自噬在高糖高脂引起心肌细胞系H9C2凋亡中的影响

Effects of autophagy on apoptosis induced by high glucose and high lipids in cardiomyocyte cell line H9C2

目的探究自噬在高糖高脂(HGHL)引起的心肌细胞系H9C2凋亡中的影响。方法不同浓度高糖高脂处理H9C2心肌细胞不同时间,HE染色并测量心肌细胞表面积;流式细胞计量术检测细胞活性和凋亡率,确定心肌细胞肥大及凋亡的最适浓度和时间;蛋白免疫印迹法(Western blot)检测自噬指标LC3Ⅱ、自噬信号通路p62/SQSTM1(p62)、Beclin-1、LAMP2蛋白表达以及自噬抑制剂氯喹预处理后cleaved caspase-3蛋白表达。结果HGHL处理后H9C2心肌细胞肥大呈浓度和时间依赖性,36 h最显著(P<0.001);细胞凋亡呈浓度和时间依赖性增高,36 h凋亡率约50%(P<0.001);与对照组相比,HGHL诱导24 h时LC3Ⅱ蛋白水平显著增加(P<0.01),Beclin-1、LAMP2蛋白水平显著下降(P<0.05),而p62蛋白水平显著升高(P<0.01);与对照组以及干预组相比,氯喹预处理1 h后cleaved caspase-3蛋白表达显著增加(P<0.01)。结论 HGHL诱导H9C2心肌细胞肥大并促进其凋亡。HGHL通过同时抑制自噬形成和降解,使自噬体异常堆积,导致细胞凋亡。这表明抑制自噬可能是促进细胞凋亡的重要原因。

Objective To investigate the effect of autophagy on the apoptosis of H9 C2 cardiomyocytes. Methods H9 C2 cardiomyocytes were incubated with different concentrations of high glucose and high lipids（ HGHL） for different time. The surface area of cardiomyocytes was measured after HE staining. The cell viability and apoptotic rate were measured by flow cytometry. Western blot was used to detect the expression levels of LC3 Ⅱ,p62,Beclin-1,LAMP2 and cleaved caspase-3. Results After treatment with HGHL,the cells appeared hypertrophy in a concentration-and time-dependent manner,the cell hypertrophy was most obvious under the condition of HGHL（ 500 μmol/L,36 h）（ P0. 001）. Cell apoptosis increased in a concentration-and time-dependent manner,the apoptotic rate was nearly 50% under the condition of HGHL（ 500 μmol/L,36 h）（ P〈0. 001）. After treatment with HGHL for 24 h,compared with the control group,the expression of LC3 Ⅱ was very significantly increased（ P0. 01）,the expression of Beclin-1 and LAMP2 were significantly decreased（ P〈0. 05）,but the expression of p62 was significantly increased（ P〈0. 01）. Compared with the control group and the intervention group,the expression of cleaved caspase-3 was significantly increased（ P〈0. 01） after 1 h of chloroquine pretreatment. Conclusions HGHL induced H9 C2 cardiomyocytes hypertrophy promote H9 C2 cardiomyocytes apoptosis in a concentration-and time-dependent manner. HGHL may inhibit autophagy formation and degradation of H9 C2 cardiomyocytes at the same time,leading to abnormal autophagy accumulation of cardiomyocytes,and promote apoptosis,suggesting that inhibition of autophagy may be an important cause for promote apoptosis.