Effects And Mechanisms Of Astragaloside ? On Kidney Injury Caused By High Insulin

Part ? Effects of Astragalus on Kidney Injury Caused by Hyperinsulinemia in early stage of Type 2 Diabetes PatientsObjective: To investigate the effects of astragalus on kidney injury caused by hyperinsulinemia in early stage of type 2 diabetes patients.Methods: After admission,sixty-eight patients,newly diagnosed type 2 diabetes,were performed short-term intensive insulin therapy with multiple daily insulin injection(MDI).The target was FPG<7mmol/L and 2h PG<10mmol/L.After reaching the target,patients were continued intensive treatment for 3 weeks.Then,fasting insulin(FINS)concentration was determined,and considered FINS levels ?15m U/L in the diabetic patients as hyperinsulinemia(HINS).These patients were randomly divided into two groups: astragalus granules(AG)group(n=20)and hyperinsulinemia(HINS)group(n=20).The next morning after grouping(T0 time point),blood samples were taken for determining FPG,2h PG,Hb A1 c,ALT,AST,TG,TC,CREA,BUN,RBP,Cys-C,MDA,GSH-Px and SOD.Specimens morning urine protein was taken for determining Ualb.Patients in group AG were treated with intensive insulin therapy plus daily administration of astragalus granules.The usage and dosage of astragalus granules was every time 4 gram,two times a day.However,patients in group HINS were treated with intensive insulin therapy only.Two groups of patients continued intensive treatment for 9 weeks.Twenty-eight patients who failed to meet the diagnostic criteria of hyperinsulinemia were excluded.After intensive treatment(T1 time point),blood and urine samples were taken for determining all the indicators at the T0 time point.Results: At the T1 time point,compared with group HINS,in group AG,the level of RBP,Cys-C,Ualb,MDA were lower,and the level of GSH-PX,SOD were higher(P<0.05).In both groups,the level of FPG and 2h PG rised gradually during the period of T0 to T1,while the level of Hb A1 c decreased gradually during the period of T0 to T1(P<0.05).In HINS group,the level of RBP,Cys-C,Ualb and MDA rised gradually during the period of T0 to T1,while the level of GSH-PX and SOD decreased gradually during the period of T0 to T1(P<0.05).Conclusion: Newly diagnosed type 2 diabetes patients were performed with intensive insulin therapy.when blood glucose met the goal,patients were continued intensive treatment for more than 3 weeks.Hyperinsulinemia may occur.If this state lasted for 9 weeks,and it can damage the kidneys by increasing the level of RBP,Cys-C,Ualb and MDA and decreasing the level of GSH-PX and SOD.Astragalus granules could prevent the increase of level of RBP,Cys-C,Ualb and suppress oxidative stress in the body,and it has protective effects on the kidneys.Part ? Effects of Astragaloside ? on Kidney Injury Caused by Hyperinsulinemia in DM RatsObjective: To investigate the effects of Astragaloside ?(AS-?)on kidney injury caused by hyperinsulinemia in a streptozotocin-induced diabetic rat model.Methods: Diabetes was induced with streptozotocin(STZ)(55 mg/kg)by intraperitoneal injection in rats.One week after STZ injection,the diabetic rats were treated with Levemir(6U/day)subcutaneously for four weeks.Diabetic rats insulin levels >30 ?U/ml were considered as hyperinsulinemia.Rats were divided in six groups(n = 8 per group): hyperinsulinemic rats(HINS),hyperinsulinemic rats treated with tempol and AS-? at 2.5,5 and 10 mg/kg/day,intragastric infusion,for 12 weeks.The normal rats were used as a nondiabetic control group.All the diabetic rats continued with s.c.insulin treatment for 12 weeks.Morning blood glucose concentrations were determined at 1 or 2-to 4-week intervals.At the end of the 12-week study period,Hb A1 c and serum insulin were determined on blood samples obtained from tail veins.CREA,BUN,ALT,AST,TG,and TC were determined on the blood samples obtained from the abdominal aorta.All rats were kept in individual metabolic cages for 24-h urine collection at the end of 4,8 and 12 weeks of drug treatment.Urinary albumin concentrations were measured for 24 h UAER.Levels of MDA,GSH-Px,SOD,IL-1?,TNF-? and COl-? in the kidney were measured.The pathologic histology of kidney in rats was observed by HE and PAS staining.The observation of basement membrane and podocyte ultrastructure in kidney were inspected by electron microscope.The expressions of p ERK1/2,Nox4 and TRPC6 proteins were measured by WB respectively.Results:1.Compared with the control group,HINS rats had higher levels of albuminuria(P<0.05).AS-? ameliorated albuminuria.This protective effect was evident at a dose as low