The role and mechanism of glucose-regulated protein 78 in fibrosis after myocardial infarction
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维普
目的 探究葡萄糖调节蛋白78(GRP78)在心肌梗死后纤维化中的作用及机制.方法 选择雄性C57BL/6J小鼠12只,分为观察组和对照组,各6只.观察组结扎冠脉左前降支建立急性心肌梗死小鼠模型.使用苏木精伊红染色试剂盒和马松三色染色试剂盒检测心肌纤维化区域.使用蛋白免疫印迹(WB)法检测心肌组织中GRP78、磷酸化蛋白激酶R样内质网激酶(p-PERK)、磷酸化真核启动因子2α蛋白(p-elF2α)和CCAAT/增强子结合蛋白同源蛋白(CHOP)相对蛋白表达量.提取心脏成纤维细胞,将心脏成纤维细胞分为血管紧张素Ⅱ(Ang Ⅱ)组、Ang Ⅱ组+阴性对照物(sh-NC)组、Ang Ⅱ组+抗GRP78短发夹RNA(sh-GRP78)组、Ang Ⅱ组+sh-GRP78+衣霉素(Tn)组及对照组.使用细胞计数试剂盒8评估心肌成纤维细胞活性.使用划痕愈合实验检测心肌成纤维细胞迁移能力.使用WB法检测心肌成纤维细胞Ⅰ型胶原蛋白(Collagen Ⅰ)、Ⅲ型胶原蛋白(Collagen Ⅲ)、α-平滑肌肌动蛋白(α-SMA)、GRP78、p-PERK、p-eIF2α和CHOP相对蛋白表达量.结果 与对照组比较,观察组小鼠心脏组织出现明显损伤及明显纤维化,GRP78、p-PERK、p-elF2α和CHOP相对蛋白表达量较高(均P＜0.001).在体外细胞模型中,与对照组比较,Ang Ⅱ组心脏成纤维细胞活性较强,迁移能力较强,Collagen Ⅰ、CollagenⅢ、α-SMA、GRP78、p-PERK、p-elF2α 和 CHOP 相对蛋白表达量较高(均 P＜0.05).与 Ang Ⅱ+sh-NC 组比较,Ang Ⅱ+sh-GRP78 组细胞活性较低,迁移能力较低,Collagen Ⅰ、Collagen Ⅲ、α-SMA、GRP78、p-PERK、p-eIF2α 和 CHOP 相对蛋白表达量较低(均P＜0.05).与Ang Ⅱ+sh-GRP78组比较,Ang Ⅱ+sh-GRP78+Tn组活性较强,迁移能力较强,CollagenⅠ、Collagen Ⅲ、α-SMA、GRP78、p-PERK、p-eIF2α 和 CHOP 相对蛋白表达量较高(均 P＜0.05).结论 GRP78 可以通过激活内质网应激来调节心肌梗死诱导的心肌纤维化中的成纤维细胞活性、迁移能力和纤维化进展.
Objective To explore the role of glucose-regulated protein 78(GRP78)in myocardial infarction fibrosis and its mechanism.Methods Twelve male C57BL/6J mice were divide into an observation group and a control group,with 6 mice in each group.Acute myocardial infarction model was established by ligating left anterior descending coronary artery in the observation group.Hematoxylin eosin staining kit and Masson tricolor staining kit were used to detect myocardial fibrosis.The relative protein expression of GRP78,phosphorylated-protein kinase RNA-like endoplasmic reticulum kinase(p-PERK),phosphorylated eukaryotic factor 2α protein(p-eIF2α)and C/EBP-homologous protein(CHOP)in myocardial tissue were detected by Western blot(WB).Cardiac fibroblasts were extracted and divided into angiotensin Ⅱ(Ang Ⅱ)group,Ang Ⅱ+sh-NC group,Ang Ⅱ+sh-GRP78 group,Ang Ⅱ+sh-GRP78+tunicamycin(Tn)group and control group.Cell counting kit 8 was used to evaluate myocardial fibroblast activity.The migration ability of myocardial fibroblasts was detected by scratch healing test.WB was used to detect the relative protein expressions of Collagen Ⅰ,Collagen Ⅲ,α-smooth muscle actin(α-SMA),GRP78,p-PERK,p-eIF2α and CHOP in myocardial fibroblasts.Results Compared with the control group,the cardiac tissue of mice in the observation group showed obvious injury and fibrosis,and the relative protein expressions of GRP78,p-PERK,p-eIF2α and CHOP were higher(all P＜0.001).In vitro cell models,compared with the control group,the cardiac fibroblasts in Ang Ⅱ group had stronger activity and migration ability,and the protein expression of Collagen Ⅰ,Collagen Ⅲ,α-SMA,GRP78,p-PERK,p-elF2α and CHOP were higher(all P＜0.05).Compared with Ang Ⅱ+sh-NC group,the cell activity and migration ability,the protein expression of Collagen Ⅰ,Collagen Ⅲ,α-SMA,GRP78,p-PERK,p-eIF2α and CHOP of Ang Ⅱ+sh-GRP78 group were lower(all P＜0.05).Compared with Ang Ⅱ+sh-GRP78 group,Ang Ⅱ+sh-GRP78+Tn group had stronger activity and better migration ability,and higher protein expression of Collagen Ⅰ,Collagen Ⅲ,α-SMA,GRP78,p-PERK,p-elF2α and CHOP(all P＜0.05).Conclusion GRP78 can regulate fibroblast activity,migration ability and fibrosis progression in myocardial infarction-induced myocardial fibrosis by activating endoplasmic reticulum stress.
Acute myocardial infarctionMyocardial fibrosisGlucose-regulated protein 78Endoplasmic reticulum stress
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维普
