Effect Of 6'-Phenylsulfonylpaeoniflorin On Renal Injury In Type 2 Diabetic Nephropathy Mice And Its Mechanism

IntroductionDiabetes is a metabolic disorder which characterized by elevated blood glucose,including type 1 diabetes and type 2 diabetes.Type 2 diabetes,also called non-insulindependent diabetes,is the most common diabetes.Type 2 diabetes is accounting for more than 90% of all cases.Insulin resistance(IR)is one of the pathological mechanisms of type 2 diabetes.Insulin resistance is characterized by a decreased sensitivity to insulin in the target tissues or cells,including the liver,fat,and muscle.Diabetic nephropathy(DN)is one of the most common microvascular complications of type 2 diabetes.It has been found that the decreased sensitivity of renal tissues to insulin in patients with DN,just manifested as IR,is one of the pathogenic factors inducing and accelerating kidney injury.Podocyte is the epithelial cells of renal follicles which attached to the outer side of the glomerular basement membrane(GBM).GBM is an important part of the glomerular filtration barrier.The occurrence and development of DN and podocytes function is closely related to the integrity of the structure.G protein coupled receptor kinases(GRKs)is an important negative regulators of G protein coupled receptors(GPCRs).In adipocyte cells,high level of GRK2 can negatively regulate insulin signaling and promote IR.However,the regulation of insulin signal by GRK2 in podocytes has not been reported.Total glucosides of paeony(TGP)is extracted from paeonia lactiflora,and its active ingredient is Paeoniflorin(Pae).In the treatment of DN,TGP can restore the expression of podocyte hiatus membrane protein(nephrin),improve the injury of podocytes,and reduce proteinuria.In addition,Pae has a certain effect on reduction of urinary albumin and blood glucose in diabetic rats,and can inhibit glomerular hypertrophy.Paeoniflorin ?(-6)'-O-benzenesulfonate(CP-25)is an aromatic esterification product obtained by esterification modification of paeoniflorin,which can effectively improve Bioavailability of Pae.The bioavailability and anti-inflammatory immunomodulatory function of CP-25 are stronger than those of paeoniflorin,and CP-25 can inhibit GRK2 the membrane transfer of GRK2.Can GRK2 regulate insulin signaling in podocytes? Does CP-25 improve kidney function in type 2 diabetes? Does CP-25 regulate GRK2,activates insulin signal and improves podocyte function? It is the content of this thesis.In this study,we investigated the effects of CP-25 on glucose metabolism and renal function in DN mice,and explored the protective effects and mechanisms of CP-25 in a high glucose-induced podocyte injury model.Objective: 1.To investigate the effect of CP-25 on kidney injury in DN mice and the possiblemechanism.2.To elucidate the role and mechanism of CP-25 in the injury of podocytes induced byhigh glucose.Method:DN mouse model(C57BL/6 mice)was induced by streptozotocin(STZ)combined with a high-sugar and high-fat diet.Normal group,DN model group,CP-25(70mg/kg)group,CP-25(70mg/kg)+ Gliclazide(20mg/kg)group,Gliclazide(20mg/kg)group,Metformin(200mg/kg)group were performed(8 mice per group).The drug was continuously administered for 5 weeks.The body weight and fasting blood glucose were measured before and after the administration.IPGTT was performed.The urine of the mice was collected for 24 hours before sacrifice and urine albumin/urinary creatinine(UAlb/UCr)was measured.After administration,orbital blood was sacrificed and serum was separated.Serum creatinine(SCr),blood urea nitrogen(BUN),total cholesterol(cholesterol,CHOL),and triglyceride(TG)were measured.Serum insulin was detected using an ELISA kit.The renal tissue was examined by HE pathology.The protein expression of nephrin,GRK2,insulin receptor(INR),phosphorylated insulin receptor substrate 1(p-IRS1),phosphoinositide-3-kinase(PI3K),glucose transporter 4(GLUT4)were detected using western blot method.In vitro study,injured podocyte was induced by high glucose.The cells were in control group,high glucose(HG 30 m M)group,HG(30m M)+ CP-25(10?M,1?M,100 n M,10 n M,1n M)group,GRK2 inhibitor group,Metformin group.Podocyte glucose uptake capacity was detected by glucose oxidase peroxidase method(GOD-POD).Podocyte viability was measured CCK-8.Apoptosis rate were analyzed by flow cytometry using Annexin V-FITC/PI dual staining.Podocyte migration function was detected using transwell chamber.co-localization was measured by laser confocal and co-immunoprecipitation.Result:1.Effects of CP-25 on glucose metabolism in DN mice.Compared with the DN model,the weight of diabetic mice increased after administration.The weight of diabetic mice in the CP-25 + Gliclazide group is higher than those in the CP-25 or Gliclazide group.After 5 weeks of administration,the glucose level is very high in the models compared with that before administration.After 5 weeks of administration,the blood glucose level of mice in the DN group remained higher than that before administration,while that of CP-25 +Gliclazide group,Gliclazide group and Metformin group significantly reduced.The FBG in CP-25 + Gliclazide group is lower than those in the Gliclazide group.In the glucose tolerance experiment,compared with the model group,the area under the curve(AUC)was decreased,and the effect of CP-25 + Gliclazide was the most significant,which was better than that of the Gliclazide group.Compared with the normal group,the insulin level(FIN)and homeostasis model assessment(HOMA-IR)of DN mice