自噬调节高浓度葡萄糖诱导的人晶状体上皮细胞上皮间质转化

Autophagy regulates high glucose-induced epithelial-mesenchymal transition in human lens epithelial cells

目的:探究自噬水平变化对高糖诱导人晶状体上皮细胞上皮间质转化(EMT)的影响及其机制。方法:人晶状体上皮细胞(HLE-B3)分为正常对照组(NC组)和高糖处理组(HG组),分别用含5.5mmol/L葡萄糖的DMEM和添加了30mmol/L葡萄糖的上述DMEM培养12、24、48h,用Western blot检测上皮间质转化标志蛋白(E-cadherin、α-SMA)和自噬标志蛋白(LC3、Beclin 1和SQSTM1/p62)的表达变化,用划痕实验观察细胞的移行能力以明确高糖对晶状体上皮细胞自噬和EMT的影响。利用雷帕霉素调节自噬水平,将细胞分为正常对照组(NC组)、高糖处理组(HG组),高糖处理细胞的同时添加DMSO溶剂组(DMSO组)和添加200nmol/L雷帕霉素的雷帕霉素组(RAPA组),处理细胞24h,用Transwell实验观察细胞的移行能力,用Western blot检测EMT、自噬标志蛋白和TGF-β信号通路蛋白(TGF-β2、Smad2/3、p-Smad2/3、Snail)的表达。用细胞免疫荧光染色观察SQSTM1/p62与Smad2/3在细胞内的表达,免疫共沉淀方法检测细胞中SQSTM1/p62与Smad2/3之间的相互结合。结果:在高糖刺激后12、24、48h,HG组细胞E-cadherin、LC3Ⅱ/Ⅰ和Beclin 1蛋白表达逐渐降低(F=67.52、163、206,均P<0.0001),而α-SMA、SQSTM1/p62蛋白表达增加(F=53.37、302.1,均P<0.0001),细胞移行也较NC组增加(均P<0.001),提示高糖刺激后细胞发生EMT,而自噬水平降低;雷帕霉素处理后,与HG组和DMSO组相比,RAPA组LC3Ⅱ/Ⅰ和E-cadherin蛋白表达水平增加,α-SMA、p-Smad2/Smad2、p-Smad3/Smad3及Snail蛋白表达降低(均P<0.05),TGF-β2表达无明显改变(均P>0.05),细胞移行被抑制(均P<0.001),提示雷帕霉素在提高自噬水平的同时下调了TGF-β信号通路分子的表达进而抑制了EMT。细胞免疫荧光染色结果显示SQSTM1/p62与Smad2/3在胞浆内存在共定位,免疫共沉淀实验证实了SQSTM1/p62与Smad2/3蛋白相互结合。结论:高糖可刺激HLE-B3细胞发生EMT,下调细胞的自噬水平;自噬通过SQSTM1/p62与Smad2/3相互作用,改变了TGF-β信号通路中Smad2/3的表达,实现对EMT的调节。

·AIM:To investigate the regulation of autophagy on high glucose-induced epithelial-mesenchymal transition(EMT)in human lens epithelial cells.·METHODS:In order to investigate the changes of EMT and autophagy induced by high glucose,HLE-B3 cells were divided into two groups.In NC group,cells were cultured in DMEM with 5.5mmol/L glucose,and in HG group,cells were treated with DMEM in addition with 30mmol/L glucose for 12h,24h,and 48h.Western blot was used to detect the expression of EMT-marker proteins(E-cadherin andα-SMA)and autophagy-marker proteins(LC3,Beclin 1 and SQSTM1/p62).Wound healing assay was conducted to observe the migration ability.To investigate the regulation of autophagy on EMT,we employed rapamycin,an agonist of autophagy.HLE-B3 cells were divided into 4 groups.Two of them were mentioned as above,and the other two groups were treated with high glucose combined with DMSO(DMSO)and high glucose combined with 200nmol/L rapamycin(RAPA),respectively.Migration ability of cells was evaluated by Transwell assay.Expressions of proteins,such as EMT marker proteins,molecules in TGF-βsignaling pathway(TGF-β2,Smad2/3,p-Smad2/3,Snail),and autophagy markers were detected by Western blot.The intracellular co-localization of SQSTM1/p62 and Smad2/3 was observed by immunofluorescence staining,and their interaction was confirmed by co-immunoprecipitation assay.·RESULTS:The expression of E-cadherin,LC3Ⅱ/Ⅰ,and Beclin 1 in HLE-B3 cells of HG group gradually decreased(F=67.52,163,206;all P<0.0001),the expressions ofα-SMA,SQSTM1/p62 increased with time(F=53.37,302.1;all P<0.0001),and cell migration also increased compared with the cells in NC group(all P<0.001),indicating that high glucose stimulated EMT and suppressed autophagy.After treatment with rapamycin,the expressions of LC3Ⅱ/Ⅰand E-cadherin increased,the expressions ofα-SMA,p-Smad2/Smad2,p-Smad3/Smad3 and Snail decreased(all P<0.05),and the expressions of TGF-β2 did not change(all P>0.05)in RAPA group compared with HG group and DMSO group,cell migration was also suppressed(all P<0.001),indicating that Rapamycin down regulated the expressions of molecules in TGF-βsignaling pathway after activation of autophagy,which resulted in inhibiting EMT.Immunofluorescence staining showed co-localization of SQSTM1/p62 and Smad2/3 in cytoplasm.Co-immunoprecipitation confirmed the combination between SQSTM1/p62 and Smad2/3.·CONCLUSION:High glucose stimulates the process of EMT and suppresses the autophagy in HLE-B3 cells.Autophagy regulates EMT by interacting with Smad2/3 via SQSTM1/p62,altering the amount of Smad2/3 which works in the TGF-βsignaling pathway.