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目的:研究前体蛋白转化酶枯草溶菌素(PACE4)对异丙肾上腺素(ISO)诱导的心肌细胞凋亡的作用及其可能的作用机制。方法:构建pFLAG-PACE4重组表达载体并转染H9c2心肌细胞。将心肌细胞分为四组:正常对照组(无任何干预因素)、ISO组(10μmol/L ISO)、ISO+pFLAG组(空载质粒pFLAG转染+10μmol/LISO)、ISO+pFLAG-PACE4组(pFLAG-PACE4重组表达质粒转染+10μmol/LISO)。采用AnnexinV-FITC/PI双染法测定心肌细胞凋亡率;蛋白免疫印迹法检测活性半胱氨酸蛋白酶(caspase)-3、caspase-12、钙网蛋白、葡萄糖调节蛋白78(GRP78)、CCAAT/增强子结合蛋白同源蛋白(CHOP)、活化转录因子4(ATF4)和PERK的表达以及真核起始因子2α(eIF2α)的磷酸化水平。结果:与正常对照组相比,ISO组中PACE4表达水平明显降低,而转染pFLAG-PACE4质粒后,其表达水平显著增加。PACE4过表达可以显著抑制ISO诱导的细胞凋亡和caspase-3以及caspase-12的蛋白表达。ISO处理显著增加内质网应激分子钙网蛋白、GRP78和CHOP的表达,而PACE4过表达则可以抑制这些蛋白的表达。ISO诱导的PERK、eIF2α和ATF4的表达可以显著被PACE4过表达抑制。结论:PACE4过表达可以抑制ISO诱导的H9c2心肌细胞凋亡,其机制可能与PERK信号通路介导的内质网应激反应有关。
Objective: To investigate the effect of PACE4 on isoproterenol-induced cardiomyocyte apoptosis and its possible mechanism. Methods: The pFLAG-PACE4 recombinant plasmid was constructed and transfected into H9c2 cardiomyocytes. The cardiomyocytes were divided into four groups: normal control group (without any intervention), ISO group (10μmol / L ISO), ISO + pFLAG group (empty plasmid pFLAG transfected + 10μmol / LISO), ISO + pFLAG-PACE4 group (pFLAG-PACE4 recombinant expression plasmid transfected +10 μmol / LISO). Cardiomyocyte apoptosis rate was determined by Annexin V-FITC / PI double staining method. The activity of caspase-3, caspase-12, calreticulin, GRP78, CCAAT / CHOP, ATF4 and PERK expression and phosphorylation of eIF2alpha. Results: Compared with the normal control group, the expression level of PACE4 in ISO group was significantly decreased, while the expression level of pFLAG-PACE4 plasmid was significantly increased after transfection. PACE4 overexpression significantly inhibited ISO-induced apoptosis and caspase-3 and caspase-12 protein expression. ISO treatment significantly increased the expression of endoplasmic reticulum stress calmodulin, GRP78 and CHOP, whereas PACE4 overexpression inhibited the expression of these proteins. ISO-induced expression of PERK, eIF2α and ATF4 can be significantly inhibited by PACE4 overexpression. Conclusion: PACE4 overexpression can inhibit ISO-induced apoptosis of H9c2 cardiomyocytes, which may be related to the ERK-mediated ER stress.