| Theoretical research:Diabetes has become one of the diseases that seriously threaten populares' physical and mental health.Diabetes incidence and disability mortality rate are increasing gradually,and bringing a huge burden to society.Therefore,it is of great significance to explore effective methods to treat diabetes.There are two main mechanisms for the pathogenesis of diabetes: first,insufficient insulin secretion by pancreatic ?-cells,second,insulin resistance in surrounding tissues.The damage of pancreatic ? cells is the key factor in the occurrence and development with type 1 or type2 diabetes.Autophagy is the main reason that cells can degrade their degenerated organelles.The purpose of autophagy is to maintain the homeostasis,protect the normal survival state,and maintain the normal function of the cells.Studies have shown that autophagy can reduce pancreatic ?-cells' apoptosis and maintain pancreatic ?-cells' stability.Pancreatic ?-cells with autophagy defects often suffer from apoptosis and cell cycle arrest,which is caused by mitochondrial dysfunction.Regulating pancreatic ?-cell autophagy plays an important role in protecting pancreatic ?-cells,maintaining their normal functions,and blocking the occurrence and the development of diabetes.Diabetes belongs to the category of "xiaoke" in tradtional Chinese medicine(TCM).TCM has great advantages in the treatment of diabetes,under the guidance of syndrome differentiation and holistic concept.In the 1980 s,Professor Xiong Manqi from our team has summed up the basic pathogenesis of diabetes is "deficiency of both qi and yin,and mutual accumulation of stasis and heat",which based on the classics of traditional Chinese medicine and clinical practice.Jiangtang Sanhuang Tablets was consisting of Taohe Chengqi Decoction and the traditional medicines which is used for replenishing qi and nourishing yin.Our team has done a lot of research and confirmed that Jiangtang Sanhuang Tablets can effectively improve the clinical symptoms of patients with type 2 diabetes,lower blood sugar and blood lipid levels,protect pancreatic ? cells,improve insulin resistance,and effectively reduce the occurrence of diabetes complications.But the mechanism of Jiangtang Sanhuang Tablets is still unclear.The network pharmacology study of Jiangtang Sanhuang Tablets suggests that it can regulate pancreatic?-cell autophagy,and its mechanism is to activate multiple autophagy-related pathways,including AMPK/mTOR signaling pathway.Studies have reported that activating the AMPK/mTOR pathway can enhance glucose uptake by skeletal muscle and other tissues and improve insulin resistance.Activating AMPK/mTOR signaling pathway can also activate autophagy.Considering that Jiangtang Sanhuang Tablets can protect pancreatic ? cells and regulate pancreatic ? cells autophagy,and activating autophagy contribute to protect pancreatic ? cells.Therefore,we speculated that Jiangtang Sanhuang Tablets may activate autophagy by activating AMPK/mTOR signaling pathway to play a protective effect on pancreatic ?-cells.The purpose of this study is to clarify the role and mechanism of autophagy in the protection of pancreatic ? cells by Jiangtang Sanhuang Tablets.Experimental research Objective:This research is divided into two parts: in vivo research and in vitro research.The first part is in vivo research: 1.To observe the effect of Jiangtang Sanhuang Tablet on the general situation,blood sugar,blood lipid and other metabolic levels with diabetic model mice(db/db mice)and insulin resistance.To evaluate the effect of Jiangtang Sanhuang Tablets on glucose and lipid metabolism in diabetic mice.2.To detect the effect of Jiangtang Sanhuang Tablets on autophagy of pancreatic tissues in diabetic mice with transmission electron microscopy technology.The second part is in vitro research: 1.To verify whether the high-sugar and highfat environment can damage pancreatic ?-cells,and whether Jiangtang Sanhuang Tablets can protect pancreatic ?-cells and reduce the pancreatic ?-cells' apoptosis which is caused by glycolipid toxicity.2.To explore whether Jiangtang Sanhuang Tablets can regulate pancreatic?-cells' autophagy,and the relationship between the effect of protection of pancreatic ?