硫酸镍对人正常肝细胞存活率及相关凋亡蛋白影响

Effect of nickel sulfate on cell survival rate and related apoptotic proteins in human normal hepatocytes

目的探讨硫酸镍对人正常肝细胞系L02细胞的存活率和凋亡的影响。方法 (1)将对数生长期的L02细胞随机分为9个组,每组6个复孔,分别以浓度为0、100、200、300、400、500、600、700和800μmol/L的硫酸镍染毒0、6、12、24、48、72 h,采用CCK-8法检测L02细胞的存活率,选择后续实验的硫酸镍染毒剂量与染毒时间。(2)将对数生长期的L02细胞随机分为对照组和100、300μmol/L剂量组,分别以浓度为0、100、300μmol/L的硫酸镍染毒12 h,蛋白免疫印迹法检测B细胞淋巴瘤/白血病-2(BCL-2)、含半胱氨酸的天冬氨酸水解酶(caspase)-3蛋白、BCL-2相关X蛋白(BAX)、磷酸化RNA依赖的蛋白激酶样内质网激酶(p-PERK)、磷酸化真核细胞翻译起始因子2α(p-eIF2α)、CCAAT增强子结合蛋白的同源蛋白(CHOP)、葡萄糖调节蛋白78(GRP78)的蛋白相对表达水平。结果 (1)硫酸镍染毒后,L02细胞的细胞存活率呈现随着染毒时间的延长、染毒剂量的增加而下降的趋势(P值均<0.01);根据硫酸镍对L02细胞的半数抑制浓度,选择后续实验中硫酸镍染毒时间为12 h,染毒剂量为100、300μmol/L。(2)与对照组比较,100、300μmol/L组L02细胞中BCL-2蛋白相对表达水平均降低(P值均<0.05),BAX和caspase-3蛋白相对表达水平、BAX/BCL-2比值均升高(P值均<0.05);与100μmol/L组比较,300μmol/L组L02细胞中BCL-2蛋白相对表达水平降低(P<0.05),BAX和caspase-3蛋白相对表达水平、BAX/BCL-2比值均升高(P值均<0.05)。与对照组比较,100、300μmol/L组L02细胞中p-PERK、p-eIF2α、CHOP和GRP78蛋白的相对表达水平均升高(P值均<0.05);与100μmol/L组比较,300μmol/L组L02细胞中p-eIF2α、CHOP和GRP78蛋白相对表达水平均升高(P值均<0.05)。结论硫酸镍可调控L02细胞凋亡蛋白和PERK信号通路相关蛋白的表达,加剧L02细胞的凋亡,降低L02细胞的细胞存活率。

Objective To investigate the effect of nickel sulfate on cell survival rate and apoptosis of normal human liver L02 cells. Methods i) L02 cells in logarithmic growth phase were divided into 9 groups, each with 6 wells. L02 cells in each group were treated with 0, 100, 200, 300, 400, 500, 600, 700 and 800 μmol/L nickel sulfate. The survival rate of L02 cells was determined by CCK-8 assay after cells were treated for 0, 6, 12, 24, 48 and 72 hours. The nickel sulfate exposure dose and exposure time for subsequent experiments were selected based on the results of CCK-8 assay. ii) L02 cells in logarithmic growth phase were divided into control group, 100 and 300 μmol/L dose groups, and were exposed to 0, 100 and 300 μmol/L nickel sulfate for 12 hours, respectively. Western blot was used to detect the relative protein expression of B cell lymphoma/leukemia 2(BCL-2), Bcl-2 related protein X(BAX), caspase-3, phosphorylated RNA-dependent protein kinase-like endoplasmic reticulum kinase(p-PERK), phosphorylated eukaryotic translation initiation factor 2α(p-eIF2α), CCAAT/enhancer-binding protein homologous protein(CHOP) and glucose regulatory protein 78(GRP78). Results i) After treatment with nickel sulfate, the survival rate of cells decreased with the increase of dose and the prolongation of exposure time(all P values were <0.01). According to the half inhibitory concentration of nickel sulfate on L02 cells, the nickel sulfate exposure time in subsequent experiments was selected as 12 hours, and the exposure concentration was 100 and 300 μmol/L. ii) Compared with the control group, the relative expression of BCL-2 protein in L02 cells in the 100 and 300 μmol/L dose groups decreased(all P values were <0.05), while the relative protein expression of BAX, caspase-3 protein and ratio BAX/BCL-2 increased(all P values were <0.05). Compared with 100 μmol/L dose group, the relative expression of BCL-2 protein in L02 cells of 300 μmol/L dose group decreased(P<0.05), while the relative expression of BAX and caspase-3 protein and the ratio of BAX/BCL-2 increased(all P values were <0.05). Compared with the control group, the relative expression levels of p-PERK, p-eIF2α, CHOP and GRP78 protein in L02 cells were increased in 100 and 300 μmol/L dose groups(all P values were P<0.05). Compared with 100 μmol/L dose group, the relative expression levels of p-eIF2α, CHOP and GRP78 protein in 300 μmol/L dose group were increased(all P values were<0.05).Conclusion Nickel sulfate can regulate the expression of apoptosis related proteins and PERK signaling pathway related proteins in L02 cells, aggravate apoptosis of L02 cells and decrease the cell survival rate.