in the model group were significantly increased,while the FIN and HOMA-IR were significantly decreased after the administration of CP-25 + Gliclazide,Gliclazide and Metformin,and the effect of CP-25 + Gliclazide group was better than that of CP-25 and Gliclazide groups.2.Effect of CP-25 on renal index in DN mice.Compared with the normal group,the kidney of DN mice is relatively large,and the renal index is increased.CP-25 + Gliclazide and Metformin group can reduce the renal index after administration.3.Effect of CP-25 on renal function and blood lipids in DN mice.Compared with the normal group,the UAlb/UCr value in the model group was increased significantly.In CP-25 + Gliclazide group,Gliclazide group and Metformin groups,it was significantly reduced after administration.Compared with the Gliclazide group,the UAlb/UCr value showed a more significant downward trend in the CP-25 + Gliclazide group.Compared with the normal group,the concentrations of BUN and SCr in the model group were increased significantly.In the CP-25 group,CP-25 + Gliclazide group,Gliclazide group,and Metformin groups,these values had showed reduced.Among them,the BUN value of CP-25 + Gliclazide group was significantly lower than that of Gliclazide group.Compared with the normal group,the CHOL and TG values were significantly increased in the model group,and were decreased in the CP-25 group,CP-25 + Gliclazide group,Gliclazide group and Metformin group.Among them,the CHOL and TG values of CP-25 + Gliclazide group were significantly lower than those of Gliclazide group.4.Effect of CP-25 on pathological of renal tissue in DN mice.Compared with the normal group,the model group mice showed increased glomerular volume,increased glomerular matrix,diffuse thickening of basement membrane,and fuzzy glomerular edges.Animals treated with CP-25 + Gliclazide,Gliclazide or Metformin can be found reduced the size of hypertrophic glomerulus,reduced extracellular matrix,and reduced diffuse thickening of glomerular basement membrane.5.Effects of CP-25 on podocyte proliferation,glucose consumption and apoptotic rate which induced by high glucose.Compared with the control group,the proliferation ability of podocyte was reduced in the high glucose group.The proliferation ability of podocyte was increased in CP-25(10?M,1?M,100 n M,10 n M,1n M)group,GRK2 inhibitor group,and Metformin group.CP-25 can improve proliferation ability of podocyte which was injuried by high glucose.Compared with the control group,the glucose uptake ability was reduced in podocyte which induced by high glucose.After CP-25,GRK2 inhibitor or Metformin treatment,the glucose consumption and glucose uptake ability of podocyte was increased.Compared with the control group,the apoptosis rate was induced podocyte which injuried by high glucose.After CP-25(10?M,1?M,100 n M,10 n M)treated,the apoptosis rate was improved.It had also been found the rate of podocyte apoptosis were reduced in the GRK2 inhibitor group and metformin group.6.Effect of CP-25 on podocyte migration which induced by high glucose.Compared with the normal group,the podocyte migration was induced after high glucose treated.In CP-25 concentration groups,GRK2 inhibitors,and Metformin groups,it had also been found that podocyte migration were improved.Among them,the effect of CP-25(1?M)is the best.7.Effects of CP-25 on the expression of nephrin,GRK2,INR,p-IRS1,PI3 K,and GLUT4 protein in kidney tissue and podocyte.The expression of nephrin,INR,p-IRS1,PI3 K,GLUT4 and membrane GLUT4 protein were significantly down-regulated in podocyte treated with the high glucose than those in cells not treated.The expression of GRK2 and menbrane GRK2 protein were abnormally increased.We didn't find any change about the expression of GLUT4.The expression of nephrin,INR,p-IRS1,PI3 K,GLUT4 and membrane GLUT4 protein were significantly up-regulated after treated with CP-25,GRK2 inhibitor,and Metformin.Meanwhile,the GRK2,membrane GRK2 protein expression were down-regulated.We also didn't find any change about the expression of GLUT4.The expression of nephrin protein in the kidney tissue cortex of mice in the DN model group was abnormally upregulated compared with normal group.The GRK2 protein was abnormally increased.And the expression of INR,p-IRS1,and PI3 K were all decreased.But in CP-25 group,CP-25 + Gliclazide group,Gliclazide group and Metformin group,the expression of nephrin,INR,p-IRS1,PI3 K were can up-regulated and the expression of GRK2 was down-regulated.8.Effects of CP-25 on the interaction of GRK2 with INRand p-IRS1 in podocyte.The expression of GRK2 was increased in the high glucose treated group using the laser confocal microscope.The colocalization of GRK2 with INR and p-IRS1 was increased.After CP-25 administration,the colocalization of GRK2 with INR and p-IRS1 was reduced.The results of co-immunoprecipitation showed that compared with the normal group,the interaction between GRK2 and INR and p-IRS1 was enhanced,after CP-25 administration,the interaction between GRK2 and INR and p-IRS1 was weakly reduced.Conclusion: 1.CP-25 can improve insulin resistace in STZ induced DN mice and show help to increase the therputic efficacy of Gliclazide.2.CP-25 can protect the kidney function in STZ induced DN mice and may relate to upregulate the insulin signal pathway.3.CP-25 can down-regulate the expression of GRK2 and membrane GRK2 in podocyte,up-regulate the insulin signaling pathway,suggesting that CP-25 can improve insulin resistance and protect the cell function in podocyte.