Heiguteng Zhuifeng Huoluo capsule alleviates osteoarthritis associated with glycerophospholipid metabolism remodeling and PI3K/AKT/mTOR-related inflammatory signaling.

Osteoarthritis (OA) is characterized by cartilage destruction, osteophyte formation, and chronic inflammation. Heiguteng Zhuifeng Huoluo Capsule (HZHC) is a traditional preparation used to relieve knee joint pain and swelling. However, its anti-osteoarthritic effects and pharmacological mechanisms remain unclear. Here, an OA rat model was established by anterior cruciate ligament transection combined with excessive running. Following HZHC intervention, OA-related pathological changes were systematically evaluated. UHPLC-Q-Orbitrap HRMS technology was used to identify the chemical constituents of HZHC and its absorbed prototypes in rat plasma. Plasma metabolomics and network pharmacology were integrated to explore metabolic responses and potential therapeutic targets. A multi-level validation, including in vivo cartilage pathway analysis, HZHC-containing serum (HCS) experiments, and representative absorbed constituent validation, was performed in IL-1β- or 740Y-P-stimulated SW1353 cells. HZHC attenuated OA-related cartilage degeneration, abnormal subchondral bone remodeling, and matrix degradation. A total of 156 chemical constituents and 35 HZHC-related prototypes were identified. Metabolomics identified glycerophospholipid metabolism as a key regulated pathway while network pharmacology highlighted inflammation-related targets and PI3K/AKT/mTOR signaling. HCS reduced IL-6, TNF-α, NO production, matrix degradation-related protein alterations, and activation of inflammatory signaling in IL-1β-stimulated SW1353 cells. Cartilage tissue validation further suggested that HZHC modulated PI3K/AKT/mTOR-associated inflammatory signaling. Representative constituents, including sinomenine, magnoflorine, gallic acid, emodin, and chlorogenic acid, attenuated 740Y-P-induced signal activation. Molecular docking predicted potential interactions between glycerophospholipid metabolites and PI3K-related proteins.. HZHC exerts its effects through regulation of glycerophospholipid metabolism and PI3K/AKT/mTOR-associated inflammatory signaling. This study provides a multi-level pharmacological framework for understanding the anti-OA effects of HZHC.