The Role Of E2F7 In High Glucose-induced EMT In Renal Tubular Epithelial Cells And The Intervention Of Resveratrol

Objective: This study aims to explore whether resveratrol can promote autophagy by up-regulating E2F7 and further inhibit EMT in NRK-52 E,so as to explore a new effective target for DM prevention and treatment.Methods:(1)The wild type C57-BL/6 mice were randomly divided into 3 groups,including NC group,DM group and Res group(8 in each group).The diabetic mice model was established by injection of STZ.After 8 weeks of successful replication of the diabetic model,resveratrol was given to the Res group by continuous gavage for 12 weeks,then the mice in each group were sacrificed to detect the relevant biochemical parameters,and the pathological changes of the kidney tissues by HE staining and Masson staining.Immunohistochemical staining was performed to observe the expression of E2F7 protein in renal tissue.The protein levels of E2F7,autophagy(Beclin1,LC3 II/I),EMT(?-SMA,E-cadherin,Snail1)and fibrotic(Collagen IV)related protein were determined by Western blot.The m RNA expression of Collagen IV,?-SMA and E-cadherin were detected by real-time PCR.(2)NRK-52 E cells were cultured with NG and HG and collected protein samples after 48 hours.The protein expression levels of E2F7,Beclin1,LC3 II/I,p62 and E-cadherin,?-SMA,Snail1 and Collagen IV were identified by western blot.In addition,the expression of E-cadherin,?-SMA and E2F7 were detected by immuno-fluorescence.(3)The NRK-52 E cells were added to Resveratrol(Res)in NG and HG groups.Samples were collected after 48 h culture.Then we detected the protein expression levels of E2F7,Beclin1,LC3 II/I,p62,E-cadherin,?-SMA,Snail1 and Collagen IV by western blot.(4)The Vector and overexpression E2F7 plasmid were respectively transfected in NG and HG groups.Samples were collected after 48 h culture.Then we detected the protein expressions of E2F7,Beclin1,LC3 II/I,p62,E-cadherin,?-SMA,Snail1 and Collagen IV by western blot.(5)The si RNA and E2F7 si RNA were transfected in NG and HG groups and then resveratrol was add to NG and HG groups for 48 h.We collected samples and detected the protein expressions of E2F7,LC3 II/I,p62,E-cadherin,?-SMA and Collagen IV by western blot.Results:(1)Diabetic nephropathy was successfully replicated.Compared with NC group,fasting blood glucose(FBG),kidney index(KI),serum creatinine(Crea),urinary microalbumin(UAER)and urine total protein(UTP)of 24 h were remarkably increased in DM group,and the results of HE and Masson indicated that renal tissue presented fibrosis in DM group.Immunohistochemical showed that: the cytoplasm of renal tubular epithelial cells in DM group showed lower expression of E2F7.The protein expression levels of E2F7,E-cadherin,Beclin1,LC3 II/I were reduced in DM group,while the levels of ?-SMA,Collagen IV,Snail1 were increased.After intervened with Res for 12 weeks,all the relevant biochemical parameters were reduced but FBG and renal fibrosis lesions were obviously alleviated.Compared with DM group,immunohistochemical showed that: the cytoplasm of renal tubular epithelial cells in Res group showed higher expression of E2F7.And the protein expression levels of E2F7,E-cadherin,Beclin1,LC3 II/I were increased in Res group,but the protein expression levels of ?-SMA,Collagen IV,Snail1 were reduced.Compared with DM group,the m RNA level of E-cadherin was increased in Res group,but the m RNA levels of Collagen IV and ?-SMA were reduced.(2)(1)Compared with NG group,the protein expression levels of p62,?-SMA,Snail1 and Collagen IV in HG group were significantly increased(P<0.05),and the protein expression levels of E2F7,E-cadherin,Beclin1 and LC3 II/I were significantly reduced(P<0.05).(2)Compared with HG group,the protein expressions of p62,?-SMA,Snail1 and Collagen IV in HG+Res group were significantly reduced(P<0.05),and the protein expressions of E2F7,E-cadherin,Beclin1 and LC3 II were increased(P<0.05).(3)(1)Compared with HG+Vec group,the expressions of p62,?-SMA,Snail1 and Collagen IV in HG+E2F7 group were significantly reduced(P<0.05),and the protein expressions of E2F7,E-cadherin,Beclin1 and LC3 II/I were increased(P<0.05).(2)The protein expressions level of p62,?-SMA and Collagen IV in HG+Res+si E2F7 group were increased compared with HG+Res group(P<0.05),and the proteinexpressions level of E2F7,E-cadherin and LC3 II/I were significantly reduced(P<0.05).Conclusions:(1)The protein expression level of E2F7 is reduced both in diabetic nephropathy kidney tissue and renal tubular epithelial cells with high-glucose cultured,the autophagy levels were inhibited.(2)E2F7 may inhibit collagen deposition and EMT generation by promoting autophagy in renal tubular epithelial cells cultured with high-glucose.(3)Resveratrol may up-regulate the expression of E2F7,restore the level of autophagy,inhibit the occurrence of EMT and reduce renal tubulointerstitial fibrosis.