衰老心肌中Omi/HtrA2表达增加可促进心肌细胞自噬

Increased expression of Omi/HtrA2 promotes myocardial autophagy in aging myocardiocytes

摘要:

目的 探讨衰老心肌中增加的Omi/HtrA2在心肌细胞自噬中的作用。方法 用D-半乳糖干预胎鼠心肌细胞系H9c2建立衰老细胞模型，β-半乳糖苷酶染色观察细胞的衰老情况，cck8试剂盒检测细胞存活率，并以分析细胞乳酸脱氢酶（Lactic Dehydrogenase，LDH）水平反映其活性；给予Omi/HtrA2特异性抑制剂ucf-101降低Omi/HtrA2活性，构建稳转Omi/HtrA2的H9c2细胞株过表达Omi/HtrA2；采用western blot法测定心肌细胞中Omi/HtrA2、beclin1及LC3-Ⅱ蛋白的表达。结果 （1）与H9c2心肌细胞相比，D-半乳糖诱导的H9c2心肌细胞内β-半乳糖苷酶染色阳性率显著升高[（87.7±3.6%） vs （18.3±2.8%），P＜0.01]；cck8结果显示，两组之间无显著性差异（P＞0.05），但D-半乳糖诱导的H9c2心肌细胞中LDH活性明显升高（7.07±0.65 vs 5.93±0.34，P＜0.01）。（2）与H9c2细胞相比，D-半乳糖诱导的H9c2心肌细胞中Omi/HtrA2蛋白表达升高（P＜0.05），而beclin1表达下降（P＜0.01）；给予ucf-101后，衰老细胞中Omi/HtrA2蛋白表达明显下降（P＜0.05），而beclin1表达则进一步降低（P＜0.01）。（3）与H9c2细胞相比，过表达Omi/HtrA2的H9c2心肌细胞Omi/HtrA2蛋白表达增高，LC3-Ⅱ蛋白表达也增高（P＜0.05）；给予过表达Omi/HtrA2的H9c2心肌细胞ucf-101后，LC3-Ⅱ表达下降（P＜0.05）。结论 衰老心肌细胞中Omi/HtrA2的表达增加可促进心肌细胞自噬。

Abstract:

Objective To investigate the effects of increased expression of Omi/HtrA2 on myocardial autophagy in aging myocardiocytes. Methods The rat embryonic myocardial cell line H9c2 was induced by D-galactose (8g/L) to establish aging model. Cell aging was detected by β-galactosidase staining. Cell viability was detected by CCK8 kits and lactic dehydrogenase (LDH) activity analysis. Ucf-101, a specific inhibitor of Omi/HtrA2, was used to inhibit the activity of Omi/HtrA2, and the H9c2 cells with stable transfection of Omi/HtrA2 was constructed to increase the expression of Omi/HtrA2. The expression of Omi/HtrA2, beclin1 and LC3-Ⅱ were detected by Western blotting. Results (1) D-galactose resulted in a significant increase in positive staining to β-galactosidase in the induced cardiacmyocytes than the H9c2 cells [(87.7±3.60)% vs (918.3±2.80)%, P＜0.01]. The cck8 results showed that there was no significant difference between the cells with and without D-galactose inducement (P＞0.05). LDH activity was increased in the induced H9c2 cells than those without (7.07±0.65 vs 5.93±3.36, P＜0.01). (2) The expression of Omi/HtrA2 was increased (P＜0.05), but the expression of beclin1 was decreased (P＜0.01) in cells induced by D-galactose, as compared with H9c2 cells. In cells treated with ucf-101, the expression of Omi/HtrA2 was significantly decreased (P＜0.05), but the expression of beclin1 was further decreased (P＜0.01).（3）The expression of LC3-Ⅱwas significantly increased in Omi/HtrA2-overexpressed cells, as compared with H9c2 cells (P＜0.05). But, ucf-101 resulted in the expression of LC3-Ⅱdecreased in the Omi/HtrA2-overexpressed cells (P＜0.05). Conclusion Increased expression of Omi/HtrA2 promotes autophagy in aging myocardiocytes.