Netrin-1对心肌细胞H9c2缺氧复氧损伤中的保护作用研究

Protective effect of Netrin-1 on H9c2 injury induced by hypoxia and reoxygenation in cardiomyocytes

目的研究Netrin-1在心肌细胞H9c2缺氧复氧损伤中的作用,并探讨其机制。方法建立心肌细胞H9c2缺氧复氧损伤模型;将A/R+Netrin-1组(转染pcDNA 3.1-Netrin-1)、A/R+Ctrl组(转染pcDNA 3.1)、A/R+Netrin-1+LY294002组(转染pcDNA 3.1-Netrin-1与抑制剂LY294002共处理)、A/R+Netrin-1+DMSO组(转染pcDNA 3.1-Netrin-1与DMSO共处理)均用脂质体法转染H9c2细胞;ELISA检测细胞中LDH、MDA、SOD的含量;MTT法检测细胞活力;WB检测细胞中Bcl-2、Bax、Cleaved caspase-3、Netrin-1、p-AKT的蛋白表达;流式细胞术检测细胞的凋亡率。结果与A/R+Ctrl组相比,A/R+Netrin-1组细胞中LDH、MDA含量明显降低,SOD含量明显升高,细胞活力显著升高,Bax、Cleaved caspase-3的蛋白表达量均显著降低,Bcl-2蛋白表达量显著升高,Netrin-1、p-AKT蛋白表达均显著升高,细胞凋亡率显著降低(P<0.05);与A/R+Netrin-1+DMSO组相比,A/R+Netrin-1+LY294002组细胞中LDH、MDA含量明显升高,SOD含量明显降低,细胞活力显著降低,Bax、Cleaved caspase-3的蛋白表达量均显著升高,Bcl-2蛋白表达量显著降低,Netrin-1、p-AKT蛋白表达均显著降低,细胞凋亡率显著升高(P<0.05)。结论 Netrin-1可通过激活Akt通路促进缺氧复氧心肌细胞活力,抑制凋亡,发挥保护作用,将可为心肌损伤的治疗提供理论依据。

Objective To investigate the role of Netrin-1 in myocardial H9 c2 hypoxia-reoxygenation injury and to explore its mechanism. Methods A model of H9 c2 hypoxia-reoxygenation injury in cardiomyocytes was established. A/R+Netrin-1 group(transfected with pcDNA 3.1-Netrin-1), A/R+ Ctrl group(transfected with pcDNA 3.1), A/R+Netrin-1 +LY294002 group(transfected with pcDNA 3.1-Netrin-1 co-treated with inhibitor LY294002) and A/R+ Netrin-1+DMSO group(transfected with pcDNA 3.1-Netrin-1 and DMSO) were transfected by liposome method. H9 c2 cells were stained;LDH, MDA and SOD were measured by ELISA;cell viability was measured by MTT assay;protein expression of Bcl-2, Bax, Cleaved caspase-3, Netrin-1 and p-AKT was measured by WesternBlot;apoptotic rate of cells were measured by cytology. Results Compared with the A/R+Ctrl group, the levels of LDH and MDA in the A/R+Netrin-1 group were significantly decreased, the SOD content was significantly increased, the cell viability was significantly increased, and the protein expression of Bax and Cleaved caspase-3 was significantly increased. The expression of Bcl-2 protein was significantly increased, the expression of Netrin-1 and p-AKT protein were significantly increased, and the apoptosis rate was significantly decreased(P<0.05). Compared with the A/R+Netrin-1+DMSO group, the LDH and MDA contents in the A/R+Netrin-1+ LY294002 group were significantly increased, the SOD content was significantly decreased, and the cell viability was significantly decreased. Bax and Cleaved caspase-3 were significantly decreased. The expression of Bcl-2 protein was significantly decreased, the expression of Netrin-1 and p-AKT protein were significantly decreased, and the apoptosis rate was significantly increased(P<0.05). Conclusion Netrin-1 can promote the hypoxia-reoxygenation myocardial cell viability, inhibit apoptosis and play a protective role by activating Akt pathway, which will provide a theoretical basis for the treatment of myocardial injury.