自噬对锌指转录因子1及高糖诱导的肾小管EMT的影响

Autophagy effected high glucose-induced EMT in renal tubular epithelial cells through zinc finger transcription factor 1

目的探讨自噬是否可以通过调控锌指转录因子1(Snail1)的降解而影响肾小管上皮细胞-间充质转化(EMT)过程。方法12只清洁级雄性SD大鼠,随机分为正常(NC)组、糖尿病(DM)组,予链脲佐菌素(STZ)53 mg/kg尾静脉注射复制大鼠Ⅰ型糖尿病模型;造模成功后第24周末处死NC和DM组大鼠,检测大鼠血糖、血尿素氮(BUN)和24 h尿白蛋白(24 hUAlb),Western blot检测肾组织自噬相关分子[自噬基因BECN 1(Beclin1)、微管相关蛋白1轻链3(LC3-Ⅱ/Ⅰ)、自噬降解底物蛋白1(p62)]、Snail1蛋白的表达水平;将大鼠肾小管上皮细胞(NRK-52E)分为正常糖组(NG组)、高糖组(HG组)及高渗组(HM组),HG组培养的NRK-52E细胞在分为全蛋白组(Input组)和IP组[IP组又为阴性对照(IgG组)和实验组(Snail1/P62)];在高糖培养的NRK-52E细胞中分别使用自噬激动剂[雷帕霉素(RAP)和自噬抑制剂氯喹(CQ)]处理48 h,分为NG组、HG组、HG+RAP组及HG+CQ组,采用双标荧光腺病毒观察各组细胞的自噬流的情况,免疫荧光化学染色和免疫共沉淀观察Snail1与p62的蛋白相互作用,Western blot检测各组NRK-52E细胞中自噬相关分子、EMT、细胞外基质(ECM)指标表达变化;用二甲基亚砜(DMSO)、RAP、CQ处理NRK-52E细胞48 h,再联合放线菌酮(CHX)和蛋白酶抑制剂(MG-132)处理0、6及10 h,分为HG+DMSO/RAP组、HG+DMSO/CQ组,采用Western blot检测自噬对Snail1蛋白衰减的调控作用。结果与NC组相比,DM组大鼠血糖、BUN及24 hUAlb均增高(P<0.05);Western blot结果显示,与NC组比较,DM组大鼠肾组织及高糖培养的NRK-52E细胞中Beclin1和LC3Ⅱ/Ⅰ蛋白表达下调(P<0.05),p62、Snail1蛋白表达上调(P<0.05);双标腺病毒实验显示,在高糖刺激的NRK-52E中自噬流受阻,与HG组相比,HG+RAP组NRK-52E细胞中E-钙黏蛋白(E-cadherin)的蛋白表达增多,α-平滑肌肌动蛋白(α-SMA)、胶原蛋白Ⅲ(Col-Ⅲ)的蛋白表达减少(P<0.05),而HG+CQ组结果相反;免疫共沉淀实验发现Snail1与p62存在蛋白互作;联合CHX和MG-132后,HG+DMSO/RAP组中Snail1蛋白衰减速度加快。结论在DM大鼠肾组织和高糖培养的肾小管上皮细胞中,自噬受抑后使Snail1通过自噬降解减少,诱导EMT及ECM沉积增多,促进了肾脏纤维化的发生。

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 typeⅠDM 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,decreasedα-smooth muscle actin(α-SMA)and collagenⅢ(ColⅢ)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.