Clarifying The Protective Effect And Mechanism Of Berberine Combined With Sennoside A On Intestinal Structure And Function And Lowering Blood Glucose In Diabetic Mice

Objective: The purpose of this study was to explore the effect and mechanism of berberine(BBR)combined with Sennoside A(SA)on the intestinal structure and function and lowering blood glucose in diabetic mice by establishing diabetic mouse model and L-cell lipotoxic model,in order to provide theoretical and experimental support for improving the application value of BBR combined with SA.Method: 1.The type 2 diabetic model mice were induced by high fat diet.OGTT,oil red staining in liver and plasma TG were detected to explore the effect of BBR combined with SA on glucose and lipid metabolism.The levels of plasma GLP-1,insulin and C peptide were detected by ELISA kits.The pathological changes of colon and the number of L cells in colon were analyzed by HE staining and immunofluorescence staining respectively.2.The level of ROS production was analyzed by extracting purified mitochondria from colonic tissue in mice.The oxidative stress state of colon was evaluated by detecting the levels of Mn-SOD,GSH and MDA.The protein expression levels of mitochondrial Complex I,II,III,IV and V were detected by western blot analysis,and the activities of Complex I,II and IV in isolated colon mitochondrial were detected by Seahorse XF24 analyzer.3.The protein expression of PINK1/Parkin pathway and LC3 b were detected by western blot analysis.The recruitment of PINK1 in the outer mitochondrial membrane of colon tissue was detected by immunofluorescence staining.The mitochondrial ultrastructure and function was evaluated by biological transmission electron microscope and Seahorse XF24 analyzer respectively.The m RNA expression of Bcl-2,Bcl-xl,Bax and Bad was detected by fluorescence quantitative PCR.4.The lipotoxicity model of L cells(NCI-H716)induced by palmitic acid was used to detect the cell viability by CCK8.The intracellular ROS production was evaluated by flow cytometry.The level of GLP-1 in the supernatant was investigated by ELISA.The protein expression of PINK1/Parkin pathway and LC3 b and GCG was investigated by western blot analysis,and the m RNA expression of Bcl-2,Bcl-xl,Bax and Bad was detected by fluorescence quantitative PCR.Results: 1.BBR combined with SA can Lose weight,improve glucose and lipid metabolism,reduce hyperinsulinemia,decrease the level of high C peptide,regulate the abnormal secretion of GLP-1,protect the normal structure of colon tissue and increase the number of colon L cells.2.BBR combined with SA decreased the production of mitochondrial ROS and MDA and increased the activity of Mn-SOD and GSH.BBR combined with SA inhibited the protein expression of mitochondrial Complex I,II,III,IV and V and the activity of Complex I,and increased the activity of Complex II in colon mitochondrial respiratory chain.3.BBR combined with SA inhibited protein expression of PINK1/Parkin pathway and LC3 b in colon tissue,reduced PINK1 recruitment around mitochondria and improved mitochondrial ultrastructure and respiratory function.In addition,BBR combined with SA increased m RNA levels of Bcl-2 and Bcl-xl,and decreased m RNA levels of Bax and Bad.4.In NCI-H716 cell model,berberine combined with Senna A did not affect cell survival,inhibit palmitic acid-induced high GLP-1 level,decrease ROS production,inhibit the activation of PINK1/Parkin pathway,increase m RNA level of Bcl-2and Bcl-xl,and decrease m RNA level of Bax and Bad.Conclusion: Berberine combined with Sennoside A can alleviate the disorder of glucose and lipid metabolism in diabetic mice,improve intestinal damage and maintain normal physical barrier structure and secretory function of intestinal tract in diabetic mice.Its mechanism may be related to the inhibition of intestinal ROS/PINK1/Parkin pathway and the protection of mitochondrial structure and respiratory function.