钙敏感受体表达增加诱发内质网应激在缺氧复氧性心肌损伤中的作用

目的：观察钙敏感受体表达增加诱发内质网应激在缺氧复氧性心肌损伤中的作用。方法:乳鼠原代心肌细胞培养4-5 d后，随机分为5组:正常对照组（N组）、缺氧/复氧组（H/Re组）、阻断剂（NiCl2,CdCl2）组、激动剂（GdCl3,NiCl2,CdCl2）组和caffeine组。采用饱和氮气pH6.8的D-Hands液培养细胞3 h，再用含20%新生牛血清的DMEM液培养细胞9 h，复制心肌缺氧/复氧模型。检测血清LDH活力，MTT检测细胞存活率，心肌细胞caspase-12和CaSR 蛋白表达水平Western blotting检测, 激光扫描共聚焦显微镜（LSCM）测定心肌细胞内游离钙的变化。结果:缺氧/复氧组和激动剂组LDH活力、细胞内游离钙浓度均高于对照组;同时，caspase-12和CaSR的表达也明显高于对照组。反之，细胞存活率低于对照组。结论:心肌缺氧/复氧过程中，钙敏感受体表达增多,从而破坏了细胞内钙稳态诱发过度的内质网应激,通过caspase-12等凋亡蛋白表达的增多等途径引起心肌细胞凋亡。

Effects of endoplasmic reticulum stress induced by increased expression of calcium-sensing receptor in myocardial hypoxia/re-oxygenation injury

AIM: To observe the effects of endoplasmic reticulum stress induced by the increase in expression of calcium-sensing receptor in myocardial hypoxia/re-oxygenation injury. METHODS: The primarily neonatal rat ventricular cardiomyocytes were incubated for 4-5 d, then randomly divided into 5 groups: (1) sham control group; (2) hypoxia/re-oxygenation group; (3) H/Re+ NiCl2, CdCl2-reperfusion group; (4) H/Re+GdCl3, NiCl2, CdCl2-reperfusion group; (5) H/Re+caffeine,GdCl3, NiCl2, CdCl2-reperfusion group. The neonatal cells were in ischemia-mimetic solution for 3 h, and re-incubated cells in normal culture medium for 9 h to establish a model of H/Re. The activity of LDH was determined, the viability and apoptosis of cardiomyocytes were assayed by MTT, the expressions of CaSR and caspase-12 in each group were analyzed using Western blotting, and the concentration of intracellular calcium was measured by laser confocal scanning microscope. RESULTS: The apoptosis index, the activity of LDH, the concentration of intracellular calcium, and quantitative expression of CaSR and caspase-12 in H/Re and activator groups were significantly higher than those in control group, while the viability and apoptosis of cardiomyocytes were lower than those in control group. CONCLUSION: In myocardial hypoxia/re-oxygenation, CaSR induces endoplasmic reticulum stress by altering the intracellular calcium homeostasis. The induction of apoptosis may be due to the increase in the expreesions of caspase-12 and other proapoptotic proteins.