Effect And Mechanism Of Regulating Autophagy In Podocyte To Improve Diabetic Kidney Disease With Yishen Decoction

Diabetic kidney disease(DKD),also known as DN(Diabetic Nephropathy),is one of the major microvascular complications of diabetes.DKD is a progressive disease caused by long-term chronic hyperglycemia.The pathogenesis of DKD is complex.At present,more and more studies have found that the abnormal autophagic behavior in podocyte is closely related to the pathogenesis of DKD.Autophagy seems to be a well-established contributor to podocyte homeostasis,and either enhanced or dysregulated autophagy might involve in podocyte injury.This study based on our previous clinical research,adopted Yishen Decoction(YSKL)with clinical curative effect to DKD,and focused on the autophagy in podocyte,especially via the two signaling pathways—PI3k/Akt/mTOR and LKB1/AMPK/Sirtl pathways.Both in vivo and in vitro experiments were conducted to observe the effect of YSKL on the intervention of DKD rats and YSKL drug-containing serum on MPC5 podocyte.Part Ⅰ:Effect of YSKL on DKD rats via the PI3k/Akt/mTOR and LKBl/AMPK/Sirtl Signal PathwaysObjective:To observe the effect of YSKL on DKD rats and to explore the possible mechanism via PI3k/Akt/mTOR and LKB1/AMPK/Sirtl signal Pathways.Methods:105 male SPF Wistar rats were used in this study.10 rats were randomly selected as normal group with ordinary diet,while others were selected as the DKD rats which were induced by low-dose STZ(30mg/kg)intraperitoneally injection with high-sugar and high-fat diet.90 DKD rats were randomly divided into the model group,the valsartan group,the metformin group,the YSKL low dose group,the YSKL middle dose group,and the YSKL high dose group,15 in each group.After 10 weeks of treatment,blood and 24-hour urine were collected for detection.Histological changes in renal tissue were observed by HE staining,Oil red O staining,Masson staining,PASM staining.Changes of glomerular and autophagy in podocyte were observed by electron microscopy.The expressions of relative proteins were detected by Western blot.Results:(1)Compared with the normal group,levels of Scr,Bun,FBG,TG,TC,24-hour urine albumin increased in the model group(P<0.05).Compared with the model group,levels of Scr,Bun,FBG,TG,TC,24-hour urine albumin decreased in the other groups(P<0.01),especially the YSKL middle dose group.(2)HE staining showed that compared with the normal group,steatosis was observed in both tubules and glomeruli of the model group,accompanied by focal tubular injury,basement membrane thickening and mesangial matrix expansion.Compared with the model group,pathological changes were reduced in the other groups,especially the YSKL middle dose group and high dose group.Oil red O staining showed that compared with the normal group,severe steatosis was observed in the model group.Compared with the model group,steatosis was reduced in the other groups,especially the YSKL groups and the metformin group.Masson staining showed that compared with the normal group,the blue deposition of collagen fibers was observed in the glomeruli and renal interstitium of the model group,accompanied by mesangial area expansion,mesangial matrix hyperplasia.Compared with the model group,the blue deposition of collagen fibers was significantly reduced in YSKL middle dose group.PASM staining showed that compared with the normal group,the glomerular basement membrane thickening,the mesangial matrix hyperplasia,renal capsule dilation,inflammatory cell infiltration,and partial renal tubular epithelial cells vacuolar degeneration were observed in the model group.Compared with the model group,the degree of glomerular basement membrane thickening and the mesangial matrix hyperplasia were reduced in both YSKL middle and high dose group.Electron microscopy showed that compared with the normal group,fusion of foot process and basement membrane thickening were observed in the modem group.Autophagosomes were observed in the YSKL groups.