Zuo Gui Wan Promotes Remyelination in Multiple Sclerosis by Attenuating MAPK-Mediated Microglial M1 Polarization, an Integrated Network Pharmacology and Experimental Validation Study

Purpose: Zuo Gui Wan (ZGW) shows clinical potential in treating multiple sclerosis (MS), but its underlying mechanism of action remains elusive. This study aims to elucidate the efficacy and molecular mechanisms of ZGW in a cuprizone (CPZ)-induced demyelination mouse model. Methods: Adult male C57BL/6J mice were randomized into four groups (n = 12 each), normal control (NC), CPZ model, CPZ + 2.8 g/kg/d ZGW (ZGW-L), and CPZ + 5.6 g/kg/d ZGW (ZGW-H). Demyelination was induced by a 0.2% CPZ diet for 9 weeks. ZGW or saline was administered intragastrically from weeks 6 to 9. Behavioral performance was assessed using the rotarod, elevated plus-maze, and tail suspension tests. Remyelination was evaluated via LFB staining, TEM, and the expression of oligodendrocyte markers (Olig2, CNPase, MOG). Microglial activation/polarization (Iba-1, iNOS, CD86, Arg-1, CD206) and MAPK pathway proteins (p-ERK1/2, p-p38, and p-JNK) were quantified by Western blot or immunofluorescence. The phytochemical profile of ZGW were analyzed using LC-MS, and target prediction was performed using TCMSP, BATMAN-TCM, and SwissTarget Prediction. Network pharmacology, protein-protein interaction (PPI) analysis, and molecular docking were employed to identify core pathways and targets. Results: ZGW treatment exhibited a dose-dependent improvement in motor coordination and alleviated anxiety- and depression-like behaviors in CPZ mice. Histological and ultrastructural analyses demonstrated significantly enhanced remyelination and increased expression of Olig2, CNPase, and MOG. Network pharmacology and molecular docking analyses identified the MAPK signaling cascade as the principal therapeutic axis, with key ZGW compounds showing high affinity for MAPK3 and TNF-α. In vivo, ZGW suppressed CPZ-induced phosphorylation of ERK1/2, p38, and JNK, reduced Iba-1 expression and M1 markers (iNOS and CD86), but did not significantly alter Arg-1 or CD206. Conclusion: ZGW promotes remyelination in CPZ-induced demyelination by inhibiting MAPK pathway overactivation and attenuating microglial M1 polarization, thereby modulating the neuroinflammatory milieu. Keywords: remyelination, microglial polarization, MAPK signaling pathway, traditional Chinese medicine formula