Astragalus And Pueraria Transdifferentiation Of Renal Tubular Epithelial Cells In Diabetic Nephropathy

Diabetic nephropathy (DN) is one of the common diabetic microvascular complications which is the main reason for death of diabetes mellitus (DM) patients.Renal interstitial fibrosis(RIF) is a serious disease of DN.RIF is characterized by tubular basement membrane components and interstitial extracellular matrix(ECM) of qualitative and quantitative changes, tubular atrophy and accumulation of myofibroblasts.RIF is a common pathological change of clinical chronic progressive kidney disease,which can lead to renal failure at the end stage. RIF is characterized by appearance of massive myofibroblats which derived from tubular epithelial cells by an epithelial to mesenchymal transition (EMT).TGF-β1 perhaps is one of the most important profibrotic cytokine and the key of EMT process caused by various factors. TGF-β1/Smads signaling pathway is one of the most important signaling causing tubular EMT.Chinese medicine has been proved by clinic that it is an effective method to treat DN,but its specific role of the mechanism is not very clear,so study the mechanism of TCM on treating DN is very necessary.Puerarin injection and Astragalus mongholicus injection were usually used to treat RIF in clinic.However it may play a multi-target role in treating RIF,so we treated KKAy mice with RIF and HK.-2 cell with EMT induced by TGF-β1 by Puerarin injection and Astragalus mongholicus injection,and investigated the mechanism of them on treating DN.ObjectiveInvestigate the changes of Smads singling pathway mediated by TGF-β1 in the process of RIF in KKAy mice and EMT in HK-2.Explore a mechanism of Puerarin injection combining Astragalus mongholicus injection to treat RIF by inhibiting EMT.Reveal the mechanism and provide a scientific basis for Chinese medicine that can prevent and treat RIF.Methods(一) In vivo(1) KKAy mice were randomly divided into two groups:model and Puerarin injection combine Astragalus mongholicus injection group.C57BL/J mice were used to be controls. Puerarin injection combine Astragalus mongholicus injection group were given by peritoneal from fourteen weeks old.They were sacrificed at 24 weeks of age respectively and observed the changes of the pathomorphology of kidney.(2) E-cadherin,a-SMA,TGF-β1 and TGFβ-R I protein were detected in renal tissue of mice by immunohisto-chemical method.(3) Smad3 and Smad7 protein were detected in renal tissue of mice by Western blot.(4) TGFP-R I and Smad3 mRNA were detected in renal tissue of mice by RT-PCR. (二) In vitro(1) HK-2 cells were divided into different groups:Control group:Cells were cultured in Dulbecco's modified Eagles medium-F-12 medium supplemented with 10% fetal bovine serum(FBS).Model group:Cells were treated with recombinant TGF-β1(8ng/ml) for 48h.Treatment group:Cells were treated with recombinant TGF-β1(8ng/ml) plus Stragalus mongholicus injection(4ul/ml) and Puerarin injection(1.6ul/ml) for 48h.Cells were detected by inverted phase contrast microscope.(2) E-cadherin and a-SMA protein were detected in HK-2 cell by immunohisto-chemical method.(3) E-cadherin,a-SMA,TGF-β1,TGFβ-RⅠ,Smad3 and Smad7 mRNA were detected in HK-2 cell by Real-time PCR.(4) TGF-β1 were detected in HK-2 cell by Western blot.Result(一) In vivo(1) Renal morphologic changes were discovered at 24 weeks of age in KKAy mice.The model group demonstrated tubulointerstitial fibrosis,inflammatory cell infiltration,vacuole in the renal tubular epithelial cells and tubular ectasia.The expression of a-SMA,TGF-β1,TGFβ-RⅠand Smad3 protein in kidney of KKAy model mice were upregulated,but E-cadherin and Smad7 protein were reduced. The expression of TGFβ-RⅠand Smad3 mRNA were upregulated in kidney of KKAy model mice.(2) After the treatment of Puerarin injection combines Astragalus mongholicus injection, the changes of pathomorphology of kidney in KKAy mice were alleviated obviously.The expression of a-SMA,TGF-β1,TGFβ-RⅠand Smad3 protein were inhibited.E-cadherin and Smad7 protein were upregulated.(3) Puerarin injection combines Astragalus mongholicus injection inhibited the expression of the TGF-β1,TGFβ-RⅠand Smad3 mRNA in kidney of KKAy mice.(二) In vitro(1) After the treatment of TGF-β1 (8ng/ml) for 48h,the morphology of cell changed from typical cobblestone morphology of the epithelial cell into elongated form,called spindle-like shape in HK-2 cell in vitro. The expression of a-SMA protein and mRNA were inhibited,but E-cadherin protein and mRNA were contrary.The expression of TGF-β1 mRNA,TGFβ-RⅠmRNA and Smad3 mRNA were increased and Smad7 mRNA were decreased.(2) After the treatment of Puerarin injection combines Astragalus mongholicus injection,the expression of a-SMA and TGF-β1 protein were upregulated and E-cadherin were inhibited in HK-2 cell.(3) Puerarin injection combines Astragalus mongholicus injection can inhibited the expression of a-SMA,TGF-β1,TGFβ-RⅠand Smad3 mRNA and increased E-cadherin and Smad7 mRNA in HK-2 cell.Conclusion(1) Puerarin injection combines Astragalus mongholicus injection can significantly ameliorate the tubulointerstitial fibrosis in KKAy mice,inhibit and reverse the process of EMT mediated by TGF-β1 in HK-2 cell.(2) The possible mechanism of treating RIF by Puerarin injection combining Astragalus mongholicus injection:Inhibiting the expression of mRNA and protein of Smads singling mediated by TGF-β1/R and also upregulate the expression of Smad7 and E-cadherin to prevent RIF in KKAy mice and EMT in HK-2 cell and restore normal renal tubular epithelial cell phenotype.