Quercetin Alleviates Neuroinflammation in Chronic Insomnia by Modulating the RAGE/NF-κB Signaling Pathway: Insights from Network Pharmacology and In Vitro Validation

Background: Chronic insomnia (CI) is increasingly recognized to be closely associated with neuroimmune dysregulation and neuroinflammation. While the dietary flavonoid quercetin exhibits known broad-spectrum anti-inflammatory properties, its specific multi-target network and underlying mechanisms concerning CI-associated neuroinflammation remain systematically unmapped. Therefore, this study integrated network pharmacology with in vitro experimental validation to elucidate the specific targets and mechanistic pathways of quercetin against neuroinflammatory responses implicated in CI. Methods: Potential targets of quercetin were predicted using the SwissTargetPrediction and SEA platforms, while CI-associated targets were curated from GeneCards, OMIM, and CTD. To bridge computational predictions with physiological relevance, protein–protein interaction (PPI) and functional enrichment analyses were integrated with molecular docking to assess the binding landscape of key candidates. Subsequently, to empirically validate these network-derived mechanistic hypotheses, in vitro experiments were conducted using an LPS-stimulated BV2 microglial model. Pro-inflammatory mediators were quantified via qRT-PCR, and the regulatory dynamics of the RAGE/NF-κB axis were evaluated by Western blotting. Results: Fifty-five overlapping targets were identified, prioritizing six hub genes (e.g., TNF, AKT1, IL6). By harmonizing the predicted network topology with experimental observations in the BV2 microglial framework, our results provide a unified mechanism linking quercetin to the suppression of central neuroinflammation. Enrichment highlighted the AGE-RAGE and IL-17 pathways as central mechanistic nodes. Molecular docking confirmed high-strength affinities between Quercetin and core targets. In the in vitro neuroinflammation model, quercetin (10, 30, and 60 μM) exerted a dose-responsive suppression of TNF-α, IL-1β, IL-6, and iNOS, while concurrently elevating anti-inflammatory IL-10 levels. Mechanistically, Quercetin significantly downregulated RAGE expression and blunted the phosphorylation of P65 and IκB, leading to significant reductions in p-P65/P65 and p-IκB/IκB ratios. Conclusions: Our findings demonstrate that Quercetin may attenuate neuroinflammatory responses associated with CI through modulation of the RAGE/NF-κB signaling axis, as indicated by network pharmacology prediction and further supported by validation in an LPS-stimulated BV2 microglial model. While these in vitro anti-inflammatory effects provide a robust mechanistic basis for targeting neuroimmune dysregulation, further in vivo behavioral studies are necessary to evaluate its direct therapeutic efficacy against chronic insomnia.