as 5 mg/ kg/day.2.Compared to the control group,MDA level was increased in the HINS group(P < 0.01);GSH-Px and SOD levels were reduced in the HINS group(P<0.01).AS-? significantly reduced the elevation of renal tissue levels of MDA,and increased the elevation of tissue levels of GSH-Px and SOD,which was evident at a dose as low as 2.5 mg/kg/day(except SOD).3.Compared to the control group,IL-1? and TNF-? levels were increaed in the HINS group(P<0.01).AS-? significantly reduced renal tissue levels of IL-1? and TNF-?,which was evident at a dose as low as 2.5 mg/kg/day(TNF-?)and 5 mg/kg/day(IL-1?).4.Compared to the control group,Col-? and LN levels were increaed in the HINS group(P<0.01).AS-? significantly reduced renal tissue levels of Col-? and LN,which were evident at a dose as low as 5 mg/kg/day.5.Compared to the control group,the HINS group showed mesangial cell slight proliferation.However,AS-? treatment significantly prevented mesangial cell proliferation when compared with the HINS group.This trend was evident at a dose as low as 5 mg/kg/day.6.Compared to the control group,the observation of basement membrane and podocyte ultrastructure by electron microscopy showed basement membrane thickening obviously and minor podocyte foot process effacement in the HINS group,whereas the hyperinsulinemic rats treated with AS-? revealed a marked decrease in podocyte foot process effacement and basement membrane thickening,which was evident at a dose as low as 5 mg/kg/day.7.Compared to the control group,the HINS group presented elevated levels of Nox4,phosphorylated ERK1/2 and decreased TRPC6 in the kidneys(P< 0.01).Treatment with AS-? significantly inhibited the Nox4,phosphorylation of ERK1/2 and elevated TRPC6 expression in the kidneys from rat models of hyperinsulinemia,which was evident at a dose as low as 2.5 mg/kg/day,5 mg/kg/day and 2.5 mg/kg/day respectively.Conclusion:1.STZ induced diabetic rats were performed with intensive insulin therapy.when blood glucose was tight control,diabetic rats were continued intensive treatment for 4 weeks.Hyperinsulinemia may occur.If this state lasted for 12 weeks,and it can damage the kidneys by increasing the level of oxidative stress.2.AS-? prevented hyperinsulinemic rats kidney injury by inhibiting oxidative stress,IL-1? and TNF-? overproduction,down-regulating Nox4 and ERK1/2 activation,and up-regulating TRPC6 expression.Part ? Mechanisms of high insulin induced MCs injury and the protective effect of astragaloside ?Objective:To investigate the effects of high insulin on human mesangial cells(MCs)proliferation and to research the protective effects of Astragalosides ?(AS-?)on high insulin induced MCs injury in vitro.To explore the molecular mechanisms of AS-? in preventing MCs injury.Methods:1.The cultured MCs were divided into 6 groups as follows: normal control group(NG),INS group(INS12.5,25,50,100,200 n M),after incubated for 12 h,24h,36 h,48h,72 h,MTT assay was adopted to examine the OD value of each group.2.The cultured MCs were divided into 5 groups as follows:(1)NG,(2)HINS(100n M)12h,(3)HINS(100n M)24h,(4)HINS(100n M)36h,(5)HINS(100n M)48h.QPCR method was used to detect the expression level of Nox4 m RNA and TRPC6 m RNA,and western blot method was adopted to detect the expression of Nox4 protein and TRPC6 protein of each group.3.The cultured MCs were divided into 6 groups as follows: set the following seven groups:(1)NG,(2)HINS(100n M)0.5h,(3)HINS(100n M)1h,(4)HINS(100n M)3h,(5)HINS(100n M)24h,(6)HINS(100n M)48h.Western blot method was used to detect the phosphorylation level of ERK1/2 of each group.4.The cultured MCs were divided into 7 groups as follows:(1)NG group,(2)NG + Tempol 100?M group,(3)NG + DPI 10?M group,(4)HINS(100n M)group,(5) HINS(100n M)+ Tempol 100?M group,(6)HINS(100n M)+ DPI 10?M group.(7)HINS(100n M)+ U0126 10?M group After incubated for 48 h,using MTT assay to examine the OD value.5.The cultured MCs were divided into 8 groups as follows:(1)NG group,(2)NG + Tempol 100?M group,(3)NG + DPI 10?M group,(4)NG +U0126 10?M group,(5)HINS(100n M)group,(6)HINS(100n M)+ Tempol 100?M group,(7)HINS(100n M)+ DPI 10?M group.(8)HINS(100n M)+ U0126 10?M group After incubated for 48 h,using the fluorescent probe DCFH-DA to detect the intracellular ROS levels of each group.6.The cultured MCs were divided into 5 groups as follows:(1)NG group,(2)HINS(100n M)group,(3)HINS(100n M)+ U0126 10?M group,(4)HINS(100n M)+ DPI 10?M group,(5)HINS(100n M)+ Tempol 100?M group,After stimulated for 48 h,the cell NADPH oxidase colorimetric assay kit was usd to examine the NADPH oxidase activity,using western blot method to detect the expression of Nox4 protein and TRPC6 protein,the phosphorylation levels of ERK1/2 protein in each group.7.The cultured MCs were divided into 5 groups as follows:(1)NG group,(2)HINS(100n M)group,(4)HINS(100n M)+ AS-? 