-cells and the regulation of cell autophagy.3.To revealed the mechanism of Jiangtang Sanhuang Tablets regulating autophagy and protecting pancreatic ? cells.Methods:1.In vivo research: Spontaneous diabetic mice db/db mice and db/m mice were raised and kept in Specific Pathogen Free environment.Fasting blood glucose were used to identify db/db mouse successfully become diabetic mouse model.Diabetic mouse were randomly divided into four groups,model group,low-dose group of Jiangtang sanhuang tablet,highdose group of Jiangtang sanhuang tablet,metformin group as positive control,and db/m mouse group served as healthy control.The experiment lasted for 6 weeks.During the experiment,mouse in each group were free to eat and drink.The weight and the fasting blood glucose level of each group were measured,before the experiment and at the 3rd and 6th week of the experiment,individually.The daily food intake and daily water intake of each group were counted every 2 weeks.After the experiment,the serum glucose and lipid metabolism indexes of each group were detected: TCHO,TG,HDL-C,LDL-C,FFA and fasting insulin levels.Transmission electron microscope was used to observe the ultrastructural morphology and the number of autophagosomes in the pancreas.2.In vitro research: MIN6 cells is a well-known cell line of pancreatic ?-cells.This study was use MIN6 cells with glucose and palmitic acid to establish a diabetic cell model,in vitro.CCK-8 was used to detect cell viability.Flow cytometry was used to detect cell apoptosis.Glycolipid concentration was screened to create the high-sugar and high-fat environment cell model by CCK-8 and flow cytometry.Drug-contained serum of Jiangtang Sanhuang Tablets were extracted from SD rats.According to the results of CCK-8 and flow cytometry,the optimal concentration of drug-contained serum of Jiangtang Sanhuang Tablets was screened to explore whether Jiangtang Sanhuang Tablets could protect pancreatic?-cells in the highsugar and high-fat environment.Cell immunofluorescence was used to detect the expression of LC3 ?,an autophagy-related marker,both in the control group,the model group and the drug-contained serum group.The results were analyzed to verify the effect of Jiangtang Sanhuang Tablets with cell autophagy.Transmission electron microscopy was used to observe the changes of autophagosomes and organelles,and Western Blot was used to detect the expression of LC3 ?,P62 and Beclin-1,a series of autophagy-related protein.Western Blot was used to detect the expression of the AMPK/mTOR signaling pathway related proteins and the expression of LC3 ?,after the use of AMPK/mTOR signaling pathway inhibitors,to explore the effect of inhibiting AMPK/mTOR signaling pathway on autophagy.Results:(1)In vivo research:1.General situation.During the experiment,compared with the model group,the mobility,coat gloss,mental state,and reactivity were significantly improved in the health control group.The excretion of stool and urinary with the health control group were significantly reduced than the model group.After medication intervention,general situation of the db/db mice were improved compared with the model group.There was no significant difference between each group,with weight,food intake,and water consumption,at the beginning of the experiment.At the end of the experiment,the body weight,food intake,and water consumption of the mice in the model group were significantly higher than those in the Jiangtang Sanhuang Tablet intervention group and the metformin intervention group.2.Glucose and lipid metabolism and insulin resistance.There was no significant difference in fasting blood glucose levels between db/db mice in each group,at the beginning of the experiment.With the progress of the experiment,the fasting blood glucose level of the model group mice increased gradually and was much higher than that of the normal group mice,and severe glucose metabolism disorders appeared.The fasting blood glucose of the mice in the Jiangtang sanhuang Tablet intervention group and the metformin intervention group was significantly lower than that of the model group,especially in the Jiangtang sanhuang Tablet high-dose group and the metformin group.Regarding lipid metabolism,the blood lipid metabolism indexes of the normal group were all within the normal range,at the end of the experiment.The TCHO,TG,LDL-C and FFA indexes of the model group were significantly higher than those of the normal group,and the HDL-C level was significantly lower than that of the normal group.The blood lipid indexes of mice in the Jiangtang Sanhuang Tablet intervention group and the metformin intervention group