(3)Western blot showed that compared with the normal group,protein expression levels of PI3k,p-Akt,p-mTOR significantly increased(P<0.01),and LKB1,p-AMPK,p-Sirtl significantly decreased(P<0.01).Compared with the model group,protein expression levels of PI3k,p-Akt,p-mTOR significantly decreased(P<0.01),and LKB1,p-AMPK,p-Sirtl significantly increased(P<0.01),especially in the YSKL middle group and the metformin group.Conclusion:YSKL could reduce the levels of Scr,BUN,FBG,TG,TC and 24-hour urine albumin of DKD rats.It might regulate PI3k/Akt/mTOR and LKB1/AMPK/Sirtl signaling pathways to relieve the renal pathological changes of DKD rats.Part Ⅱ:Effect and mechanism of YSKL on Glucose and Lipid Metabolism,Liver and Pancreas of DKD RatsObjective:To obeserve the possible effect and mechanism of YSKL on glucose and lipid metabolism,liver and pancreas of DKD rats.Methods:70 male SPF Wistar rats were used in this study.10.rats were randomly selected as normal group with ordinary diet,while others were selected as the DKD rats which were induced by low-dose STZ(30mg/kg)intraperitoneally injection with high-sugar and high-fat diet.60 DKD rats were randomly divided into the model group,the metformin group,the YSKL low dose group,the YSKL middle dose group,the YSKL high dose group,12 in each group.After 10 weeks of treatment blood samples were collected for detection.The body weight,liver weight,and pancreatic weight of the rats were measured.Liver tissues were collected for hepatic HE and Oil red O staining,and pancreas tissues were collected for pancreas HE staining.Results:(1)Compared with the normal group,levels of HbAlc,FBG,FIN,and HOMA-IR significantly increased(P<0.01)in the model group,while levels of HOMA-β and HOMA-IS significantly decreased(P<0.01).Compared with the model group,levels of HbA1c,FBG and HOMA-IR significantly decreased(P<0.01),while levels of FIN,HOMA-β and HOMA-IS significantly increased(P<0.01)in the YSKL middel group and the metformin group.Compared with the normal group,levels of ALT and AST significantly increased(P<0.01)in the model group.Compared with the model group,levels of ALT and AST significantly decreased(P<0.01)in the YSKL groups and the metformin group.Compared with the normal group,levels of TG,TC,LDL-c and HDL-c significantly increased(P<0.01)in the model group.Compared with the model group,levels of TG,TC,LDL-c and HDL-c significantly decreased(P<0.01)in the YSKL groups and the metformin group.(2)HE staining and Oil red staining showed that compared with the normal group,severe steatosis was observed in both liver and pancreas of model group.Compared with the model group,steatosis was reduced in the other groups,especially the YSKL middle dose group.Conclusion:YSKL could protect the liver and pancreas of DKD rats by regulating glucose and lipid metabolism and reducing insulin resistance.Part III:The Experimental Study of YSKL Drug-containing Serum on Regulating Autophagy In Podocyte to Relieve Podocyte Injury stimulated by High GlucoseObjective:To observe the effect of autophagy of MPC5 podocytes stimulated by high glucose with YSKL drug-containing serum.Methods:MPC5 podocytes were stimulated by high glucose in vitro.Podocytes treated for 48h with high glucose concentrations(35mM)in the high glucose model group,and YSKL drug-containing serum(15%optimal serum concentration)and metformin-containing serum were used for intervention.Western blot was used to detect the protein expression of podocytes and autophagy.Results:(1)Compared with the control group,protein expression levels of Nephrin,Podocin,LC3-Ⅱ/Ⅰ,Beclin-1 and p-AMPK decreased,while p-mTOR,p-Akt and P62 increased.Compared with the model group,protein expression levels of Nephrin,Podocin,LC3-Ⅱ/Ⅰ,Beclin-1 and p-AMPK increased,while p-mTOR,p-Akt and P62 decreased.(2)MDC staining showed that after 48 hours of high glucose stimulation,the autophagic activity of podocytes decreased and fluorescence decreased in model group.Metformin group and YSKL group could restore podocyte autophagy.The fluorescence activity of MDC staining was increased in Metformin group and YSKL group.Lyso-tracker staining showed that compared with the control group,the fluorescence activity was decreased in the model group.Compared with the model group,the fluorescence activity was increased in the metformin group and the YSKL group.DAPI staining showed that compared with the control group,the fluorescence activity was increased in the model group and the podocyte apoptosis could be observed.Compared with the model group,the fluorescence activity was decreased in metformin group and YSKL group,and podocyte apoptosis was reduced.(3)Electron microscopy showed that the organelle structure of the podocyte was clearly visible,with little autophagosomes in the control group.Compared with the control group,the damage of the podocyte structure was obvious in the model group,with many black particulate matters after degradation.Compared with the model group,the degree of podocyte damage in the YSKL group was reduced.Conclusion:YSKL drug-containing serum could restore the defective autophagy and relieve podocyte injury stimulated by high glucose in vitro.