HSP27在热打击致F98细胞氧化应激损伤及细胞凋亡中的作用

Effect of HSP27 on heat stress-induced oxidative stress injury and apoptosis in F98 cells

目的探讨热休克蛋白27(HSP27)在热打击(HS)致F98细胞氧化应激损伤及细胞凋亡中的作用。方法以43℃热刺激60min建立F98细胞热打击模型,在热打击后的不同时间点(0、3、6、9、12h)收集细胞,Western blotting检测HSP27及其各丝氨酸位点的磷酸化水平;采用丝裂原活化蛋白激酶(MAPKs)通路抑制剂(SB203580、SP600125、PD98059)及腺病毒转染特异性抑制相关信号通路,探索F98细胞HSP27的信号调节途径;继而通过磷酸化位点抑制性突变腺病毒转染观察HSP27各位点磷酸化对F98细胞活力、活性氧(ROS)及凋亡水平等的影响。结果给予热打击后,F98细胞HSP27在复温早期即轻度升高,其3个丝氨酸位点(Ser15,Ser78,Ser82)均发生磷酸化,以Ser78位点磷酸化为主。经MAPKs抑制剂预处理后,HS+SB203580组HSP27的3个位点磷酸化水平较对照组明显下降(P<0.05);MAPK激活蛋白激酶2(MK2)特异性抑制剂2'-氟-N-(4-羟基苯基)-[1,1'-联苯]-4-丁酰胺(CMPD-1)及腺病毒Ad-MK2(A)的干预,使HSP27丝氨酸位点磷酸化水平较HS组明显降低(P<0.05)。将F98细胞分别转染HSP27磷酸化位点的抑制性突变腺病毒后,与HS组比较,Ad-Ser78-HSP27组细胞活力增强,ROS水平、Caspase-3活性及细胞凋亡率下降(P<0.05);而AdSer15-HSP27、AdSer82-HSP27组与HS组比较无明显差异(P>0.05)。结论 p38MAPK/MK2途径主要通过磷酸化HSP27的Ser78位点上调ROS,促进F98细胞凋亡。

Objective To explore the effect of HSP27 on heat stress-induced oxidative stress injury and apoptosis in F98 cells. Methods The F98 cells were stress at 43 degrees for 60 minutes. After the stress, cells were collected at different time points(0, 3, 6, 9, 12 h), the phosphorylation levels of HSP27 at various sites were examined by Western blotting. To explore the function of mitogen-activated protein kinase(MAPKs) pathway on HSP27 phosphorylation, MAPK inhibitors(SB203580, SP600125, PD98059) and adenoviruses expressing MAPK-activated protein kinase 2(MK2) were employed. To decipher the mechanism of HSP27 phosphorylation on cell apoptosis, adenoviruses carrying HSP27 phosphorylation resistant mutations were transduced into the cell. Then, cell viability test kit, reactive oxygen species(ROS) test kit, apoptosis test kit, and caspase-3 activity test kit were used to detect the cell viability, ROS activities, and apoptosis of F98 cells. Results After heat stress, the expression of HSP27 increased slightly and its three Serine sites were all phosphorylated, with Ser78 being the main phosphorylation site. After pretreatment with MAPKs inhibitors, cells pretreated with SB203580 showed the lowest level of phosphorylation on HSP27 phospho-sites(P<0.05). Interestingly, either repressing MK2 activity by 2’-fluoro-N-(4-hydroxyphenyl)-[1,1’-biphenyl] 4-butylamide(CMPD-1) or upregulation MK2 using adenoviruses, the phosphorylation levels at three sites of HSP27 were significantly lower than those in the HS(heat stress) group(P<0.05). The activity of ROS, caspase-3 activity and apoptosis rate were decreased in the Ad-Ser78-HSP27(A) group(P<0.05), but there were no significant changes in the Ad-Ser15-HSP27(A) and Ad-Ser82-HSP27(A) groups(P>0.05). Conclusion The p38 MAPK/MK2 pathway regulates the apoptosis of F98 cells through the phosphorylation of HSP27 Ser78 site.