Background Heat shock proteins (HSPs) are highly conserved proteins that are induced in cells when confronted with a wide variety of proteotoxic stresses.HSP27 has a high degree of similarity with α-crystallin protein.The abnormality of HSP27 structure and expression are closely related to the formation of cataracts.Our previous study showed sodium salieylate has the protective effect on H2O2-induced lens damage.Objective This study was to investigate the roles of MAPK signal pathway in sodium salicylate-induced the expression of HSP27 in human lens epithelial cells (LECs) in vitro.Methods Human LECs were incubated in the fresh media containing sodium salicylate at different concentrations (0-55 mmol/L) for different times (1-5 hours) and allowed to be recovered in fresh medium without sodium salicylate for 1-24 hours with or without pretreatment with P38MAPK inhibitor (SB203580), ERK1/2 inhibitor (PD98059) and JNK/SAPK inhibitor ( SP600125). The expressions of P38MAPK, EBK1/2, JNK/SAPK, phosphorylated P38MAPK, phosphorylated ERK1/2, phosphorylated JNK/SAPK and HSP27 were detected by Western blot. HSP27 mRNA was detected by RT-PCR. The expression of HSP27 was also detected by immunohistochemistry. Results There was only weak expression of HSP27 in normal human LECs.After stimulation of 35-55 mmol/L sodium salicylate was removed and human LECs were cultured again for 6 hours,the expression of HSP27 in LECs were significantly increased ( F= 509. 953,P<0. 01). HSP27 was absent expressed in human LECs in 55 mmol/L sodium salicylate stimulation for 1-5 hours groups, but LECs were re-cultured for 3,6 hours after removed the stimulation, the expression of HSP27 was elevated (F = 452. 534, P<0. 01). Activation of P38 M APK occurred after sodium salicylate stimulation 30 minutes and 1 hour ( F = 865.68, P<0. 01). However, ERK 1/2 was expressed after sodium salicylate was eliminated for 1-6 hours ( F = 388.84, P<0. 01). JNK/SAPK was inactived by sodium salicylate. The expression of HSP27 could be down-regulated with the pretreatment of SB203580 and PD98059. Conclusion Sodium salicylatc can induce the expression of HSP27 in human (LECs) . The effects are mediated,at least in part ,through the activation of P38MAPK and ERK1/2 signaling pathway .%背景 热休克蛋白(HSPs)是生物体在各种应激情况下产生的高度保守蛋白,HSP27与人晶状体中α-晶状体蛋白在氨基酸和基因水平上高度同源,其结构和表达的异常与白内障的形成密切相关.此研究先前的研究证实水杨酸钠对H2O2造成的人晶状体上皮细胞(LECs)损伤具有保护作用.目的 探讨水杨酸钠诱导的人LECs中HSP27的表达及与丝裂素蛋白激活激酶(MAPK)信号途径的关系.方法 人LECs.B3 系在含质量分数15%胎牛血清的DMEM中进行培养,将不同浓度的水杨酸钠(0～55 mmoL/L)加入培养基中刺激人LECs不同时间,然后去除刺激再分别培养.在阻断实验中,分别给予P38 MAPK、ERK1/2及JNK/SAPK信号通路特异性阻断剂SB20350(10 μmol/L)、PD98059(20 μmol/L)及SP600125(10 μmol/L)预孵育细胞1 h,在给予水杨酸钠刺激后检测相关指标,采用Western blot法检测各种处理情况下人LECs中HSP27的表达,用逆转录聚合酶链反应(RT-PCR)法检测HSP27 mRNA的表达;免疫组织化学法检测HSP27蛋白在人LECs中的表达.结果正常人LECs仅有微弱的HSP27表达,35～55 mmoL/L水杨酸钠刺激人LECs 1 h去除刺激再培养6 h后可使HSP27表达显著增加,与正常对照组比较差异有统计学意义(F=509.953,P<0.01);55 mmol/L水杨酸钠刺激人LECs 1～5 h不能诱导HSP27的表达(F=2.119,P>0.05),而去除刺激再分别培养6 h后可诱导HSP27表达,差异有统计学意义(F=452.534,P<0.01);55 mmol/L水杨酸钠刺激人LECs 1 h后去除刺激再培养3 h后HSP27表达明显增加,6 h达到高峰,直至24 h恢复到基础水平,差异有统计学意义(F=419.234,P<0.01).水杨酸钠刺激30 min时磷酸化p38MAPK表达增加,1 h表达显著,各组总体差异有统计学意义(F=865.680,P<0.01);磷酸化ERK 1/2在水杨酸钠刺激时间内未见升高;去除水杨酸钠刺激再培养1 h后开始增加,6 h达到高峰,各时间点总体差异有统计学意义(F=388.840,P<0.01);水杨酸钠刺激1 h及去除刺激再培养过程中未见到磷酸化JNK的表达;加入P38MAPK、ERK1/2特异性阻断剂预处理人LECs 1 h后可使水杨酸钠诱导的HSP27表达受到显著抑制,加入JNK特异性阻断剂未见对水杨酸钠诱导的HSP27表达产生影响.结论水杨酸钠可诱导人LECs中HSP27的表达,P38MAPK和ERK1/2信号途径促进水杨酸钠诱导的HSP27在人LECs中的表达.
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