Renal Protective Effects Of Astragaloside ? Combined With Ligustrazine In Diabetic Rats And Its Mechanism Based On Akt/mTOR

Objectives:To investigate the effects of Astragaloside IV combined with Ligustrazine on the renal protective effect and Akt/mTOR signal pathway in diabetic rats induced by STZ plus high fat diet,providing experimental basis for the combination of these two drugs for prevention and treatment of diabetes and its renal complications.Methods:1.Experimental animals:Sixty-eight male SD rats of SPF were randomly divided into normal group(n=8)and diabetic model group(n=60)rats according to their body weight after one week of adaptive feeding.The model group rats were injected 30mg kg-1 of STZ through caudal vein,and fed with high sugar and fat feeds on the same day.The normal rats were injected equal volume of citric acid buffer through caudal vein,and fed with ordinary standard feeds.Four weeks later,after 12 hours' fasting,blood glucose were determined and 40 diabetic rats with FPG?16.7mmol·L-1 and obvious symptoms of excessive drinking,eating and diuresis were selected for experiment.2.Drug intervention:According to blood glucose and weight,forty diabetic model rats were randomly divided into 5 groups as the diabetic model control(DM)group,Astragaloside IV(Ast IV)group,Ligustrazine(Lig)group,Astragaloside IV combined with Ligustrazine(Ast IV+Lig)group and valsartan positive control group with each group 8 rats.Ast IV group,Lig group,Ast IV+Lig group and valsartan group were orally given corresponding drugs(drug dose/body weight)of 20mg·kg-1150mg·kg-1?20mg·kg-1+150mg·kg-1 and 25mg·kg-1?diabetes model control and normal control group were given normal saline.The dose volume was 10ml·kg-1(drug/weight),once a day,for 6 weeks.3.Irndex detection:After drug intervention,the general signs of experimental rats were observed.The blood was taken regularly from tail vein to measure the fasting plasma glucose(FPG)and glycosylated hemoglobin.The body weight,food intake and water consumption of rats were measured regularly,and 24h urine was collected by metabolic cage to measure the urine volume and 24h urinary protein.At the end of the experiment,cardiac blood samples were collected to measure total cholesterol(CHOL),triglyceride(TG),low density lipoprotein(LDL-C)and other biochemical parameters,such as serum creatinine(Scr),blood urea nitrogen(BUN),Microalbumin(ALB)and total protein(TP);the kidneys were stripped and the renal index was calculated;pathological changes of rat kidney were observed by HE staining;and the expression level of AKT/mTOR protein in kidney was detected by Western blot analyses.Results:1.Compared with normal rats,the glycosylated hemoglobin and blood sugar were significantly increased;the body weight was obviously decreased;diet,drinking water and urine volume,as well as CHOL,TG,LDL-C,were increased in diabetic rats,which were consistent with the symptoms of type 2 diabetes.Simultaneously,the serum level of BUN,Scr and 24h urinary protein increased significantly,with the renal index increased,the glomerular volume of rat kidney and mesangial matrix increased,and the renal tubular epithelial changed to vacuolation,which accorded with the diagnosis of early renal injury in diabetic rats.AST IV at the dose of 20mg·kg-1 could improve HDL-C,decrease Scr and ALB,but fail to reduce blood sugar and 24h urinary protein.Lig at the dose of 150mg·kg-1 could reduce FPG significantly,but could not influence LDL-C and kidney-related indicators such as BUN?Scr and 24h urinary protein.Meanwhile,in Astragaloside IV combined with Ligustrazine(20mg·kg-1+150mg kg-1)group,HbAlc and fasting blood glucose levels,food and water intake were reduced,body weight was increased,TG?LDL-C,Scr,BUN,ALB,TP and urinary protein in 24 hours were reduced,kidney index was reduced and renal pathological was improved compared with model control.The results showed that the hypoglycemic and renal protection effects of drug combination were signigicantly higher than single drug treatements.Besides,compared with Valsartan(25mg·kg-1)group,Astragaloside ? combined with Ligustrazine group was similar in the control of blood sugar,but was better on the renal impairment index such as BUN and Scr.2.Western blot results showed that there was no significant difference in total Akt protein expression of each group,but the expression levle of phosphorylated Akt protein in renal tissue of DM group was significantly higher than that in normal group.As a result,the expression of phosphorylated Akt protein in the renal tissue of treated groups was lower than that in DM group,and the change was significant in Lig Group and Ast IV+Lig group.In addition,there was no remarkable difference in the expression of total mTOR protein in the kidney tissue of the experimental groups,but the phosphorylation mTOR protein expression level in renal tissues of DM group was higher than that of normal control group,and Ast ?+Lig rats was significantly lower than that in DM group.These results indicated that Ast ?+Lig might play an important role in the protection of diabetic nephropathy by inhibiting the Akt/mTOR signaling pathway.Conclusions:1.Astragaloside ? combined with Ligustrazine can improve the general physical sign condition of STZ and high fat diet-induced diabetic rats,it can also reduce the glycosylated hemoglobin and fasting blood glucose levels,improve the lipid metabolism-related indicators TG and LDL-C,and all mentioned above is superior to the single use of Astragaloside ? or Ligustrazine.2.Astragaloside ? combined with Ligustrazine can improve plasma indicators of kidney damage such as Scr and BUN,reduce 24 hours urine protein,improve renal function,reduce kidney indexes and ameliorate pathological changes,and the renal protective effect is superior to the single use of Astragaloside IV or Ligustrazine.3.Astragaloside ? combined with Ligustrazine can inhibit kidney Akt/mTOR signaling pathway activation in diabetic rats,which might be one of its mechanisms for improving diabetic nephropathy.