Wogonin Inhibits Oxidative Stress in Hypoxia-Induced hCMEC/D3 Cells Through Suppressing CXCL8-Mediated TLR4/NF-κB Pathway

Abstract

High-altitude cerebral edema (HACE) is a critical neurological disorder with limited treatment options. Wuwei Shaji San (WWSHS), a traditional Chinese medicine formula, demonstrates therapeutic potential for HACE, yet its mechanisms remain unclear. This study explores the molecular mechanism of WWSHS in HACE treatment. Network pharmacology was performed to identify core active ingredients and targets of WWSHS against HACE. Protein-protein interaction (PPI) networks, functional enrichment analysis, and molecular docking were performed. Hypoxia-induced injury in hCMEC/D3 cells was used to validate wogonin's effects. Apoptosis, oxidative stress indicators (ROS, H₂O₂, MDA, SOD, LDH), and inflammation factors (IL-6, IL-1β, TNF-α) were evaluated via flow cytometry, biochemical assays, and qPCR. CXCL8 overexpression and TLR4 inhibitor (Resatorvid) were applied to investigate mechanisms. Network analysis revealed 63 shared targets between WWSHS and HACE, with CXCL8 as a central target and wogonin as the core ingredients. Molecular docking confirmed strong binding between wogonin and CXCL8 (−7.6 kcal/mol). In vitro studies revealed that wogonin effectively mitigated hypoxia-induced cytotoxic effects, apoptotic responses, and oxidative stress in hCMEC/D3 cells. The mechanistic investigation demonstrated that wogonin downregulated CXCL8 expression, consequently attenuating TLR4/NF-κB signaling activation. Notably, CXCL8 overexpression counteracted wogonin's cytoprotective properties against hypoxic damage. Complementary experiments showed that co-administration with resatorvid, a pharmacological TLR4 inhibitor, potentiated wogonin's therapeutic efficacy. Our findings suggest that wogonin may serve as a potential therapeutic agent for HACE by targeting CXCL8 and modulating the TLR4/NF-κB pathway, thereby reducing inflammation and oxidative stress.