The Mechanism Of Grape Seed Oligomeric Procyanidins In The Treatment Of Experimental Autoimmune Encephalomyelitis Mice

Objective:This study was designed to investigate the mechanism of grape seed oligomeric procyanidins（GPC）in the treatment of experimental autoimmune encephalomyelitis（EAE）mice,in order to provide pharmacodynamic basis for drug development in the treatment of multiple sclerosis（MS）.Methods:1.The constituents which have nerve protective effect in GPC were collected through literature retrieval.We used Pharm Mapper and STITCH database to predict drug targets,Uniprot database to standardize target genes,Gene Cards and OMIM database to predict MS-related genes.Targets of GPC treating MS were obtained from intersected targets between drug and disease.The protein-protein interaction（PPI）network was constructed by the STRING database and Cytoscape software.The GO functional enrichment and KEGG pathway enrichment analysis of potential targets were performed by DAVID database,and important biological processes and pathways were screened out.2.To design animal experiments to verify the mechanism of GPC treatment of EAE mice based on the results of network pharmacology.C57BL/6 female mice,aged for10-12 weeks,were used to establish EAE mouse model.Eighteen mice were divided into the EAE control group（n=6）and the GPC treatment group（n=6）.From day 3post-immunization（p.i.）to day 28 p.i.,the GPC treatment mice group were intragastrically administered with GPC(50 mg·kg^-1·day^-1).The control mice received vehicle only in a similar manner.Animals were evaluated for clinical scores every other day in a double-blinded fashion.On the 28 p.i.,hematoxylin/eosin（HE）,Luxol fast blue（LFB）and immunofluorescence staining were used to observe the infiltration of inflammatory cells and myelin loss in mice spinal cords.Enzyme-linked immunosorbent assay（ELISA）were used to detect oxidative stress and inflammatory response indicators in mice spinal cords.Western Blot（WB）were used to detect the expressions of protein phosphorylation related to spinal inflammatory signaling pathway in mice.Graph Pad Prism7.0 statistical analysis software is used for experimental data analysis.Results:1.We collected seven active ingredients of GPC.Forty-two targets were discovered to be related to the treatment of GPC in MS.GO analysis showed that the mechanism of GPC in treating EAE mice was closely related to cell proliferation,regulation of transcription,signal transduction,protein phosphorylation,apoptotic process,and so on.KEGG enriched a total of 32 pathways involving inflammation,oxidative stress,cancer,virus infection,and so on.2.Animal experiments showed that GPC reduced the clinical score of EAE mice.HE staining showed that GPC reduced the infiltration of inflammatory cells in the spinal cord of mice（P<0.05）.LFB staining showed that GPC reduced the myelin loss in mice spinal cords（P<0.01）.Immunofluorescence staining and WB were used to detect MBP expression.The results showed that MBP average fluorescence intensity and relative protein expression of the GPC treatment group were higher than those of EAE control group（P<0.05）.ELISA results showed that GPC treatment inhibited the expressions of malondialdehyde（MDA,P<0.01）,reactive nitrogen species（RNS,P<0.01）,reactive oxygen species（ROS,P<0.001）,interferon-γ（IFN-γ,P<0.05）,interleukin-17（IL-17,P<0.01）,interleukin-1β（IL-1β,P<0.01）,interleukin-6（IL-6,P<0.001）and tumor necrosis factor-α（TNF-α,P<0.01）.WB results showed that GPC treatment inhibited the phosphorylation of Akt（P<0.05）,JNK（P<0.05）and ERK（P<0.05）.Conclusion:In this study,the method of combining network pharmacology and experiment was used to analyze and verify the active ingredients,targets and pathways of GPC.On the basis of network pharmacology research,animal experiment results found that GPC improves the clinical symptoms of EAE mice,inhibits the infiltration of inflammatory cells into the spinal cords,and reduces the myelin depigmentation.GPC also inhibits the expression of indicators of oxidative stress and inflammatory responses in the spinal cords.The therapeutic effect of GPC in EAE mice is associated with the suppression of MAPK and PI3K-Akt signaling pathways,providing a theoretical basis for the application of GPC in MS treatment.