25?M group(7)HINS(100n M)+ AS-? 50?M group,(8)HINS(100n M)+ AS-? 100?M group.After incubated for 48 h,MTT assay was adopted to examine the OD value of each group.8.The cultured MCs were divided into 6 groups as follows:(1)NG group,(2)HINS(100n M)group,(3)HINS(100n M)+ AS-? 25?M group,(4)HINS(100n M)+ AS-? 50?M group,(5)HINS(100n M)+ AS-? 100?M group,(6)HINS(100n M)+ Tempol 100?M group,After incubated for 48 h,the fluorescent probe DCFH-DA was used to detect the intracellular ROS levels of each group.9.The cultured MCs were divided into 6 groups as follows:(1)NG group,(2)HINS(100n M)group,(3)HINS(100n M)+ AS-? 25?M group,(4)HINS(100n M)+ AS-? 50?M group,(5)HINS(100n M)+ AS-? 100?M group,(6)HINS(100n M)+ OAG 100?M group.After incubated for 48 h,using real-time PCR method to detect the expression level of TRPC6 m RNA,using western blot method to detect the expression of TRPC6 protein in each group.10.The cultured MCs were divided into 6 groups as follows:(1)NG group,(2)HINS(100n M)group,(3)HINS(100n M)+ AS-? 25?M group,(4)HINS(100n M)+ AS-? 50?M group,(5)HINS(100n M)+ AS-? 100?M group,(6)HINS(100n M)+ DPI 10?M group.After incubated for 48 h,the cell NADPH oxidase colorimetric assay kit was usd to examine the NADPH oxidase activity,and QPCR was used to detect the expression level of Nox4 m RNA.western blot method was used to detect the expression of Nox4 protein.11.The cultured MCs were divided into 6 groups as follows:(1)NG group,(2)HINS(100n M)group,(3)HINS(100n M)+ AS-? 25?M group,(4)HINS(100n M)+ AS-? 50?M group,(5)HINS(100n M)+ AS-? 100?M group,(6)HINS(100n M)+ U0126 10?M group.After incubated for 48 h,western blot was adopted to detect the expression of p-ERK1/2 and ERK1/2.Results:1.HINS significantly stimulated MCs proliferation in comparison to NG condition.MCs in HINS condition for 48 h resulted in a significant increase in NADPH activtion and in ROS generation compared with NG.The HINS actin in MCs was notably abolished by the treatment of NADPH oxidase inhibitor,ROS inhibitor and ERK signaling pathway inhibitor.2.The expression level of Nox4 protein and m RNA were upregulated significantly in HINS condition.After Inhibition of Nox4 protein expression,the HINS-induced NADPH activtion and ROS generation in MCs were inhibited obviously,suggesting that the effect of HINS on promoting cell growth might be closely related to Nox4 upregulation and ROS generation.3.High insulin induced ERK1/2 activation as manifested by that the relative amount of phosphorylated ERK1/2 are significantly higher than that of control cells.Treatment of NADPH oxidase inhibitor(DPI,10 ?M)or ROS inhibitor(Tempol,100 ??)blocked ERK1/2 activation in MCs by high insulin,indicating that NADPH oxidase activation by high insulin is upstream of ERK1/2 in MCs.U0126 is a ERK pathway inhibitor,failed to inhibit Nox4 expression and ROS generation,and it could inhibit cell abnormal proliferation only.NADPH oxidase might be upstream of ERK pathway in the high insulin stimulated signaling pathway in MCs.4.Incubation of MCs with high insulin for 48 h markedly decreased the expression level of TRPC6 protein and m RNA compared with NG.The down-expression of TRPC6 induced by high insulin was obviously abrogated by Nox4 or ERK1/2 inhibitors.Furthermore,in the presence of inhibitor of signaling molecules,which are U0126,DPI,Tempol,HINS-induced TRPC6 down-expression was markedly abolished.These results suggested that HINS might induce the contractile dysfunction of MCs by down-regulating the TRPC6 expression through NADPH oxidase/ROS/ERK signaling pathway.5.Administration of AS-? in the concentration range of 25-100 ?M showed in significant inhibition of cell proliferation,ROS generation,Nox4 upregulation,phosphorylation of ERK1/2,downregulation of TRPC6 in MCs stimulated by high insulin.These results suggested that AS-? might inhibit HINS-induced proliferation in MCs under HINS condition through inhibiting NADPH oxidase/ROS/ERK signaling pathway.Conclusion:1.High insulin could induce glomerular MCs proliferation and downexpression of TRPC6 protein by promoting Nox4 upregulation,ROS generation,ERK pathway activation.NADPH oxidase and ROS generation could be upstream of ERK pathway in the HINS-stimulated signaling pathway in MCs.2.AS-? treatment might prevent damage of MCs in HINS through inhibiting NADPH oxidase/ROS/ERK signaling pathway.