Clarifying The Mechanisms Of Compatibility Of Berberine And Sennoside A In The Treatment Of Type 2 Diabetes Mellitus By Targeting Gut Microbiota Related SCFA/GPR43/GLP-1 Pathway

Objective: In this study,we aimed to illustrate the function and mechanism based on gut microbiota related SCFA/ GPR43 /GLP-1 pathway of berberine(BBR)combined with sennoside A(SA)in the treatment of type 2 diabetes mellitus(T2DM),so as to provide important basis for increasing the application value of the BBR and SA in T2DM.Methods: 1.T2DM mice model was induced by high fat diet(HFD).Based on 454 pyrophosphate sequencing method,the diversity of 16 s r RNA gene V4 area was detected.The richness and diversity of gut microbiota were analyzed by alpha diversity analysis.The changes of Bacteroides,Firmicutes and butyric acid-producing bacteria were compared by the taxonomic composition analysis.The metabolic functions of gut microbiota were predicted.The correlation between correlation factors and gut microbiota were clarified through RDA redundancy analysis.2.The contents of fecal SCFAs were detected by gas chromatograph.Plasma GLP-1 level and serum insulin level were detected by ELISA kit.The pathological changes in liver were analyzed by HE and Oil Read O staining.The number of colonic GLP-1 positive L cells were detected by immunohistochemical analysis.RT-PCR was used to detect gene levels of GPR43,GLP-1 and hepatic gluconeogenesis.Western blot was used to detect protein levels of GPR43 and GLP-1.3.The pathological changes in colon were analyzed by HE and AB-PAS staining.Colonic ATP contents were detected by ATP test kit.The ultrastructure of mitochondria was observed by electron microscopy.The mitochondrial respiratory oxygen consumption rate of isolated colon and the activity of Complex I,II and IV were detected by Seahorse XF24 analyzer.Western blot was used to detect AMPK pathway proteins.4.The lipotoxic NCI-H716 cell model was induced by palmitic acid(PA).Cell proliferation was detected by CCK 8.The mitochondrial membrane potential and apoptosis of the cell were detected by flow cytometry.The function of cellular mitochondrial respiration was studied by Seahorse XF24 analyzer.RT-PCR was used to detect cellular gene levels of GPR43,GLP-1and insulin signaling pathway.Western blot was used to detect cellular protein levels of GPR43,GLP-1 and AMPK pathway.Results: 1.BBR combined with SA can decrease the ratio of Firmicutes to Bacteroides,and increase contents of butyric acid-producing bacteria,thus improving the production of butyric acid,activating GPR43 receptor,and promoting GLP-1 secretion.2.The compatibility of BBR and SA can not only improve insulin resistance and hepatic steatosis,but also avoid constipation induced by berberine.It can improve the intestinal atrophy induced by HFD,with protecting the colonic mucosal structure and L cells.3.BBR combined with SA can inhibit the mitochondrial Complex I dependent respiratory chain but promote Complex II and IV dependent respiratory chain in colon of T2DM mice,thus inhibit ATP overheating,activate AMPK,and protect the mitochondrial structure and function.4.In PA-induced lipotoxic NCI-H716 cell,BBR combined with SA can also upregulate GPR43 /GLP-1 pathway,inhibit ATP overheating,and activate AMPK,thus improving cell vitality and mitochondrial function.Conclusion: BBR combined with SA can increase the level of butyric acidproducing bacteria and activate SCFA/GPR43/GLP-1 pathway through adjusting the gut microbiota of T2DM mice.Furthermore,it can reduce ATP overheating,activate AMPK,improve mitochondrial structure and function,protect the intestinal mucosa and L cells,thus upregulating GLP-1 secretion.Finally,it can improve insulin resistance,thereby playing a significant role in the treatment for T2DM.