Identification of Kaempferol as a Critical Component in Mahuang Fuzi Xixin Decoction for Protecting Against Demyelination in Multiple Sclerosis via Matrix Metalloproteinase‐3 Inhibition

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Multiple sclerosis (MS) is an inflammatory demyelinating disease affecting the central nervous system (CNS). Chronic demyelination is a pathological feature of this disease. Mahuang Fuzi and Xixin Decoction (MFXD) is a traditional Chinese medicine prescription used for treating multiple sclerosis (MS) and has a long history. However, conclusive research on its effectiveness and mechanisms of action remains limited. Our study aims to elucidate the pharmacodynamic role of MFXD in combating MS and to explore potential mechanisms through a combination of component analysis of Chinese medicine in the bloodstream and transcriptome sequencing. The composition of MFXD particles was analyzed using high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Experimental autoimmune encephalomyelitis (EAE) serves as a model for MS. The Multiple Sclerosis Severity Score (MSSS) was employed to estimate disease severity. Serum samples from treated animals were collected for metabolomics analysis of Chinese medicine components in the blood, while peripheral blood mononuclear cells (PBMCs) were extracted for transcriptomic analysis. Morphological changes in myelination were assessed using hematoxylin-eosin staining, Luxol Fast Blue staining, and transmission electron microscopy. Immunofluorescence was utilized to examine the expression and localization of Matrix Metalloproteinase 3 (MMP3), IL-17, P65, and p-IkBa in nerve cells. Molecular docking studies and Surface Plasmon Resonance (SPR) predicted the binding affinity between MMP3 and kaempferol. The results indicated that both MFXD and kaempferol delayed the progression of EAE symptoms. Furthermore, kaempferol was found to bind to MMP3 in neurons, thereby reducing the expression levels of MMP3 and IL-17 and inhibiting the P65/IkBa pathways, subsequently reducing demyelination. Thus, these results demonstrate that kaempferol is a significant component of MFXD that delays the progression of EAE.