were improved to varying degrees compared with the model group.Regarding serum insulin levels and insulin resistance index,mice in the model group showed significant insulin resistance,and after drug intervention,insulin resistance was improved.3.Transmission electron microscope observation of mouse pancreatic cells.The mitochondria in the pancreatic cells of the normal group were normal,and the morphology of various organelles was clear.There were many autophagosomes with double-layer membrane structure in the cells,which indicates that the pancreatic tissue cells of the normal group are undergoing autophagy.The cell structure of pancreatic tissue in the model group was not clear,some mitochondria appear abnormal in morphology,and the number of autophagosomes was sharply reduced,which indicates that the degree of autophagy in the model group is low.Compared with the model group,the destruction of the pancreatic cells' structure in the drug intervention group was reduced,the cell structure was intact,some organelles were swollen and deformed,and the number of autophagosomes increased compared with the model group.(2)In vitro research:1.The cytotoxicity of glycolipid with pancreatic ?-cells show that high sugar environment can cause damage to MIN6 cells,and the degree of damage is positively correlated with sugar concentration.High-fat environment will cause damage to MIN6 cells,and the degree of damage is positively correlated with lipid concentration.High-sugar and high-fat environment will aggravate the damage of MIN6 cells,together.2.The results showed that different concentrations of Jiangtang Sanhuang Tablets drugcontained serum can improve the cell viability and cell apoptosis with MIN6 cells in a highsugar and high-fat environment.3.The study on the regulation of pancreatic ?-cell autophagy by Jiangtang Sanhuang Tablets shows that high-sugar and high-fat environment will reduce the fluorescence of autophagy marker LC3 ?,and Jiangtang Sanhuang Tablets can increase the fluorescence of autophagy marker LC3 ?.Western Blot of autophagy-related protein showed that high-sugar and high-fat environment would decrease the expression of autophagy-related proteins LC3 ? and Beclin-1,and increase the expression of P62 protein,in MIN6 cells.Jiangtang Sanhuang Tablets can increase the expression of LC3 ? and Beclin-1,and decrease the expression of P62 protein,in MIN6 cells.4.The study on the protection of pancreatic ?-cells and the regulation of autophagy by Jiangtang Sanhuang Tablets shows that Jiangtang Sanhuang Tablets can increase the viability and autophagy,decrease the cell apoptosis of MIN6 cells.Chloroquine,an autophagy inhibitor,can counteract the autophagy promotion effect of Jiangtang Sanhuang Tablets on MIN6 cells.After the use of autophagy inhibitors,the Jiangtang Sanhuang Tablets reduced autophagy promotion and protective effects on MIN6 cells.5.The study on the mechanism of Jiangtang Sanhuang Tablets regulating pancreatic ?-cells autophagy showed that the AMPK/mTOR signaling pathway was inhibited in a highsugar and high-fat environment in MIN6 cells.After the intervention of Jiangtang Sanhuang Tablets,the signal pathway was partially restored.6.After using the AMPK/mTOR signaling pathway inhibitor,the AMPK/mTOR signaling pathway was blocked,and the effect of Jiangtang Sanhuang Tablets in activating cell autophagy was reversed.After the AMPK/mTOR signaling pathway was inhibited,cell autophagy is subsequently inhibited.Conclusion:1.Studies show that Jiangtang Sanhuang Tablets can regulate pancreatic ?-cells autophagy and regulate multiple autophagy-related signaling pathways.2.Jiangtang Sanhuang Tablets can improve the general living condition of db/db diabetic mice and the symptoms of diabetic,such as,polydipsia,polyphagia,obesity.It can improve the state of glucose and lipid metabolism disorder and insulin resistance in diabetic mice,and can also activate the autophagy of pancreatic ?-cells in db/db mice,and improve the ultrastructure of pancreatic cells.3.High-sugar and high-fat environments can cause the damage of MIN6 cells and promote cell apoptosis.Jiangtang Sanhuang Tablets can protect MIN6 cells,reduce cell apoptosis,and improve autophagy of MIN6 cells.Jiangtang Sanhuang Tablet protects MIN6 cells by improving cell autophagy.The mechanism of activating autophagy is to activate the AMPK/mTOR signaling pathway. |
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