Effects Of Exercise And Metformin On Mitochondrial Autophagy And Oxidative Stress In Type 2 Diabetic Kidney

Research objectives:The purpose of this study was to investigate the effects of exercise and metformin alone or combined intervention on the autophagy and oxidative stress levels of renal mitochondria in diabetic rats,and to explore the effects of three methods on their related signaling pathways.The results of different intervention methods were compared to provide a theoretical source for the effective comprehensive prevention and treatment of diabetic nephropathy.Research methods:Male SD rats fed with high fat diet and streptozotocin injection were randomly divided into diabetic model group(B,n=8),diabetic exercise group(C,n=8),diabetic metformin group(D,n=8),diabetic exercise + metformin group(E,n=8),and normal group(A,n=8).Group C ran the treadmill for 8 weeks;Group D was given metformin intragastric administration for 8 weeks,and group E was given metformin intragastric administration for 8 weeks.The body weight and other conditions of the rats were measured and recorded weekly,and the blood glucose(GLU)and insulin resistance index were measured to evaluate the blood glucose control of the rats.Blood urea nitrogen(BUN)and creatinine(CRE)levels were used to assess renal function.HE staining was used to evaluate the degree of renal tissue damage,and Masson staining was used to analyze the status of renal interstitial fibrosis.The ultrastructure of kidney was observed by transmission electron microscopy.The protein contents of AMPK,p-AMPK,ULK1,p-ULK1,TFEB,LC3-I,LC3-II and p62 were detected by Western Blot to evaluate the mitochondrial autophagy level.Nrf2,SOD1 and NOX4 protein content as well as the total antioxidant capacity(T-AOC)and malondialdehyde(MDA)levels of kidney were detected to evaluate the level of oxidative stress.Then analyze and compare the data differences of each group to draw a conclusion.Research results:1.The rats in group A had good mental state,normal body weight and glossy hair;The rats in group B had dry hair,slow activity,listless spirit,emaciation,polydipsia,polyuria.After 8 weeks of exercise and metformin alone or combined intervention,the above symptoms of rats in group C,D and E were improved,body weight was reduced,and the mental state was better.2.Compared with group A,GLU and insulin resistance index of rats in groups B,C,D and E were significantly higher than those in group A,with significant differences(P < 0.01).After 8 weeks of exercise and metformin alone or combined intervention,GLU and insulin resistance index in groups C,D and E were significantly decreased compared with group B,and had significant differences(P < 0.05 or P < 0.01);In addition,compared with group C,GLU expression level of group E was lower than that of group C,and the difference was significant(P < 0.05),indicating that the effect of combined intervention of group E was better than that of exercise alone,but there was no significant difference between the effect of combined intervention of group E and metformin alone(P > 0.05).3.From the measurement results of BUN and CRE,the levels of BUN and CRE in kidney of rats in group B were the highest,which had A significant difference compared with that in group A(P < 0.01).After 8 weeks of exercise and metformin alone or combined intervention,the levels of BUN and CRE in the kidney of groups C,D and E were significantly decreased,and the expression levels of BUN and CRE in the kidney of rats in group E were the lowest,which had a significant difference compared with that in group B(P < 0.01).After intervention,the expression levels of BUN and CRE in kidney of group E were similar to those of group A,but there was no significant difference between group A and group E(P >0.05).4.Kidney morphological indexes of rats in different groups:(1)The results of HE staining showed that the kidney structure of rats in group A was complete and clear,and the glomerular cells were arranged regularly without pathological changes.In group B,mesangial hyperplasia,basement membrane thickening,glomerular volume increasing,renal tubule atrophy and lumen stenosis were observed.The renal lesions in groups C and D were significantly less severe than those in diabetic control group,and there was an obvious recovery,and the vacuolar degeneration of renal tubular epithelial cells was reduced.In group E,mesangial hyperplasia and tubule atrophy were obviously recovered,tubule dilation was reduced,and the renal structure was complete and clear,which was obviously better than that of group B and similar to that of group A.(2)Masson staining of rat kidneys showed that the morphology of rat kidneys in group A was clear and complete;In group B,the renal tissue structure was severely abnormal,a large number of blue collagen fiber hyperplasia could be seen in the renal interstitium,the damage of the renal tubule interstitium presented tubular dilatation,and the fibrosis of the glomeruli and renal tubules was obvious.Compared with group B,the degree of fibrosis in group C and group D was reduced,and the blue collagen fibers were relatively reduced.Only a few blue collagen fibers could be seen in the renal interstitium of group E.(3)The results of renal transmission electron microscopy showed that the renal structure of rats in group A was clear,the glomerular basement membrane thickness was normal and uniform,the morphology of podiocytes was complete,the arrangement was regular,and no obvious foot process fusion was observed.In group B,the glomerular basement membrane was thickened,and the number of podocytes was sharply reduced,without obvious structure.The morphology of podocytes in group C was clear and the fusion phenomenon was reduced.In group D,the thickness of glomerular basement membrane decreased and the fusion of foot process decreased.The kidney of rats in group E showed obvious recovery and the shape of the foot process was relatively complete.5.Western Blot analysis showed that compared with group A,autophagy marker proteins related to p-AMPK,p-ULK1,TFEB and LC3 were significantly down-regulated,while autophagy degradation substrate protein p62 was up-regulated in group B.After 8 weeks of exercise alone or combined with metformin,the protein contents of p-AMPK,p-ULK1,TFEB and LC3 in the kidney of rats in groups C,D and E were significantly increased and p62 was significantly downregulated compared with those in group B.Compared with C group,the contents of P-AMPK /AMPK and LC3-II/LC3-I in kidney of group E were significantly increased,while the content of p62 was significantly decreased,and the difference was significant(P < 0.05 or P < 0.01).The contents of P-ULK1 /ULK1 and TFEB in group C were higher than those in group C,but there was no significant difference(P > 0.05).Compared with group D,the protein contents of P-AMPK /AMPK,p-ULK1/ULK1,TFEB and LC3-II/LC3-I in kidney of group E were increased,while the content of p62 was decreased,but there was no significant difference(P > 0.05).6.By testing related indexes of renal oxidative stress of rats in different groups,it can be seen that,compared with rats in group A,the expressions of Nrf2,SOD1 and T-AOC in kidney of rats in group B are significantly decreased,while NOX4 and MDA are significantly increased,with significant differences(P <0.01).After 8 weeks of exercise and metformin alone or combined intervention,the levels of Nrf2,SOD1 and T-AOC in groups C,D and E were significantly increased compared with those in group B(P < 0.05 or P < 0.01);NOX4 and MDA are down-regulated,but the down-regulated MDA of group C and group D is not significantly different from that of group B(P > 0.05),and the down-regulated MDA of group E and group C is significantly different from that of group B(P < 0.01).Conclusion:1.All three interventions can improve blood glucose metabolism and renal structure and function in diabetic rats.And compared with exercise group,the combined intervention effect of the two is better.After exercise or metformin alone or in combination,the degree of renal lesions was reduced and collagen fibers were reduced.2.The mitochondrial autophagy level of kidney in type 2 diabetic rats decreased and the oxidative stress level increased.8 weeks of exercise and metformin alone or in combination can protect the kidney by up-regulating the autophagy level of kidney mitochondria and inhibiting the oxidative stress level of diabetic rats.This may be related to the activation of AMPK-related signaling pathway,which leads to the activation of mitochondrial autophagy and the decrease of oxidative stress level.The specific mechanism needs further study.