EPO对慢性缺氧心肌细胞线粒体生物合成的影响

Effect of erythropoietin on mitochondrial biogenesis in cardiomyocytes during chronic hypoxia in vitro

目的观察促红细胞生成素(erythropoietin,EPO)在慢性缺氧条件下对心肌线粒体生物合成的影响及其可能的机制。方法将H9c2心肌细胞株置入缺氧培养箱7 d(94%N2,5%CO2,1%O2),建立H9c2心肌细胞株慢性缺氧模型;采用5、10、20 U/mL不同浓度的rhEPO(recombinant human erythropoietin;重组人促红细胞生成素)干预处理后,用荧光探针标记线粒体,观察线粒体数目的变化;RT-PCR技术检测线粒体DNA的相对拷贝数变化;Western blot技术检测Akt、eNOS及其磷酸化蛋白水平的变化。结果 rhEPO干预7 d后线粒体数目及拷贝数增加(P<0.05);Akt、eNOS蛋白磷酸化水平增强(P<0.05),Akt、eNOS总蛋白无明显变化(P>0.05)。结论 EPO增强了慢性缺氧心肌细胞的线粒体生物合成,Akt、eNOS可能是EPO增强线粒体生物合成的重要信号通路。

Objective To determine the effect of erythropoietin （EPO） on the mitochondrial bio- genesis in rat cardiomyocytes under the condition of chronic hypoxia. Methods Rat cardiomyocyte-derived H9c2 cells were deprived of oxygen for 7 d to establish the chronic hypoxia model （94% N2, 5% CO2and 1% 02 ）. Then the obtained H9c2 cells were treated by recombinant human EPO （rhEPO, 5, 10 and 20 U/ml） for 3 or 7 d. Fluorescent probe was used to mark mitochondria in the cells for the amount of mitoehondria. The pro- tein levels of Akt, phospho-Akt （ serine473 ）, endothelial nitric oxide synthase （ eNOS）, and phospho-eNOS （serine1177） were determined with Western blotting. RT-PCR was used to detect the relative copy number of mitoehondrial DNA. Results rhEPO treatment for 7 d resulted in a significant increase in the amount and copy number of mitochondria （ P 〈 0. 05 ）. The phosphorylation levels of Akt and eNOS were also enhanced, but those of akt and eNOS had no change （ P 〉 0. 05 ）. Conclusion EPO improves mitochondrial biogenesis in the cardiomyocytes exposure to chronic hypoxia, probably through Akt and eNOS signal transduction.