Network Pharmacological Study and Monomer Experimental Validation of Buyang Huanwu Decoction(补阳还五汤)in the Treatment of Multiple Sclerosis
Objective:To explore the mechanism of Bushen Huoxue Decoction(BYHWD)(补阳还五汤)in the treatment of multiple sclerosis(MS)and verify the therapeutic effects of its monomeric components based on network pharmacology,molecular docking technology,and ani-mal experiments.Methods:Active ingredients of BYHWD were collected from the TCMSP and HERB databases and screened with oral bio-availability(OB)≥30％,drug-likeness(DL)≥0.18,and blood-brain barrier permeability(BBB)≥-0.3.The SwissADME and SwissTar-getPrediction platforms were used to predict target proteins of active ingredients,with UniProt providing standardized gene names for the tar-gets.Disease-related targets of MS were obtained from GeneCards,OMIM,and DisGeNET databases.The intersection targets of active ingre-dients and MS-related disease targets were visualized using a Venn diagram.A"drug-active ingredient-disease target gene"network was con-structed using Cytoscape 3.9.1 software.GO and KEGG pathway enrichment analyses of the intersection targets were performed through the Metascape database,and were visualized by microbiome platform.The analysis suggested JAK-STAT pathway to be the key action pathway of active components of BYHWD.A"drug key action target-target pathway"map was established.A protein-protein interaction network(PPI)was constructed using the STRING 11.5 database and Cytoscape 3.9.1 software.Based on the results of the PPI network,molecular docking technology was used to detect the spontaneous binding of the active component of small molecule drugs,formononetin(FMN),to receptor pro-teins.The active ingredient FMN was tested for its inhibitory effect on demyelination using a cuprizone(CPZ)-induced demyelination model.Thirty male C57BL/6 mice were randomly divided into a normal control group,a model group,and an FMN group,with 10 mice in each group.Mice in the normal control group received normal feed,while those in the model control group and FMN group were fed with feed containing 0.2％CPZfor 6 weeks.From the 5th week,the FMN group received intraperitoneal injections of FMN for 2 weeks.Behavioral tests,including elevated plus maze and open field tests,were used to evaluate the effects of FMN on mouse behaviors.Black gold and immunofluorescence staining were used to detect demyelination in the corpus callosum area and changes in the expression of MBP,IBA1,and GFAP protein in each group.The content of TNF-α,IL-1β,IL-6,and IL-10 in brain tissue fluid was detected by ELISA,and the content of LPO and GSH was measured by micro enzyme labeling method.Results:A total of 162 key targets for the treatment of MS by BYHWD were identified.KEGG enrichment analysis revealed three pathways related to MS,i.e.,Janus kinase(JAK)/signal transducer and activator of transcription(STAT)signaling pathway,nuclear factor κB(NF-κB)signaling pathway,and peroxisome proliferators-activated receptors(PPARs)signaling path-way.The primary pathway was the JAK-STAT pathway,with key targets including STAT3,STAT1,and JAK2.Molecular docking identified FMN as an effective small molecule drug.Animal experiments demonstrated that the FMN improved behavioral manifestations of CPZ-induced mania and anxiety,inhibited demyelination,reduced levels of TNF-α,IL-1β,IL-6,and LPO in brain tissue ofCPZmodel,increased levels of IL-10 and GSH,promoted microglial activation,and inhibited astrocyte activation.Conclusion:Through network pharmacology analysis,the mechanism of action of BYHWD was elucidated,and FMN was predicted as an effective monomer by molecular docking.For the first time,an-imal experiments confirmed that FMN significantly improved CPZ-induced behavioral abnormalities and demyelination,possibly by inhibiting inflammation and oxidative stress ofCPZmodel,and regulating the activation of microglia and astrocytes.
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