Protective Effect And Mechanism Of Coptidis Rhizoma Extract Higenamine On Colitis Induced By Dextran Sulfate Sodium In Mice

Background:Ulcerative colitis(UC)is a recurrent chronic non-specific inflammatory bowel disease.At present,there is still a lack of drugs with good efficacy and few side effects for the treatment of UC.Coptidis Rhizoma has been reported to improve UC.Higenamine,the structural class of protoberberines extracted from Coptidis Rhizoma,has antioxidant,anti-inflammatory and immunomodulatory effects.Recent studies have found that higenamine has a protective effect on intestinal ischemia-reperfusion injury,allergic rhinitis,rheumatoid arthritis and other diseases.However,whether higenamine can treat UC is still unclear.Objectives:To explore the relieving effect of higenamine on UC,and clarify its mechanism of improving UC.Methods:1.Network pharmacology:the active ingredients of Coptidis Rhizoma were retrieved from TCMSP and SymMap;PharmMapper was used to predict the target of active components of Coptidis Rhizoma;UC-related disease targets were obtained from OMIM and Genecards databases,and Venny2.1 was used to intersected with Coptidis Rhizoma and higenamine respectively to obtain common targets;Kyoto Encyclopedia of Genes and Genomes(KEGG)and Gene Ontology(GO)were2.performed on the common targets using R 4.0.2.String,Cytoscape3.9.0 were used to construct a protein interaction network for the intersection target.3.Animal experiment:Male C57BL mice were randomly divided.The mice in the normal group were given pure water daily,and the mice in the other groups were given 3.5%Dextran Sulfate Sodium(DSS)freely to induce acute colitis.The treatment groups were given daily intraperitoneal injection of higenamine.The Disease activity index(DAI)was assessed daily.On the 7th day of the experiment,the mice were sacrificed and the colon was collected,and the colon length was measured.Elisa was used to measure the levels of IL-6,TNF-α,CXCL1,CXCL2 and myeloperoxidase(MPO)in the colon.Ly6G immunofluorescence staining was used to observe neutrophil infiltration.The protein expressions of ZO-1 and Occludin in colon were detected by Western blot and immunohistochemistry.Goblet cells were labeled with PAS staining.Western blot was used to detect the expression of AKT and p-AKT.4.Cell experiment:Caco-2 cells were used to simulate intestinal epithelial cells,and lug/ml Lipopolysaccharide(LPS)was used to stimulate the inflammation of Caco-2 cells,and the corresponding concentration of higenamine was given at the same time.CCK8 was used to detect the cytotoxicity of higenamine The mRNA expression levels of IL-6,TNF-α were detected by qPCR.The expression levels of AKT and p-AKT were detected by Western blot.Results:1.We obtained 210 common targets of Coptidis Rhizoma and UC,and obtained 162 common targets of higenamine and UC.KEGG showed that higenamine may play a role in UC through PI3K-AKT signaling pathway.2.Animal experiments showed that DAI score,colonic inflammation and intestinal barrier of DSS-induced colitis mice were significantly improved after higenamine treatment.Cell experiments showed that higenamine can promote proliferation and reduce inflammation.In addition,both in vito and in vivo showed that higenamine affects AKT phosphorylation and regulates PI3K-AKT signaling pathway.Conclusion:Higenamine alleviates DSS-induced colitis in mice,possibly by regulating the PI3K-AKT signaling pathway,thereby inhibiting colon inflammation,reducing colon neutrophilic infiltration,and improving the intestinal barrier.