N-乙酰半胱氨酸及salubrinal对全反式视黄酸诱导视网膜色素上皮细胞凋亡的抑制作用

Inhibitory effects of N-acetylcysteine and salubrinal on apoptosis of retinal pigment epithelial cells induced by all-trans-retinoic acid

目的观察抗氧化剂N-乙酰半胱氨酸(NAC)及选择性内质网应激反应抑制剂salubrinal在全反式视黄酸(ATRA)诱导视网膜色素上皮细胞(RPE)凋亡中的作用。方法取ARPE-19细胞系进行培养,并将其分为正常对照组、模型对照组、NAC处理组、salubrinal处理组、NAC+salubrinal组,分别采用完全培养基、含10μmol/L ATRA、含10μmol/L ATRA+5 mmol/L NAC、含10μmol/L ATRA+40μmol/L salubrinal、含10μmol/L ATRA+5 mmol/L NAC+40μmol/L salubrinal的完全培养基培养细胞24 h。采用流式细胞仪检测各组细胞凋亡比率、多重含半胱氨酸的天冬氨酸蛋白水解酶(Multicaspase)阳性比率、活性氧簇(ROS)水平。Western blot法检测各组细胞血管内皮生长因子A(VEGF-A)、C/EBP同源蛋白(CHOP)、剪切含半胱氨酸的天冬氨酸蛋白水解酶3(cleaved-caspase 3)的表达水平。结果流式细胞检测结果显示,各处理组细胞凋亡比率、Multicaspase阳性比率及ROS水平总体比较差异均有统计学意义(F=113.23、602.41、160.39,均P<0.001),其中正常对照组、NAC处理组、salubrinal处理组及NAC+salubrinal组细胞凋亡比率、Multicaspase阳性比率、ROS水平均明显低于模型对照组,差异均有统计学意义(均P<0.05)。Western blot检测结果显示,各组VEGF-A、CHOP、cleaved-caspase 3相对表达量总体比较,差异均有统计学意义(F=24.62、36.35、60.25,均P<0.001),其中正常对照组、NAC处理组、salubrinal处理组及NAC+salubrinal组VEGF-A、CHOP、cleaved-caspase 3蛋白相对表达量明显低于模型对照组,差异均有统计学意义(均P<0.05)。结论ATRA诱导RPE细胞产生氧化应激及内质网应激损伤并导致细胞凋亡,NAC及salubrinal可通过抑制应激反应,有效减轻细胞凋亡水平。

Objective To observe the effect of the antioxidant N-acetylcysteine(NAC)and selective endoplasmic reticulum stress response inhibitor salubrinal on the apoptosis of retinal pigment epithelial cells induced by all-trans-retinoic acid(ATRA).Methods Human ARPE-19 cell line was used as the experimental cell line,and was divided into normal control group cultured with complete medium,model control group cultured with complete medium containing 10μmol/L ATRA,NAC treatment group cultured with complete medium containing 10μmol/L ATRA+5 mmol/L NAC,salubrinal group cultured with complete medium containing 10μmol/L ATRA+40μmol/L salubrinal,NAC+salubrinal group cultured with complete medium containing 10μmol/L ATRA+5 mmol/L NAC+40μmol/L salubrinal.After 24-hour culture,apoptosis rate,multicaspase level and reactive oxygen species(ROS)level of ARPE-19 cells were detected by flow cytometry.The expressions of vascular endothelial growth factor A(VEGF-A),C/EBP-homologous protein(CHOP),cleaved-caspase 3 in cells were detected by Western blot.Results There were significant differences in the apoptosis rate,multicaspase and ROS levels among the five groups(F=113.23,602.41,160.39;all at P<0.001).The apoptosis rate,multicaspase and ROS levels of normal control group,NAC treatment group,salubrinal group and NAC+salubrinal group were significantly lower than those of model control group(all at P<0.05).There were significant differences in the expression levels of VEGF-A,CHOP and cleaved-caspase 3 among the five groups(F=24.62,36.35,60.25;all at P<0.001).The protein expression levels of VEGF-A,CHOP and cleaved-caspase 3 of normal control group,NAC treatment group,salubrinal group and NAC+salubrinal group were significantly lower than those of model control group(all at P<0.05).Conclusions ATRA can induce RPE cells to produce oxidative stress and endoplasmic reticulum stress injury,which leads to apoptosis.NAC and salubrinal can effectively reduce the RPE cell apoptosis by inhibiting stress response.