| ObjectiveTo investigate the expression of Caveolin-1(Cav-1)and Adenosine5'-monophosphate(AMP)-activated protein kinase(AMPK)and eNOS proteins in blood vessels of type 2 diabetic rats.By high-glucose culture of endothelial cells in vitro to further investidate investigate the relationship between Cav-1 and AMPK signaling.Methods1.Establishment of the rats model with Type 2 diabetes mellitus: 15 male Wistar rats(weight about 180-200 g)at 6 weeks of age were adapted to the feeding for 1 week and randomly divided into control group(Control,n=5)and model group(Model,n=5),metformin group(Met,n=5).The control group was given normal feed throughout the feeding period.The model group and metformin group were fed with high-fat and high-sugar diets.After 7 weeks,streptozotocin(STZ 40mg/kg)was injected int raperi-toneally.Fasting blood glucose was measured after 3 days.Subsequent fasting blood glucose ?16.7 mmol/L suggests a successful model of type 2 diabetes.After the model was successfully established,Metformin(Met,200mg/kg/d)was given to the metformin group once a day for 4 weeks and the body weight and fasting blood glucose were measured weekly.After the end of the experiment,relevant biochemical indicators in serum were detected.2.Abdominal aorta was stained with Hematoxylin-eosin(H&E): To observed,the morphological changes of vascular endothelium in Abdominal aorta of the rats.3.Immunohistochemical method for detection of signal molecules: The expression of Cav-1 and AMPK proteins were detected by immunohistochemistry method in Abdominal aorta tissues of the rats.4.Western blotting: Detected the protein expression and of Cav-1 and AMPK inAbdominal aorta tissues of the rats by western blot.5.In vitro cell experiments: Human umbilical vein endothelial cells(HUVECs)were cultured in vitro and the cells were passaged for 2 to 3 generations for experiments.Glucose-containing culture medium was used to culture endothelial cells for 24 to 48 hours.The number of cell growth reached 70% to 80%.They were randomly divided into control group,high glucose group(HG)and metformin group(Met)and given si-RNA-Cav-1 respectively.Protein interference treatment for 6 h,after the end of time,after the replacement of different concentrations of glucose culture solution,while the metformin group was given drug-containing glucose mixed culture medium,culture 48 h Proteins were extracted and the expression of Cav-1,AMPK and eNOS proteins was detected by Western Blot.Results1.General characteristics of all rats : The model group and metformin group were fed with high-sugar and high-fat diets.At the end of the 14 th week,compared with the control group,the body weight of the model group and metformin group increased by16.7% and 15.6% respectively,and the difference was statistically significant(p<0.001);There was no significant difference in body weight between the metformin group and the model group(p=0.6135).In the 15 th week,the model group and metformin group were reduced in body weight after the successful modeling.The weight of the rats in each group was measured at the end of the 18 th week(the end of the experiment).Compared with the control group,the weights of the model group and metformin group were reduced by 6.9%,4.3%,the difference was statistically significant(p<0.001,p=0.013);metformin group than the model group increased by 2.8%,the difference was statistically significant(p = 0.006).Rats fed high-fat and high-sugar diets,on the 14 th weekend,compared with the control group,there was no significant difference in fasting blood glucose between the model group and metformin group(p>0.05).At the end of the 14 th week after injection of STZ,the blood glucose level of the model group and metformin group began to increase.Compared with the control group,the fasting blood glucose of the model groupincreased significantly at the time points of 15 w,16w,17 w,and 18 w,which were respectively increased by 228.1% and 278.14%.214.5% and 230.06%,the difference was statistically significant(p<0.001).Fasting blood glucose in the metformin group increased by 275.7%,225.5%,181.6%,and 180.0%,respectively,with a statistically significant difference(p<0.001).There was no significant difference between the metformin group and the control group(p>0.05)..Pearson correlation was used to analyze the relationship between body weight and fasting blood glucose in rats: After 14 weeks of feeding in each group,there was no correlation between body weight and fasting glucose in each group at the end of the 14th(r=-0.0849,p=0.7634).After STZ injection,blood glucose in the model group and metformin group began to increase,and body weight decreased gradually.There was a negative correlation between body weight and fasting glucose in each group at the end of the 18 th week(r=-0.7829,p<0.001).Changes in serum and blood lipids at the end of 18-week-old rats: Compared with the control group,the fasting blood glucose(FBG)in the model group increased by374.99%,the triglyceride(TG)increased by 276.13%,and the total cholesterol(TC)increased by 107.44%.High-density lipoprotein(HDL)increased 102.43%,very low-density lipoprote-in(VLDL)increased 277.5%,and HbA1 C increased 110.6%,the difference was statisti-cally significant(p<0.001);and metformin group FBG increased by 346.89%,TG increas-e d by 133.52%,TC increased by 52.43%,HDL increased by50.3%,VLDL increased by 132.5%,HbA1 C increased by 96.7%,the difference was statistically significant(p<0.01);Compared with the group,the FBG in the metformin group decreased by 5.9%,TG decreased by 37.9%,VLDL decreased by 38.4%,HbA1 C decreased by 6.6%,and the difference was not statistically significant(p>0.05);TC decreased by 26.5% and HDL decreased by 25.72%(p<0.05),the difference was statistically significant.2.Changes in