P38有丝分裂素激活蛋白激酶信号转导通路介导过氧化氢诱导的人晶状体上皮细胞中热休克蛋白27表达的研究

目的研究P38有丝分裂素激活蛋白激酶(MAPK)在H2O2诱导的人晶状体上皮细胞热休克蛋白质27(HSP27)表达中的作用。方法使用100和200μmol/LH2O2分别以不同时间刺激培养的人晶状体上皮细胞系B3,在阻断实验中加入特异性P38MAPK阻断剂SB203580阻断P38MAPK信号转导通路。采用逆转录聚合酶链反应检测刺激不同时间的人晶状体上皮细胞HSP27mRNA的表达情况,采用免疫印迹法(Westernblotting)检测细胞中HSP27和磷酸化P38MAPK的表达情况。结果(1)H2O2刺激后的人晶状体上皮细胞中HSP27mRNA及其蛋白的表达显著增加;(2)100和200μmol/LH2O2刺激30min,人晶状体上皮细胞应激活化蛋白激酶的活性(平均灰度值)分别增强至800±081和825±050,6h后恢复至基线水平(P<001);磷酸化P38MAPK的表达随H2O2浓度的增高而增加,且在H2O2刺激后15min达到峰值,随后逐渐下降;(3)加用特异性P38MAPK阻断剂SB203580预处理后,200μmol/LH2O2刺激6h的人晶状体上皮细胞HSP27表达(平均灰度值)从3600±082降至875±126,受到显著抑制(P<001)。结论P38MAPK信号转导途径参与H2O2诱导的人晶状体上皮细胞HSP27的表达。(中华眼科杂志,2005,414751)

Oxidative stress induced HSP27 expression in human lens epithelial cells is regulated by P38 map kinase

OBJECTIVE: To investigate the role of P38MAPK signaling pathway in hydrogen peroxide (H2O2) induced the expression of heat shock protein 27 in human lens epithelial cell line HLE-B3. METHODS: Human lens epithelial cell line HLE-B3 was cultured and then co-incubated with H2O2 (100 micromol/L, 200 micromol/L) for different time with or without pretreatment with Specific P38MAPK inhibitor SB 203580. RT-PCR and Western blotting were used to detect HSP(27) mRNA and protein expressions at different time points after H2O2 stimulation in human lens epithelial cells. Western blotting was used to detect the expression of phosphorylated P38MAPK. RESULTS: The expression of HSP(27) mRNA and protein was increased significantly after the H2O2 stimulation. The activity of stress-activated protein kinase was increased at 30 minutes after treatment with H2O2 and decreased to base level at 6 hours (P < 0.01). The expression of phosphorylation of P38MAPK was increased after treatment of H2O2 and reached the highest level at 15 minutes after stimulation and then decreased. The expression of HSP(27) was down-regulated with the pretreatment of SB 203580 compared with that in the H2O2 group (P < 0.01). CONCLUSIONS: H2O2 induces the expression of heat shock protein 27 in human lens epithelial cell line. The effects are mediated, at least in part, through the activation of P38MAPK signaling pathway.