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目的观察血管紧张素Ⅱ(AngⅡ)刺激心肌细胞后,与心肌细胞增殖密切相关的细胞内信号物质细胞外信号调节激酶(ERK)的变化情况,探讨ERK入核机制。方法以原代培养的心肌细胞为研究对象,实验分6组:AngⅡ组、AngⅡ加缬沙坦组、AngⅡ加AngⅡ2型受体(AT2R)拮抗剂CGP42112A组、AngⅡ加MEK(MAPK激酶)抑制剂PD98059组、AngⅡ加蛋白合成抑制剂亚胺环己酮(CHX)组和AngⅡ加蛋白酶体抑制剂硼替佐米(LLnL)组,每组又分为5～8个不同时相点。以细胞免疫荧光检测磷酸化ERK(pERK)核转位,Westernblot法检测pERK表达情况。结果 AngⅡ可以使pERK表达迅速增加,该作用可被缬沙坦、PD98059所抑制;CHX作用1h左右,可见有pERK入核现象,随作用时间延长,未出现明显pERK聚集胞核中的现象;LLnL作用后显示pERK核聚集现象,与AngⅡ作用相比,LLnL组pERK核聚集明显。结论 ERK主要以活化或磷酸化形式入核,AngⅡ可使ERK发生核聚集,用CHX抑制蛋白质的合成可阻断上述核聚集过程,而用LLnL抑制蛋白的降解则可增强AngⅡ引起的ERK核聚集。
Objective To observe the changes of extracellular signal-regulated kinase (ERK), which is closely related to the proliferation of cardiomyocytes, after stimulating cardiomyocytes with angiotensin Ⅱ (AngⅡ), and to explore the mechanism of ERK entry. Methods Primary cultured cardiomyocytes were divided into 6 groups: Ang Ⅱ group, Ang Ⅱ plus valsartan group, Ang Ⅱ plus AngⅡ2 receptor (AT2R) antagonist CGP42112A group, Ang Ⅱ plus MEK (MAPK kinase) inhibitor PD98059 group, Ang Ⅱ plus protein synthesis inhibitors imine cyclohexanone (CHX) group and Ang Ⅱ plus proteasome inhibitor bortezomib (LLnL) group, each group is divided into 5 to 8 different time points. The nuclear translocation of phosphorylated ERK (pERK) was detected by immunofluorescence and the expression of pERK was detected by Western blot. Results AngⅡcan rapidly increase the expression of pERK, and this effect could be inhibited by valsartan and PD98059. When CHX was treated for about 1 h, pERK entry into the nucleus was observed. With the prolongation of action time, no significant accumulation of pERK was observed. LLnL The results showed pERK nuclear aggregation phenomenon, compared with Ang Ⅱ role, LLnL group pERK nuclear aggregation significantly. Conclusion ERK mainly enters the nucleus through activation or phosphorylation. AngⅡcan cause nuclear ERK to aggregate. Inhibition of protein synthesis with CHX can block the process of nuclear aggregation. Inhibition of protein degradation by LLnL enhances AngⅡ-induced ERK nuclear aggregation .