vascular morphology: At the end of the 18 th week,HE staining of the abdominal aorta was performed.The vascular endothelial cells in the control group were arranged uniformly and neatly.The nucleus was regular and the intravascular elasticmembrane was structurally complete with clear wavy layers.The model group had vascular endothelium.Under the thickening of the cells,endothelial cell defects,irregular arrangement,irregular cell shape,internal elastic membrane structure is irregular,the level is unclear,metformin group than the model group endothelial cells better than before.Immunohistochemistry showed positive expression of Cav-1,located in the membrane,positive expression of AMPK protein,located in the cytoplasm.3.Expression of protein in rat blood vessels: Western blotting was used to detect the expression of Cav-1 protein in abdominal aortic tissue: Compared with the control group,the expression of Cav-1 protein in the model group was reduced by 44%,and the difference was statistically significant(p<0.001),Metformin treatment,Cav-1protein expression increased significantly,compared with the model group increased by70.14%,the difference was statistically significant(p<0.001),compared with the control group,metformin group Cav-1 protein expression A decrease of 4.79% was not statistically significant(p=0.563).AMPK(Thr172)protein expression: compared with the control group,the expression of AMPK protein in the model group was decreased by 39.34%,the difference was statistically significant(p=0.002),metformin group AMPK protein expression increased by 31.36% compared with the control group,and The model group increased by 116.56%,the difference was statistically significant(p=0.046,p=0.001).eNOS protein expression: compared with the control group,the eNOS protein expression in the model group decreased by 36.78%,the difference was statistically significant(p<0.001);metformin group eNOS protein expression was reduced by 21.55%compared with the control group,the difference was statistically significant Significance(p<0.05),compared with the model group,increased by 24.09%,and the difference was not statistically significant.4.Using Pearson correlation analysis:Correlation Analysis of Cav-1,AMPK,and eNOS Proteins and Glycolipids: Cav-1 in the Tissue and Fasting Blood Glucose(r=-0.5487,p=0.0341),Triglycerides(r=-0.5885,p=0.0210),Total Cholesterol(r=-0.7426,p=0.0015)was negatively correlated.AMPK protein expression wasnegatively correlated with fasting plasma glucose(r=-0.5214,p=0.0462),triglyceride(r=-0.6333,p=0.0113),total cholesterol(r=-0.7207,p=0.0024).The eNOS protein expression was negatively correlated with fasting plasma glucose(r=-0.5865,p=0.0216),triglyceride(r=-0.5885,p=0.0210),total cholesterol(r=-0.7426,p=0.0015).Correlations between protein expressions: There was a positive correlation between the expression of Cav-1 in the abdominal aorta and the expression of AMPK protein in each group(r=0.7670,p<0.001);there was no correlation with the expression of eNOS protein(r=0.3671,p=0.1783);There was a positive correlation between AMPK protein and eNOS protein expression(r=0.7670,p<0.001).5.Protein expression in cells:(1)Cav-1 protein expression: Compared with the control group,the expression level of Cav-1 protein in the high glucose group was decreased by 9.5%(p=0.011),and the high glucose metformin group increased by 24.4%.The difference was statistically significant(p=0.016);however,the high-sugar metformin group was increased by 37.6%compared with the high-sugar group,and the difference was statistically significant(p=0.005).AMPK protein expression: compared with the control group,the high glucose group and high glucose metformin group protein expression levels were reduced by31.7% and 19.8%,respectively,the differences were statistically significant(p<0.001,p=0.0159);after the administration of metformin,The expression of AMPK protein in the high-metabolic metformin group was increased by 14.9% compared with the high-sugar group,and the difference was not statistically significant(p=0.088).eNOS protein expression: compared with the control group,high glucose group protein expression increased by 36.7%,the difference was statistically significant(p<0.001),the high-metabolic metformin group decreased by 3.12%,the difference was not statistically significant(p>0.05).After admini-stration of metformin,the expression of eNOS protein in the high-meta metformin group was lower by 29.2% than that in the high-sugar group(p<0.001).(2)Cav-1 protein silencing: Compared with negative interference(si-NC+GH),the expression of Cav-1 protein in the si-Cav-1 + high glucose group was reduced by 34.7%,and the difference was statistically significant.The significance(p=0.001),but the expre-ssion of AMPK protein and eNOS protein decreased by 56.0% and 24.4%,respectively,the difference was statistically significant(p<0.05).After Cav-1 protein was silenced and metformin was administered,Cav-1 protein expression was reduced by 49.59% in the si-Cav-1+HG+Met),after interference with the negative interference(si-NC+GH+Met).The expression of AMPK protein and eNOS protein was reduced by 42.4% and 21.1%,respectively,and the difference was statistically significant(p<0.05).Conclusions1.In the state of type 2 diabetes,decreased expression of Cav-1 in blood vessels may downregulate AMPK-mediated glucose and lipid metabolism signaling pathways and downstream eNOS protein signaling pathways,resulting in disorders of glucose and lipid metabolism and structural abnormalities of vascular endothelial.2.The decreased expression of Cav-1 can down-regulate the expression of AMPK protein and affect the downstream signal transduction pathways mediated by AMPK and endothelial dysfunction. |
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