雷帕霉素对人晶状体上皮细胞侵袭、迁移及炎症因子TNF-α、 IL-6、MIP-1α的影响

Effects of rapamycin on invasion and migration of human crystalline epithelial cells and inflammatory cytokines TNF-α,IL-6 and MIP-1α

目的探讨雷帕霉素(RAPA)对过氧化氢(H_(2)O_(2))诱导的人晶状体上皮细胞侵袭、迁移,以及对肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)、巨噬细胞炎症蛋白-1α(MIP-1α)的影响。方法体外培养人晶状体HLE-B3细胞系,分为Con组、H_(2)O_(2)组、H_(2)O_(2)+RAPA组、Y组、RAPA+Y组和RAPA+A组。采用细胞计数试剂盒-8(CCK-8)测定细胞活力;采用酶联免疫吸附试验(ELISA)检测TNF-α、IL-6和MIP-1α表达水平;采用Transwell小室法检测细胞侵袭、迁移能力;采用实时荧光定量PCR(RT-qPCR)检测上皮间质转化(EMT)相关基因、蛋白的相对表达量;采用蛋白免疫印迹(WB)法测定EMT和转化生长因子-β(TGF-β)/Smads信号通路相关蛋白的相对表达量。结果细胞活力实验结果显示,选择50μmol/L的H_(2)O_(2)作为造模条件进行后续实验,选择20 nmol/L RAPA加入TGF-β1/Smads信号通路抑制剂LY2109761和激活剂SRI-011381进行TGF-β1/Smads信号通路验证实验。与Con组比较,H_(2)O_(2)组TNF-α、IL-6、MIP-1α表达水平,细胞迁移与侵袭能力,N-钙粘蛋白(N-cadherin)、波形蛋白(Vimentin)及纤维粘连蛋白(FN)的mRNA、蛋白相对表达量,TGF-β1、p-Smad3蛋白相对表达量均显著升高(P<0.05),E-钙粘蛋白(E-cadherin)的mRNA、蛋白相对表达量和Smad7蛋白相对表达量均显著降低(P<0.05);与H_(2)O_(2)组比较,H_(2)O_(2)+RAPA组和Y组TNF-α、IL-6、MIP-1α表达水平,细胞迁移与侵袭能力,N-cadherin、Vimentin及FN mRNA、蛋白相对表达量,TGF-β1、p-Smad3蛋白相对表达量均显著降低(P<0.05),E-cadherin mRNA、蛋白相对表达量和Smad7蛋白相对表达量均显著升高(P<0.05);与H_(2)O_(2)+RAPA组比较,RAPA+Y组TNF-α、IL-6、MIP-1α表达水平,细胞迁移与侵袭能力,N-cadherin、Vimentin及FN mRNA、蛋白相对表达量,TGF-β1、p-Smad3蛋白相对表达量均显著降低(P<0.05),E-cadherin mRNA、蛋白相对表达量和Smad7蛋白相对表达量均显著升高(P<0.05),RAPA+A组TNF-α、IL-6、MIP-1α表达水平,细胞迁移与侵袭能力,N-cadherin、Vimentin及FN mRNA、蛋白相对表达量,TGF-β1、p-Smad3蛋白相对表达量均显著升高(P<0.05),E-cadherin mRNA、蛋白相对表达量和Smad7蛋白相对表达量均显著降低(P<0.05)。结论RAPA可显著抑制H_(2)O_(2)诱导的人晶状体上皮HLE-B3细胞的侵袭、迁移能力,抑制EMT进程,并且缓解炎症反应,其作用机制可能与抑制TGF-β1/Smads通路的信号转导有关。

Objective To investigate the effect of rapamycin(RAPA)on the invasion and migration of human crystalline epithelial cells induced by hydrogen peroxide(H_(2)O_(2)),tumor necrosis factor-α(TNF-α),interleukin-6(IL-6)and macrophage inflammatory protein-1α(MIP-1α).Methods Human crystalline lens HLE-B3 cell line was cultured in vitro and divided into the Con group,H_(2)O_(2) group,H_(2)O_(2)+RAPA group,Y group,RAPA+Y group and RAPA+A group.The cell viability was determined by cell counting Kit-8(CCK-8).The expression levels of TNF-α,IL-6 and MIP-1αwere detected by enzyme-linked immunosorbent assay(ELISA).The Transwell chamber assay was used to detect the abilities of cell invasion and migration.The expression levels of epithelial-mesenchymal transition(EMT)related genes and proteins were detected by real-time fluorescent quantitative PCR(RT-qPCR).The expressions of EMT and TGF-β1/Smads signaling pathway related proteins were determined by Western blotting(WB).Results The cell viability experiment results showed that 50μmol/L H_(2)O_(2) was selected as the modeling condition for conducting subsequent experiments.20 nmol/L RAPA was selected to add TGF-β1/Smads signaling pathway inhibitor LY2109761 and activator SRI-011381 for conducting TGF-β1/Smads pathway validation experiment.Compared with the Con group,the expression levels of TNF-α,IL-6 and MIP-1α,cell migration and invasion ability,mRNA and protein relative expression levels of N-cadherin,Vimentin and fipronectin(FN),and protein relative expression levels of TGF-β1 and p-Smad3 in the H_(2)O_(2) group were significantly increased(P<0.05),while the mRNA and protein relative expression levels of E-cadherin and the protein relative expression level of Smad7 were significantly decreased(P<0.05).Compared with the H_(2)O_(2) group,the expression levels of TNF-α,IL-6 and MIP-1α,cell migration and invasion ability,mRNA and protein relative expression levels of N-cadherin,Vimentin and FN,and protein relative expression levels of TGF-β1 and p-Smad3 in the H_(2)O_(2)+RAPA group and Y group were significantly decreased(P<0.05),while the mRNA and protein relative expression levels of E-cadherin and protein relative expression level of Smad7 were significantly increased(P<0.05).Compared with the H_(2)O_(2)+RAPA group,the expression levels of TNF-α,IL-6 and MIP-1α,cell migration and invasion ability,mRNA and protein relative expression levels of N-cadherin,Vimentin and FN,and protein relative expression levels of TGF-β1 and p-Smad3 in the RAPA+Y group were significantly decreased(P<0.05),while the mRNA and protein relative expression levels of E-cadherin and protein relative expression of Smad7 were significantly increased(P<0.05).Compared with the H_(2)O_(2)+RAPA group,the expression levels of TNF-α,IL-6 and MIP-1α,cell migration and invasion ability,mRNA and protein relative expression levels of N-cadherin,Vimentin and FN,and protein relative expression levels of TGF-β1 and p-Smad3 in the RAPA+A group were significantly increased(P<0.05),while the mRNA and protein relative expression levels of E-cadherin and protein relative expression level of Smad7 were significantly decreased(P<0.05).Conclusion Rapamycin could significantly inhibit H_(2)O_(2)-induced invasion and migration of human crystalline lens epithelial HLE-B3 cells,inhibit the EMT process,and alleviate the inflammatory response,and its mechanism may be related to the inhibition of TGF-β1/Smads pathway signal transduction.