Study On The Hypoglycemic Effect And Mechanism Of Glimepiride/Metformin Cocrystal

BackgroundType 2 diabetes mellitus(T2DM)is the most common type of diabetes.Increasing insulin production,improving immunity and reducing blood glucose level are the key goals for the treatment of diabetes.Although there are many types of hypoglycemic drugs in clinical use,their practicability is limited due to their side effects and limited therapeutic effects.Therefore,the development of new hypoglycemic drugs has always been a hot spot in the medical field.Glimepiride/metformin cocrystal(GM)is a non-covalent bond adduct co-crystal synthesized from glimepiride and metformin at a molar ratio of 1:1.It is a candidate drug with independent intellectual property rights developed by the College of Pharmacy of Chongqing Medical University.This study is mainly to research the hypoglycemic effect of GM,and preliminarily explore its mechanism of action.Methods1.The hypoglycemic effect of GM1.1.Db/db mice were selected as experimental animal model to research the hypoglycemic effect of GM in vivo.1.1.1.During the experiment,the body weight,food intake and blood glucose level of mice were detected.1.1.2.The four items of blood lipids and glycosylated hemoglobin in db/db mice were detected by biochemical methods.1.1.3.The histopathological changes of pancreas and liver in db/db mice were detected by HE staining method.1.1.4.RT-qPCR method was used to detect the mRNA expression level of insulin of pancreatic tissue in db/db mice.1.1.5.Immunohistochemical and ELISA assay were used to detect insulin secretion level of pancreatic tissue in db/db mice.2.The hypoglycemic mechanism of GM2.1.Construct a high-glucose injury model on rat insulinoma cells INS-1 and mouse insulinoma cells MIN-6 to simulate the high-glucose environment in vivo.2.1.1.MTT assay was used to detect the effect of different concentrations of glucose on the viability of rat insulinoma cells INS-1 and mouse insulinoma cells MIN-6.2.1.2.RT-qPCR method was used to detect the mRNA expression level of Bax.2.1.3.Western blotting was used to detect the protein expression levels of cleaved-caspase3,caspase3,Bax and bcl-2.2.2.Preliminary exploration of the hypoglycemic mechanism of GM in vitro under the high glucose condition.2.2.1.ELISA assay was used to detect the insulin secretion levels of INS-1 and MIN-6 cells under the high glucose condition.2.2.2.RT-qPCR method was used to detect the mRNA expression levels of insulin and TXNIP in INS-1 and MIN-6 cells under the high glucose condition.2.2.3.Western blotting was used to detect the effect of GM on the protein expression levels of AMPK,p-AMPK,TXNIP,p-STAT3,STAT3 and MaFA in INS-1 and MIN-6 cells under the high glucose condition.And to detect the effect of GM on AMPK,p-AMPK,TXNIP protein in INS-1 and MIN-6 cells under the high glucose condition after adding AMPK agonist and inhibitor.2.3.Verification of the hypoglycemic mechanism of GM in vivo:2.3.1.RT-qPCR method was used to detect the mRNA expression level of TXNIP in pancreas and liver tissues of db/db mice.2.3.2.Western blotting was used to detect the effects of GM on AMPK,p-AMPK,TXNIP,p-STAT3,STAT3 and MaFA(Only expressed in the pancreas,so it is not detected in the liver)protein in the pancreas and liver tissues of db/db mice.Results1.GM shows a great hypoglycemic effect1.1.GM could effectively reduce blood glucose and glycosylated hemoglobin levels in db/db mice,and significantly reduce the food intake and body weight of db/db mice in the 4th week.1.2.GM could effectively improve the pathological changes of liver tissue in db/db mice.1.3.The results of immunohistochemistry and ELISA showed that GM can effectively promote insulin secretion in pancreatic tissues of db/db mice.1.4.RT-qPCR results showed that GM can significantly increase insulin mRNA levels in pancreatic tissues of db/db mice.2.GM plays a hypoglycemic effect by activating AMPK/TXNIP/MaFA signaling pathway2.1.The results of MTT,RT-qPCR and Western blotting showed that when the glucose concentration is 33.33 mmol/L,a high-glycemic injury model can be successfully constructed for subsequent experimental studies.2.2.The results of RT-qPCR,ELISA and Western blotting showed that GM can significantly increase insulin secretion in INS-1 and MIN-6 cells under high glucose conditions.2.3.RT-qPCR and Western blotting results showed that under high glucose conditions,GM could significantly up-regulate the protein expression of p-AMPK,p-STAT3 and MaFA,and down-regulates the protein and mRNA expression of TXNIP.After treatment with AMPK agonists and inhibitors,the degree of up-regulation of p-AMPK and down-regulation of TXNIP in INS-1 cells by GM was basically the same as that of AMPK agonists,and could reverse the up-regulation effect of AMPK inhibitors on TXNIP.2.4.RT-qPCR and Western blotting results showed that GM could significantly up-regulate the protein expression of p-AMPK and p-STAT3 in the pancreas and liver tissues of db/db mice,and significantly up-regulate the expression of MaFA protein in the pancreas of db/db mice,significantly down-regulate the protein and mRNA expression of TXNIP in pancreas and liver tissues of db/db mice.ConclusionGM can effectively reduce blood glucose and glycosylated hemoglobin levels,and improve liver histopathological changes in db/db mice.The mechanism may be related to the activation of AMPK/TXNIP/MaFA signaling pathways,thereby increasing the level of insulin secretion.