目的 探讨内质网应激(ERS)阻滞剂Salubrinal对人晶状体上皮细胞(HLEC)的保护作用及机制.方法 实验研究.应用H2O2诱导HLEC B3系细胞(HLE-B3)建立氧化应激模型,诱导ERS的发生.分别在H2O2干预和不干预的情况下,加入不同浓度的Salubrinal(10、15、20、25、30、35 μmol/L)培养24 h,采用细胞计数试剂盒(CCK-8)检测各组HLE-B3的增殖活性.实验分组为3组,A组(正常对照组)、B组(H2O2200μmol/L组)、C组(H2O2200μmol/L+Salubrinal 25μmol/L组).采用原位缺口末端转移酶标记法(TUNEL)和流式细胞仪(FCM)检测药物作用48 h后HLE-B3的凋亡情况.Westernblot检测不同时间点下各组葡萄糖调节蛋白质78(GRP78)、C/EBP同源蛋白(CHOP)、半胱氨酸蛋白酶12(Caspase-12)、真核细胞翻译起始因子2α(eIF2α)的磷酸化型p-elF2α的变化.单因素方差分析用于组间均数的多重比较,当方差齐时组间两两差异比较采用Dunnett t检验,当方差不齐时Dunnett's T3用作两两比较.结果 CCK-8试剂盒结果显示,没有H2O2干预时,不同浓度的Salubrinal对HLE-B3的细胞活性无明显抑制作用,存活率分别为(98.6±3.3)％,(98.7±2.6)％,(99.4±3.2)％,(98.6±1.9)％,(98.8±2.5)％,(99.3±3.2)％,(99.5±2.4)％,差异无统计学意义(F=0.09,P=0.10);应用H2O2干预时,随着Salubrinal浓度的升高,HLE-B3存活分别为(52.9±4.7)％,(65.0±3.6)％,(72.9±3.8)％,(84.5±3.6)％,(91.6±2.1)％,(93.1±2.9)％,(92.0±3.3)％,与对照组相比,差异有统计学意义(P值均＜0.01),当Salubrinal浓度大于25 μmol/L时,HLE-B3细胞的存活率增加不明显(P=0.56,0.88).流式细胞仪检测3组的细胞凋亡率分别为(1.9±0.7)％、(8.8±0.5)％、(4.3±0.3)％,各组之间差异有统计学意义(F=396.26,P＜0.01;与A组比较P值均＜0.01).TUNEL染色结果分别为A组(7.7±1.0)％,B组(36.9±1.0)％,C组为(16.7±2.2)％,凋亡指数差异有统计学意义(F=618.39,P＜0.01,与A组比较P＜0.01).Western blot结果表明:随着H2O2作用时间的延长,B组中p-elF2α在6h较初始时间点增加(2.16±0.38)倍,GRP78在12h较初始时间点增加(2.56±0.15)倍,CHOP在12h开始升高,持续至24 h,后又逐渐降低,在48 h升高量达初始时间点的(2.49±0.23)倍,Caspase-12在48 h表达明显增加,是初始时间点的(3.53±0.30)倍;C组与B组相比,GRP78、CHOP、p-elF2α表达量增加,而Caspase-12表达降低(GRP78:F=37.85,P＜0.01;CHOP:F=61.09,P＜0.01;Caspse-12:F=22.27,P＜0.01;p-eiF2α:F=15.11,P＜0.01).结论 Salubrinal可以通过抑制内质网应激的凋亡通路,对H2O2诱导的HLE-B3凋亡起保护作用.%Objective To study the protective effect of Salubrinal on human lens epithelial cells and its mechanism in endoplasmic reticulum stress (ERS).Methods Hydrogen peroxide (H2O2 200 μmol/L) was used to intervene in the cultured human lens epithelial cells B3 (HLE-B3) so as to create an oxidative stress model and induce ERS in the model.Different concentration of Salubrinal (10,15,20,25,30 and 35 μmol/L) were added to the cultured HLE-B3 with or without H2O2intervention.Then the cells were cultured for 24 hours.The cell counting kit (CCK-8) assay was used to test the viability of HLE-B3.The HLE-B3 cells were divided into three groups:Group A (normal control group),Group B (H2O2 200 μmol/L group),and Group C (H2O2 200 μmol/L+ Salubrinal 25 μmol/L group).After 48 h,TUNEL and flow cytometry assay (FCM) were used to examine the effect of Salubrinal on HLE-B3 apoptosis.The expression of glucoseregulated protein 78(GRP78),C/EBP homologous protein (CHOP),cysteinyl aspartate specific proteinase 12 (Caspase-12) and phosphorylation eukaryotic translation initiation factor 2α (p-elF2α) were tested by western blot at different points in time.Data from different groups was analyzed by one-way analysis of variance (ANOVA) while Dunnett t test was used under an equal condition,Dunnett's T3 for the unequal variances.Results CCK-8 results showed that without the intervention of H2O2,different concentrations of Salubrinal had no inhibitive effect on HLE-B3 viability,and that survival rates were (98.6±3.3)％,(98.7±2.6)％,(99.4± 3.2)％,(98.6 ± 1.9)％,(98.8 ± 2.5)％,(99.3 ± 3.2)％ and (99.5 ± 2.4)％.There was no statistically significant difference between them (F=0.09,P=0.10).With the increasing of Salubrinal concentration,the survival rates of HLE-B3 in the presence of H2O2 intervention were (52.9±4.7)％,(65.0±3.6)％,(72.9±3.8)％,(84.5±3.6)％,(91.6±2.1)％,(93.1 ±2.9)％,(92.0±3.3)％.There was statistically significant difference from the control group (all P＜0.01).However,the survival rates no longer increased (P=0.56,0.88) if the Salubrinal concentration was greater than 25 μmol/L.FCM results indicated that apoptosis rates of Group A,B and C were (1.9±0.7)％,(8.8±0.5)％,(4.3±0.3)％,respectively and the differences were statistically significant (F=396.26,P＜0.01,comparing with Group A,all P＜0.01).TUNEL results showed that apoptosis indexes of Group A,B,and C were (7.7 ± 1.0)％,(36.9± 1.0)％,(16.7 ± 2.2)％,respectively and the differences were statistically significant.(F=618.39,P＜0.01,comparing with Group A,all P＜0.01).Results of western blotting in group B at different points in time (0,12,24,36,48 h) showed that p-elF2α had increased by (2.16±0.38) times at 6 h;GRP78 had increased by (2.56±0.15)times at 12 h;CHOP started to rise after 12 h until it dropped after 24 h,and its amount had increased by (2.49±0.23) times at 48 h;Caspase-12 had increased significantly by (3.53 ±0.30)times at 48 h.The expression of GRP78,CHOP and p-elF2α in group C was greater than that in Group B,but the expression of Caspase-12 in Group C was lower than that in Group B (GRP78:F=37.85,P＜0.01;CHOP:F=61.09,P＜0.01;Caspse-12:F=22.27,P＜0.01;p-elF2α:F=15.11,P＜0.01).Conclusion Salubrinal might protect HLE-B3 against H2O2-induced apoptosis by inhibiting ERS related apoptosis pathways.
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