The Study On Mechanism Of Yupingfeng Granule’s Anti-inflammatory Effect In Contact Ulcerative Colitis Mice Based On Network Pharmacology

Objective:To explore the anti-inflammatory effect and immune mechanism of Yupingfeng granule and its influence on the intestinal barrier,and to clarify the potential mechanism of its anti-inflammatory effect.Methods:1.TCMSP database was used to collect active components and predict targets of Yupingfeng granules.The related targets of ulcerative colitis were obtained from Gene Cards,OMIM and Drugbank databases.The intersection targets were obtained by venny2.1.0 and imported into String database to construct the protein interaction network model,which was visualized by Cytoscape software.Go function and KEGG pathway enrichment analysis of intersection targets were analyzed through DAVID.Auto Dock software was used to dock the molecules of the absorbed ingredients of Yupingfeng granules in blood with the key targets.2.The LPS concentration of 1 mg/m L was used to stimulate RAW264.7 cells to establish an in vitro inflammation model.The safe concentration range of Yupingfeng granules for administration of RAW264.7 cells was screened by MTT method;NO content was determined by Griess method;the content of TNF-a and IL-1β was determined by Elisa method to evaluate the effect of Yupingfeng granules on the secretion of inflammatory factors;Western-blot detected the expression levels of proteins related to PI3K-AKT/NF-κB/MAPK signaling pathway.3.The mouse model of chronic colitis was established by drinking dextran sodium sulfate(DSS)freely,and the mice were treated by gavage with different concentrations of Yupingfeng granules.The effects of Yupingfeng granules on the inflammatory phenotype were comprehensively evaluated from the indicators of mouse body weight,colon length,feces,cytokines and colon histopathological sections.Detection of PI3K-AKT/NF-κB/MAPK signaling pathway and tight junction-related proteins by Western-blot and immunohistochemistry.Results:1.38 potential active ingredients and 116 key targets of Yupingfeng Granules in the treatment of ulcerative colitis were screened.The targets mainly involved the regulation of apoptosis,transcriptional regulation,angiogenesis,inflammatory reaction and other biological processes,mainly through PI3K/Akt,NF-κB and MAPK signaling pathways to play a role in the treatment of ulcerative colitis.2.After LPS-induced RAW264.7 cells were treated with different concentrations of Yupingfeng granules,the secretion of NO,TNF-a and IL-1β in the cell supernatant was significantly reduced and dose-dependent.Compared with the model group,Western-blot results showed that AKT,p65 and ERK did not change significantly after Yupingfeng granule intervention,but their phosphorylation levels were inhibited.It indicated that Yupingfeng granules may exert its anti-inflammatory effect by inhibiting the activation of AKT/NF-κB/ERK signaling pathway.3.Yupingfeng granules can improve chronic colitis model mice.Compared with the model group,after the intervention of Yupingfeng granules,the weight reduction of the mice treated with Yupingfeng granules was significantly slowed down,the spleen index decreased,the length of the colon increased,the expression of inflammation-related factors TNF-a and IL-1β decreased and the destruction of the colonic crypt was alleviated.Western-blot and immunohistochemical results showed that Yupingfeng granules could inhibit the phosphorylation expression of P-AKT,P-p65 and P-ERK,inhibit the expression of PKC,and restore the expression of tight junction proteins Occludin and Claudin-1.Conclusion:Yupingfeng granules can improve DSS-induced ulcerative colitis,and its mechanism may be achieved by inhibiting the activation of PI3K/Akt,NF-κB and MAPK signaling pathways,and has a certain protective effect on the intestinal mucosal barrier.