Effect Of Panax Notoginseng Saponisns On EMT Of Rat Renal Proximal Tubular Epithelial Cells Induced By High Glucose/TGF-?₁

Objectives: In this study,renal tubular epithelial cells were cultured in vitro,induced by high glucose or TGF-?1,and interfered by SIRT1 agonist(resveratrol,RSV),SIRT1 inhibitors(EX527),panax notoginseng saponins(PNS)respectively,by observing the occurrence of EMT and the changes of various indicators.To investigate the effect of panax notoginseng saponins on the occurrence of EMT in renal tubular epithelial cells and the mechanism based on the SIRT1/TGF-?1/Smad signaling pathway,in order to provide a new therapeutic method and experimental basis for diabetic nephropathy.Methods: 1?Effect of Panax Notoginseng Saponisns on EMT of Rat Renal Proximal Tubular Epithelial Cells induced by High Glucose.NRK-52 E were cultured in DMEM medium with 10% fetal bovine serum,and divided into blank group(5.5mmol·L-1glucose),high glucose group(30mmol·L-1 glucose),resveratrol group(30mmol·L-1 glucose +50 mg·L-1 RSV),PNS group(30mmol·L-1 glucose +100 mg·L-1 PNS),EX527 group(30mmol·L-1 glucose +10?mol·L-1 EX527),and EX527+PNS group(30mmol·L-1 glucose +10?mol·L-1 EX527,and after one hour,added 100 mg·L-1 PNS then collected cells after 48 hours of drug intervention.The expression of the ?-SMA and E-cadherin were also examined by Immunohistochemistry.In addition,SIRT1,?-SMA,E-cadherin,TGF-?1,Smad3 and Smad4 gene expression were analyzed by Real-time-PCR.The protein expressions of SIRT1,?-SMA,E-cadherin,TGF-?1 were determined by Western blot.The binding level of SIRT1 and Smad3 was detected by immunoprecipitation.2?Effect of Panax Notoginseng Saponisns on EMT of Rat Renal Proximal Tubular Epithelial Cells induced by TGF-?1.Thawing Cells.NRK-52 E were cultured in DMEM medium with 10% fetal bovine serum,and divided into blank group(5.5mmol·L-1 glucose),TGF-?1 group(5?g·L-1 TGF-?1),resveratrol group(5?g·m L-1 TGF-?1+50mg·L-1 RSV),EX527 group(5?g·L-1 TGF-?1+10?mol·L-1 EX527),PNS group(5?g·L-1 TGF-?1+100mg·L-1 PNS),EX527+PNS group(5?g·L-1 TGF-?1+10?mol·L-1 EX527,and after one hour,added 100 mg·L-1 PNS),then collected cells after 48 h of drug intervention.The expression of ?-SMA,Ecadherin,SIRT1,TGF-?1,Smad3,Smad4 m RNA in each group were detected by Real-time PCR.The protein expression of ?-SMA,E-cadherin,SIRT1 and TGF-?1 were detected by Western blot.Result: 1?Effect of Panax Notoginseng Saponisns on EMT of Rat Renal Proximal Tubular Epithelial Cells Induced by High Glucose(1)Immunohistochemical showed that in the blank group,the expression of E-cadherin on the cell membrane was obviously,and the expression was decreased after high glucose intervention,while the expression of ?-SMA in the cytoplasm of NRK-52 E cells was significantly increased.(2)Fluorescence quantitative PCR results showed that compared with the blank group,the expression of E-cadherin decreased(P<0.01),and the expression of ?-SMA m RNA increased(P<0.05)in high glucose group.Compared with high glucose group,PNS and RSV intervention resulted in the expression of E-cadherin and SIRT1 increased(P<0.01),while the expression of ?-SMA,TGF-?1,Smad3 and Smad4 decreased(P<0.05),and the expression of all indexes in the EX527+PNS group was the same as that in the high glucose group.(3)Western-blot results showed that compared with the blank group,the expression of E-cadherin and SIRT1 protein decreased and the expression of ?-SMA,TGF-?1 protein increased in the high glucose group.Compared with high glucose group,the expression of E-cadherin and SIRT1 protein increased(P<0.01)and the expression of ?-SMA,TGF-?1 protein decreased(P<0.01)in PNS and RSV group.(4)Immunoprecipitation results showed: There is an interaction between Smad3 and SIRT1,and the SIRT1 can directly deacetylate Smad3.2?Effect of Panax Notoginseng Saponisns on EMT of Rat Renal Proximal Tubular Epithelial Cells Induced by TGF-?1(1)Fluorescence quantitative PCR results showed that compared with the blank group,the expression of E-cadherin m RNA decreased significantly(P<0.01)and ?-SMA m RNA increased obviously(P<0.05)in TGF-?1 group.Compared with TGF-?1 group,the expression of E-cadherin m RNA increased significantly(P<0.01)in resveratrol and PNS group while the ?-SMA decreased(P<0.01)and EMT was inhibit.Meanwhile,the expression of SIRT1 m RNA increased(P<0.01)while the expression of TGF-?1,Smad3,and Smad4 m RNA decreased(P<0.01).Under the intervention of SIRT1 blocker EX527,PNS could not play a significant inhibitory role on the cells.(2)Western-blot results showed that compared with the blank group,the expression of ?-SMA increased(P<0.01),the expression of Ecadherin decrease(P<0.01),the expression of SIRT1 decreased significantly(P<0.01)and the expression of TGF-?1 increased(P<0.01)in TGF-?1 group.Compared with TGF-?1 group,the expression of E-cadherin,SIRT1 protein increased significantly(P<0.01)and the expression of ?-SMA,TGF-?1 protein decreased(P<0.01)in RSV and PNS group.and the group of EX527+PNS's expression was the same as TGF-?1 group.Conclusion:1.The expression of ?-SMA was increased significant and the expression of Ecadherin was decreased of rat renal tubular epithelial cells induced by high glucose or TGF-?1.It indicates that EMT occurs in cells.2.The expression of ?-SMA decreased and the expression of E-cadherin was increased in the intervention with resveratrol and Panax Notoginseng Saponisns,and EMT was inhibited,suggesting that Panax Notoginseng Saponisns has a protective effect on renal tubular epithelial cells induced by high glucose and TGF-?1.3.Under the intervention of SIRT1 blocker EX527,then add Panax Notoginseng Saponisns to intervention,we found that it couldn't reverse the occurrence of EMT in renal tubular epithelial cells induced by high glucose or TGF-?1.It suggested that Panax Notoginseng Saponisns reduce the occurrence of EMT by activating SIRT1 and inhibiting the TGF /Smad pathway,In turn,it has a protective effect on the renal tubular epithelial cells.