From multi-omics discovery to experimental validation: GPC4 mediates the anti-neuroinflammatory effects of Buyang Huanwu decoction via TLR4/NF-κB signaling in ischemic stroke with Qi deficiency and blood stasis syndrome

ETHNOPHARMACOLOGICAL RELEVANCE: Buyang Huanwu Decoction (BYHW) is a classical Chinese herbal formula derived from Wang Qingren's "Medical Forest Corrections" during the Qing Dynasty. Research has consistently demonstrated that BYHW possesses substantial traditional usage evidence as an ethnic medicine for treating ischemic stroke (IS) with Qi deficiency and blood stasis (QDBS) Syndrome. AIM OF THE STUDY: Identifying key differentially expressed proteins in BYHW treatment for IS with QDBS Syndrome and its anti-neuroinflammatory mechanism of action. MATERIALS AND METHODS: An IS with QDBS Syndrome rat model was established using a combination of fasting, weight-bearing swimming, and MCAO/R. BYHW efficacy was evaluated through neurological function scoring, TTC staining, histopathology, blood rheology, and coagulation function assessment. Integrated proteomics, transcriptomics, and network pharmacology screened key targets and pathways, validated through molecular docking, molecular dynamics simulations, Western blot, RT-qPCR, and immunofluorescence. Furthermore, knockdown of GPC4 using siRNA demonstrated its involvement in the regulation of the TLR4/NF-κB signaling pathway. RESULTS: BYHW effectively improved neurological deficits, reduced infarct size, corrected blood rheology abnormalities, and restored coagulation function in model rats. Multi-omics integration analysis identified GPC4 as a key differentially expressed protein, consistently pointing to the TLR4/NF-κB signaling pathway. Mechanistic studies revealed co-expressed of GPC4 and TLR4 in astrocytes. BYHW significantly suppressed GPC4 and TLR4 expression as well as NF-κB phosphorylation, thereby downregulating the transcription and protein levels of downstream pro-inflammatory factors TNF-α and IL-1β. Molecular docking and molecular dynamics simulations suggested that the active components of BYHW (Calycosin, calycosin-7-glucoside, formononetin, Ononin, and Paeoniflorin) exhibit strong binding affinity with GPC4. Moreover, knockdown of GPC4 suppressed the TLR4/NF-κB signaling pathway and reversed the pro-inflammatory effects, establishing that the therapeutic action of BYHW is mediated through this axis. CONCLUSIONS: This study first identifies GPC4 as a key differentially expressed protein in IS with QDBS Syndrome and reveals that BYHW alleviates neuroinflammation by targeting the TLR4/NF-κB signaling axis, providing scientific rationale for its clinical application.