丝裂素蛋白激活激酶信号途径介导水杨酸钠诱导的人晶状体上皮细胞中HSP27表达

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造成的人品状体上皮细胞（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（0～55mmol／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P38MAPK、ERK1／2及JNK／SAPK信号通路特异性阻断剂SB20350（10μmol／L）、PD98059（20μmol／L）及SP600125（10μmol／L）预孵育细胞1h，在给予水杨酸钠刺激后检测相关指标，采用Westernblot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HSP27mRNA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，35～55mmol／L水杨酸钠刺激人LECs1h去除刺激再培养6h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）；55mmol／L水杨酸钠刺激人LECsI～5h不能诱导HSP27的表达（F=2．119，P〉0．05），而去除刺激再分别培养6h后可诱导HSP27表达，差异有统计学意义（F：452．534，P〈0．01）；55mmol／L水杨酸钠刺激人LECs1h后去除刺激再培养3h后HSP27表达明显增加，6h达到高峰，直至24h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30min时磷酸化p38MAPK表达增加，1h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ERK1／2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1h后开始增加，6h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1h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LECs1h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。

Implication of MAPK in sodium salicylate-induced heat shock protein 27 expression in human lens eplthelial Cells in vitro

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 .