Multiple sclerosis (MS) is a chronic autoimmune disorder of the central nervous system. Huangqi Guizhi Wuwu Decoction (HGWD), a classic traditional Chinese medicine formula, exerts protective effects against MS, yet the underlying mechanism involving mitophagy and microglial pyroptosis remains poorly elucidated. This study investigated the ameliorative effects of HGWD on experimental autoimmune encephalomyelitis (EAE) and its molecular mechanism. HGWD components were identified via UHPLC-Q-Orbitrap HRMS, and potential anti-MS targets were predicted by network pharmacology and molecular docking. EAE mice and LPS-stimulated microglia were treated with HGWD, with mitophagy inhibitor 3-MA and PINK1 siRNA used for mechanism validation. Assessments included clinical scoring, histopathology, Western blotting, immunofluorescence, ELISA, transmission electron microscopy and mitochondrial function assays. A total of 59 constituents were identified; network pharmacology revealed core pyroptosis-related targets (IL-1β, IL-18, NLRP3, CASP1) in MS, and molecular docking confirmed strong binding affinities between key active components and these proteins. HGWD significantly alleviated EAE clinical severity and reduced demyelination by enhancing PINK1/Parkin-mediated mitophagy, improving mitochondrial function, and inhibiting microglial pyroptosis, which was validated in vitro. Notably, both 3-MA and PINK1 silencing abolished HGWD's protective effects. This study first confirms that HGWD treats EAE via the PINK1/Parkin mitophagy-pyroptosis axis, providing theoretical evidence for its clinical translation and quality control.
