Study On The Mechanism Of Yitangkang Regulating AMPK-ROS Pathway To Improve IR In T2DM Mic

Purpose: To observe the effects of Yitangkang on fasting blood glucose,serum insulin and other glucose metabolism related indexes,SOD and MDA levels,and H2O2 expression in skeletal muscle and liver of type 2 diabetic mice by establishing diabetic mouse model.By establishing insulin resistant Hep G2 cell model,the effects of Yitangkang drug-containing serum on survival rate,glucose consumption,AMPK,PGC-1α,SIRT1,FOXO1 protein and m RNA expression of Hep G2 cells were detected.To study the improvement effect of Yitangkang on energy metabolism,oxidative stress and insulin resistance of type 2 diabetic mice,accumulate new experimental evidence for the treatment of type 2 diabetes with TRADITIONAL Chinese medicine,and strive to explore new targets for the treatment of type2 diabetes.Material and methodEssay 1:Methods: 30 C57BL/6 mice were divided into blank group(n = 10)and model group(n = 20)according to average body weight.The blank group was fed with normal diet,and the mice in the modeling group were fed with high-sugar and high-fat diet by INTRaperitoneal injection of STZ for modeling.After 8 weeks of feeding,20 mice in the model group were divided into model group and Yitangkang group according to their average body weight by number table method,with 10 mice in each group.Normal group and model group were given normal saline intragastric treatment,yitangkang was given compound Yitangkang decoction intragastric treatment,once a day.Three weeks later,mice were anesthetized with 10%chloral hydrate by intraperitoneal injection.Blood was taken from abdominal aorta and transferred to anticoagulant tube for testing.The mitochondria were extracted from fresh skeletal muscle and liver tissue of mice,and total protein was extracted from other liver tissue.The levels of fasting blood glucose and insulin in serum were detected and insulin resistance index was calculated,and the contents of SOD and MDA in serum were detected.The relative expression levels of AMPK and PGC-1 α protein in liver of mice in each group were detected by Western blot,and the H2O2 content in mitochondria of liver and skeletal muscle was detected by immunofluorescence method.Essay 2:Twenty SPF male SD rats were divided into blank group(n = 20)and Yitangkang group(n =10)by random number table method.Normal saline 10ml/kg and Yitangkang decoction10ml/kg were given intragastric administration once a day.After 7 days,abdominal aorta blood was taken to prepare blank serum and yitangkang drug-containing serum.Hep G2 cells were cultured and induced by certain concentration of insulin to establish insulin-binding cell model.The cells were randomly divided into blank group,model group and Yitangkang group.Blank group and model group were given blank rat serum,and Yitangkang group was given yitangkang drug-containing serum intervention.Cck-8 kit was used to measure cell viability,glucose oxidase method was used to measure glucose consumption,western-blot method was used to detect AMPK,PGC-1α,SIRT1 and FOXO1 protein expression levels and their phosphorylation,AND RT-PCR was used to detect AMPK,SIRT1 and FOXO1 gene expression levels.Results:Essay 1:1.Compared with blank group,fasting blood glucose,serum insulin level and insulin resistance index of model group were significantly increased(P<0.01).Compared with model group,fasting blood glucose,serum insulin level and insulin resistance index in Yitangkang group were significantly decreased(P<0.01).2.Compared with blank group,MDA in model group and Yitangkang group was significantly increased(P < 0.01),SOD was significantly decreased(P < 0.01);Compared with model group,MDA in Yitangkang group was significantly decreased(P < 0.01)and SOD was significantly increased(P < 0.01).3.H2O2 level in mitochondria of skeletal muscle and liver tissues of mice in each group: The results showed that compared with blank group,H2O2 level in liver and skeletal muscle of mice in each group was significantly increased(P < 0.01);Compared with model group,H2O2 level in liver and skeletal muscle of mice in Yitangkang group was significantly decreased(P < 0.01).4.Protein expression of mice in each group,the results showed as follows: compared with blank group,the levels of AMPK and PGC-1α in liver tissues of mice in each group were significantly decreased(P < 0.01);Compared with model group,the expression of AMPK and PGC-1α in Yitangkang group was significantly increased(P < 0.01).Essay 2:1.Compared with blank,the activity and glucose consumption of Hep G2 cells in each group were significantly decreased(P<0.01).Compared with model group,the activity and glucose consumption of Hep G2 cells in Yitangkang group were significantly increased(P<0.01).2.Hep G2 cells: changes of AMPK,SIRT1 and Fox O1:Compared with normal group,the m RNA expression levels of AMPK,PGC-1α and SIRT1 in model group were significantly decreased(P < 0.01),the m RNA expression level of Fox O1 was significantly increased(P < 0.01),and the protein expression levels of AMPK,PGC-1αand SIRT1 were significantly decreased(P < 0.01).The expression level of Fox O1 phosphorylated protein was significantly increased(P < 0.01).Compared with model group,the m RNA expression levels of AMPK,PGC-1 α and SIRT1 in Yitangkang group were significantly increased(P < 0.01),the m RNA expression levels of Fox O1 were significantly decreased(P < 0.01),and the protein expression levels of AMPK,PGC-1α and SIRT1 were significantly increased(P < 0.01).Fox O1 protein expression level was significantly decreased(P < 0.01).Conclusion:1.Yitangkang can improve the energy metabolism disorder of type 2 diabetic mice.2.Yitangkang may reduce oxidative stress response and improve insulin resistance of type 2diabetic mice by removing reactive oxygen species in cells.3.Yitangkang drug-containing serum can improve the glucose intake of insulin-induced Hep G2 cells and improve cell activity.4.Ytangkang acts as an activator of AMPK/PGC-1 pathway,and regulates the expression of SIRT1 and FOXO1 proteins downstream of this signaling pathway to promote energy metabolism,reduce oxidative stress and improve insulin resistance,thus treating type 2diabetes.