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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(10 umol/L ISO)、ISO+pFLAG组(空载质粒pFLAG 转染+10 umol/L ISO)、ISO+ pFLAG-PACE4 组(pFLAG-PACE4 重组表达质粒转染 +10 umol/L ISO)。采用Annexin V-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2alpha(eIF2alpha) 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、eIF2-alpha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。

英文摘要:

Objective:To investigate the effect of paired basic amino acid-cleaving enzyme 4 (PACE4) on the apoptosis in H9c2 cardiomyocyte induced by isoprenaline (ISO) and the underlying mechanism.Methods:The recombinant expression vector pFLAG-PACE4 was established and transfected into the cardiomyocytes. Then cardiomyocytes were divided into four groups: control group (without treatment), ISO group (10 umol/L ISO), ISO+pFLAG group (pFLAG empty vector+10 umol/L ISO), ISO+ pFLAG-PACE4 group (pFLAG-PACE4+10 umol/L ISO). Cell apoptosis was analyzed by Annexin V-FITC/PI method. Protein levels of caspase-3, caspase-12, calreticulin, glucose regulated protein 78kDa (GRP78), CCAAT/enhancer binding protein homologous protein (CHOP) and activating transcription factors4 (ATF4) were detected by western blot. Meanwhile, the phosphorylated protein levels of PERK and eukaryotic initiation factor 2alpha(eIF2-alpha) were measured by Western blot.Results:Compared with the control group, the expression of PACE4 was significantly decreased by ISO treatment which was markedly increased after pFLAG-PACE4 transfection. PACE4 overexpression significantly attenuated cell apoptosis and the protein expression of caspase-3 and caspase-12 induced by ISO treatment. ISO significantly induced the protein expression of ERS markers including calreticulin, GRP78 and CHOP which was markedly decreased by PACE4 overexpression. Furthermore, the increased protein expression of PERK, eIF2-alpha and ATF4 was also repressed by PACE4 overexpression.Conclusion:PACE4 overexpression could alleviate ISO-induced apoptosis in H9C2 cardiomyocyte and the underlying mechanismmight be associated with the inhibition of PERK signaling pathway-mediated endoplasmic reticulumstress.

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