论文部分内容阅读
目的探讨calpain是否通过改变细胞自噬水平而参与心肌缺血再灌注(I/R)损伤。方法原代乳鼠心肌细胞分离培养,建立原代心肌细胞缺氧/复氧模型(H/R)模拟心肌I/R损伤,分为对照组,H组(缺氧12小时),HR组(缺氧12小时后复氧3小时),HR+ALLN组(calpain抑制剂ALLN预处理30min后缺氧12小时复氧3小时),HR+CQ组(自噬流抑制剂氯喹预处理30min后缺氧12小时复氧3小时),HR+ALLN+CQ组(氯喹和ALLN预处理30min后缺氧12小时复氧3小时)。检测各组calpain活性,心肌细胞损伤指标乳酸脱氢酶(LDH),心肌细胞活力(MTT法),WesternBlot检测自噬相关蛋白LC3的表达,以LC3II/LC31比值作为自噬程度指标,以氯喹干预检验自噬流。结果缺氧使calpain激活(p<0.001),心肌细胞LDH释放增加(p<0.001),心肌活力下降(p<0.001),同时自噬上调(p<0.05),复氧时calpain活性更高(p<0.005),LDH量更高(p<0.001),心肌活力更低(p<0.001),自噬更增强(p<0.01),而自噬流未受阻。ALLN抑制calpain激活(p<0.001),使H/R心肌细胞LDH释放减少(p<0.001),改善细胞活力(p<0.001),这种改善伴随着自噬上调(p<0.01),自噬流未受影响,提示calpain有抑制H/R心肌细胞的自噬的作用。结论calpain通过抑制心肌细胞的自噬反应而参与I/R损伤。
Objective To investigate whether calpain participates in myocardial ischemia-reperfusion (I / R) injury by altering the level of autophagy. Methods Primary neonatal rat cardiomyocytes were isolated and cultured to establish primary cardiomyocytes hypoxia / reoxygenation model (H / R) to simulate myocardial I / R injury and divided into control group, H group (hypoxia for 12 hours), HR group Reoxygenation for 12 hours after hypoxia for 3 hours), HR + ALLN group (reoxygenation for 3 hours after hypoxia 12 hours after pretreatment with calpain inhibitor ALLN for 3 hours), HR + CQ group Oxygen 12 hours reoxygenation 3 hours), HR + ALLN + CQ group (chloroquine and ALLN preconditioning for 30min after 12 hours of anaerobic reoxygenation 3 hours). The activity of calpain in each group, the indexes of cardiomyocyte injury, such as lactate dehydrogenase (LDH) and cardiomyocyte viability (MTT), the expression of autophagy-related protein LC3 were detected by Western Blot. The LC3II / LC31 ratio was taken as the index of autophagy. Test autophagy flow. Results Hypoxia activated calpain (p <0.001), increased LDH release (p <0.001), decreased cardiac viability (p <0.001), increased autophagy (p <0.05) and increased calpain activity during reoxygenation p <0.001), higher LDH levels (p <0.001), lower myocardial viability (p <0.001), higher autophagy (p <0.01), and autophagic flow unobstructed. ALLN inhibited calpain activation (p <0.001), reduced LDH release (p <0.001) and improved cell viability (p <0.001) in H / R cardiomyocytes, which was accompanied by upregulation of autophagy (p <0.01) Flow was not affected, suggesting that calpain has an inhibitory effect on H / R cardiomyocytes autophagy. Conclusion calpain participates in I / R injury by inhibiting autophagy in cardiomyocytes.