甘氨酸改善缺氧性MDCK细胞损伤的作用依赖于ERK1/2、Akt及p38MAPK信号通路

ERK,Akt and p38MAPK are involved in the cytoprotective effect of glycine on hypoxic impair in MDCK cells

目的:建立犬肾细胞(Madin-Darby canine kidney,MDCK)细胞缺氧模型,进一步阐明甘氨酸对缺氧细胞增殖活性的影响及其作用机制。方法:将MDCK细胞置于体积分数为95%N2和5%CO2的有机玻璃调节性密闭容器中,分别培养24、36、48、72或84 h,用四甲基偶氮唑盐(MTT)法检测甘氨酸对缺氧性损伤MDCK细胞增殖活性的影响。将MDCK细胞分为正常组、缺氧组和甘氨酸处理组,加药后孵育1、2或3 h后,收集细胞总蛋白,用Western blot检测细胞外信号调节激酶(extracellular signal-regulated kinase 1 and 2,ERK1/2)、p38MAPK和Akt的磷酸化活性。结果:在所观察的所有缺氧时段内,MDCK细胞MTT活性均较正常对照组明显下降(P<0.01)。加入甘氨酸后,缺氧24、36或48 h后细胞的增殖能力比缺氧组有明显增强,差异有统计学意义。在缺氧72或84 h后,甘氨酸未能显示明显的保护作用。缺氧时ERK1/2和Akt的磷酸化活性明显降低,p38MAPK的磷酸化活性明显增高。将甘氨酸加入到缺氧细胞中,ERK1/2和Akt又重新被激活,p38MAPK被抑制。结论:甘氨酸可保护MDCK细胞免于早期缺氧性损伤,该作用可能通过激活ERK1/2和Akt,抑制p38MAPK而实现。

Objective：To investigate the effects of glycine on hypoxic impair in cells. Methods：MDCK cells were cultured in a modular incubator chamber full of 95% N2 and 5% CO2 in the presence or absence of glycine for 24 h,36 h,48 h,72 h and 84 h. We observed the effects of glycine on cellular proliferation under hypoxic conditions by MTF method. Western blot was used to detect the phosphorylation activities of ERK1/2,p38MAPK and Akt in cells. Results：The proliferation of MDCK cells cultured under hypoxic conditions decreased significantly,while the proliferation increased after glycine treatment. Protective effect of glycine on cells was found at 24 h,36 h and 48 h of hypoxia. Activation of p38MAPK and depression of ERK1/2 and Akt in MDCK cells were detected when oxygen was deprived. Treatment with glycine antagonized the hypoxia-induced phosphorylative changes in ERK1/2,Akt and p38MAPK. Conclusion：Glycine could protect MDCK ceils from early hypoxic injury. Activation of ERK1/2 and Akt but suppression of p38MAPK may contribute to the cytoprotective mechanisms of glycine on hypoxic impair in MDCK cells.