Effects Of Shhj On Glucose And Lipid Metabolism And AMPK/mTOR-TFEB Autophagy Signaling Pathway With T2DM With NAFLD

ObjectiveMultiple batches of basic research have confirmed that SHHJ has a variety of effects such as reducing IR,reducing glycemia and lipids,antioxidant damage,and reducing inflammatory factors.Based on the previous research of our team,this experiment further established a T2DM with NAFLD model through in vivo and in vitro tests,and explored the relationship between the AMPK/mTOR-TFEB pathway and T2DM-NAFLD.The aim is to provide a new therapeutic target for diabetes combined with liver injury,to provide preliminary experimental evidence for the clinical treatment of SHHJ and traditional Chinese medicine compound for diabetic liver injury,and to lay a certain experimental foundation for the further research and development of traditional Chinese medicine.MethodsPart I:Effect of SHHJ on glycolipid metabolism in T2DM-NAFLD rats and its mechanismForty male SD rats were randomly selected from the normal group according to body weight.The remaining 30 SD rats were fed with high-fat and high-sugar diet for 42 days,and then intraperitoneally injected with STZ to establish a model of T2DM-NAFLD.They were randomly divided into model group,DMBG group and SHHJ group.Rats in each group were intragastrically administered for 28 days,and body weight and fasting blood glucose were measured every 7 days.After gavage,the rats were euthanized.Abdominal aortic blood and liver tissues were taken.Glycosylated serum protein（GSP）,insulin（INS）,triglyceride（TG）,and total cholesterol（TC）were measured in the serum of each group,high-density lipoprotein（HDL-C）,low-density lipoprotein（LDL-C）,alanine aminotransferase（ALT）,aspartate aminotransferase（AST）content;HE staining to observe changes in liver tissue morphology;immunohistochemical observation of TFEB expression;Western Blot detection of AMPK/mTOR-TFEB signaling pathway-related protein expression.Part II:The effect and mechanism of SHHJ-containing serum on IR-fatty HepG2 cellsHepG2 cells were cultured in DMEM high glucose medium+10%FBS+1%Penicillin（37℃,5%CO₂）.Using Cell Counting Kit-8 detects and screens the most suitable model concentration of Palmitic acid and time.The glucose oxidase method（GOD-POD）was used to investigate the concentration and duration of HepG2 palmitic acid to establish a stable HepG2IR-adipocyte model.Using Cell Counting Kit-8 to detect the proliferation of HepG2 cells by the SHHJ-containing serum,determine the concentration and time of administration,and test the effect of drug-containing serum on glucose consumption of IR-fatty HepG2 cell model.Oil red O staining was used to observe the degree of steatosis of liver cells and the effect of treatment.Western Blot was used to detect the influence of autophagy in the SHHJ-containing serum and the expression of AMPK/mTOR-TFEB signaling pathway related proteins in each group of cells.ResultsPart I:In vivo experiment:1.Glucose metabolism:Compared with the model group,FBS、GSP and insulin secretion of rats in the SHHJ group and the DMBG group were significantly improved（P<0.01）.2.Lipid metabolism:Compared with the model group,the blood lipids of the rats in the SHHJ group and the DMBG group were improved,the HDL-C was significantly improved（P<0.05）,and the TC,TG,and LDL-C were significantly improved（P<0.01）.3.AST and ALT:Compared with the normal group,the AST and ALT of the model group were significantly increased（P<0.01）,only the ALT of the DMBG group and the SHHJ group was significantly increased（P<0.01）;Compared with the model group,both AST and ALT in the DMBG group and the SHHJ group were significantly reduced（P<0.01）。It shows that liver function has improved significantly.4.HE staining results showed that both SHHJ and metformin could imProve the liver morphology of T2DM combined NAFLD rats to a certain extent.The cell morphology of SHHJ group was closer to that of the normal group.5.Immunohistochemical results showed that:P-AMPK and TFEB were not significantly expressed in the model control group（P<0.01）;compared with the model group,the expression in the normal control group was increased（P<0.01）;the metformin group and the SHHJ group were significantly increased（P<0.01）,and most were expressed in vascular endothelial cells.6.Western Blot results showed that both SHHJ and metformin can phosphorylate AMPK（P<0.05）,inhibit mTOR（P<0.05）,activate autophagy,and increase expression of TFEB and LC3B（P<0.01）.Part II:In vitro experiment:1.250μM PA exhibited steatosis after 24 h in HepG2 cells,and had obvious insulin resistance.The model was successfully established.2.2.5%SHHJ-containing serum can significantly increase glucose metabolism in IR-fatty HepG2 cells.（P<0.01）.3.2.5%SHHJ-containing serum can significantly improve the steatosis of IR-fatty HepG2cells.4.2.5%SHHJ-containing serum can activate autophagy,increase LC3II expression（P<0.01）,and decrease P62 expression（P<0.01）.5.Western Blot results showed that 2.5%SHHJ-containing serum could increase the P-AMPK/AMPK ratio（P<0.05）,the expression of TFEB protein（P<0.01）,and down-regulate the expression of mTOR protein（P<0.01）.ConclusionBoth in vivo and in vitro experiments showed that SHHJ can reduce cytotoxicity and insulin resistance by activating AMPK/mTOR-TFEB autophagy signaling pathway,and improve liver function and pathological morphology.Therefore,SHHJ may be a promising way against the disorder of glucose and lipid metabolism,which needs further clinical verification.