrophage crosstalk amplified inflammatory cytokine levels by 2-3 fold compared with monoculture, yet quercetin still significantly attenuated this amplified cytokine storm and reversed THP-1 M1 polarization. These findings were further validated in primary NHBE cells. CETSA confirmed direct binding of quercetin to p65 (ΔTm = +4.3°C) and TLR3 (ΔTm = +3.3°C) in intact cells. Comparative experiments showed that quercetin exhibited significantly superior anti-inflammatory efficacy over isorhamnetin and kaempferol at equimolar concentrations. Quercetin attenuates cytokine storm in viral pneumonia through multi-target inhibition of the TLR3-NF-κB-IL-6 signaling axis, with both preventive and therapeutic efficacy demonstrated in epithelial monoculture, epithelial-macrophage co-culture, and primary cell models. Genetic knockdown and CETSA experiments provide direct evidence for pathway specificity and target engagement, providing scientific evidence for the clinical application of Houttuynia cordata.