生长停滞特异性基因6对大鼠心肌细胞缺血再灌注的保护机制

Gas6-induced cardioprotection during cardiac ischemia and reperfusion

目的初步探讨生长停滞特异性基因6(Gas6)在缺血预适应(IPC)中,对大鼠心肌细胞缺氧/复氧(I/R)损伤的保护作用及机制。方法选择大鼠胚胎源性H9C2心肌细胞株培养48 h后,随机分为5组:NC组、I/R组、IPC组、Gas6组、Gas6+LY组为Gas6+磷脂酰肌醇3激酶(PI3K)阻断剂LY294002培养,各组按分组条件培养后,测乳酸脱氢酶(LDH)活性;流式细胞术测细胞凋亡率;实时荧光定量PCR法测NC组、I/R组和IPC组Gas6 mRNA表达;Western blot法测磷酸化蛋白激酶B(p-Akt),磷酸化叉头蛋白O亚家族(p-Foxo3a)蛋白水平。结果与NC组比较,I/R组LDH活性和细胞凋亡率明显升高;与I/R组比较,IPC组Gas6 mRNA表达增加,IPC组和Gas6组LDH活性及细胞凋亡率明显减少,p-Akt、P-Foxo3a蛋白明显增加,与Gas6组比较,Gas6+LY组LDH活性和细胞凋亡率明显升高,p-Akt和p-Foxo3a蛋白表达明显下降(P<0.05)。结论 Gas6通过PI3K/Akt/Foxo3a通路在IPC中起心肌细胞保护作用。

Objective to study the protective effect and mechanism of Gas6-induced cardioprotection with ischemic preconditioning mediated by the rat embryonic heart-derived cell line, H9C2 cells. Methods The H9C2 cells were randomly divided into five groups： normal control, IR,ischemic pre-conditioning（IPC） ,Gas6 and Gas6＋LY294002 group. Serum LDH was detected with kit; cell apoptotic rate was evaluated by flow cytometry; the Gas6 mRNA was examined by the real-time PCR,and the protein expressions of p-Akt and p-Foxo3a were examined by Western blot. Results Compared with I/R group, Gas6 mRNA expression in IPC group was significantly increased （P〈0.05） ,serum LDH level and the cell apoptotic rate were significantly decreased in IPC group and Gas6 group（P〈0.05）,meanwhile protein expressions of p-Akt and p-Foxo3a were significantly increased（P〈0.05）;in IPC group serum LDH level,the cell apoptotic rate and protein expressions of p-Akt and p-Foxo3a were not different from that in Gas6 group（P〉0.05） ,but serum LDH level and the cell apoptotic rate were significantly increased in Gas6 ＋ LY294002 group, meanwhile p-Akt and p-Foxo3a protein expressions significantly decreased（P〈0.05）. Conclusion Gas6 showed a protective effect in ischemia preconditioning, that may be mediated by Phosphatidylinositol3-Kinase/Akt/Foxo3a Survival Pathway.