Objective To investigate whether autophagy can affect epithelial-mesenchymal transition(EMT)of renal tubular epithelial cells by regulating the degradation of zinc finger transcription factor 1(Snail1).Methods Twelve male SD rats of clean grade were randomly divided into normal control(NC)group and diabetes mellitus(DM)group.The rats were injected with 53 mg/kg streptozotocin(STZ)through tail vein to replicate type Ⅰ DM model.The rats in NC and DM groups were sacrificed at the end of the 24th week after successful modeling.Rat blood glucose,blood urea nitrogen(BUN)and 24-hour urine albumin(24 hUAlb)were measured.Western blot was used to detect the protein expression levels of autophagy-related molecules[(autophagy gene BECN1(Beclin1),microtubule-associated protein 1 light chain 3-Ⅱ/Ⅰ(LC3-Ⅱ/Ⅰ),autophagy substrate p62 and Snail1 protein]in renal tissues.Rat renal tubular epithelial cells(NRK-52E)were divided into normal glucose group(NG group),high glucose group(HG group)and hypertonic group(HM group).NRK-52E cells in HG group was further divided into whole protein group(Input group)and IP group(IP group was also divided into a negative control group(IgG group)and experimental group(Snail1/P62).NRK-52E cells were treated with autophagy agonists[rapamycin(RAP)]or autophagy inhibitors[chloroquine(CQ)]for 48 hours,and divided into NG group,HG group,HG+RAP group and HG+CQ group.Dual fluorescent labeled adenovirses were used to observe autophagic flows of cells in each group.Immunofluorescence staining and immunoprecipitation(IP)were applied to observe the interaction between Snail1 and p62.Western blot was used to detect the expression changes of autophagy-related molecules,EMT and extracellular matrix(ECM)indicators in NRK-52E cells of each group.NRK-52E cells were treated with DMSO,RAP and CQ for 48 hours,then with cycloheximide(CHX)or protease inhibitor(MG-132)for 0,6,and 10 h,and divided into HG+DMSO/RAP and HG+DMSO/CQ groups.Western blot was used to detect the regulatory effect of autophagy on Snail1 protein attenuation.Results When compared with NC group,DM group exhibited the increases in blood glucose,BUN and 24 hour UAlb in rats(P＜0.05).Western blot results showed that when compared with NC group,the protein expression levels of Beclin1 and LC3Ⅱ/Ⅰwere downregulated(P＜0.05),while the protein expression levels of p62 and Snail1 were upregulated in rat renal tissues of DM group and high glucose-cultured NRK-52E cells(P＜0.05).Dual fluorescence labeled adenovirus experiment showed that autophagic flow was blocked in high glucose-stimulated NRK-52E cells.When compared with HG group,HG+RAP group displayed increased E-cadherin protein expression,decreased α-smooth muscle actin(α-SMA)and collagen Ⅲ(Col Ⅲ)protein expressions in NRK-52E cells(P＜0.05),while the results in HG+CQ group were opposite.Co-Immunoprecipitation experiments revealed that Snail1 interacted with p62.After combining CHX and MG-132,the decay rate of Snail1 protein was increased in HG+DMSO/RAP group.Conclusion In rat renal tissues of DM model and high glucose-cultured renal tubular epithelial cells,inhibiting autophagy reduces Snail1 degradation through autophagy,induces the increased depositions of EMT and ECM and promotes the occurrence of renal fibrosis.
维普
万方数据
