解偶联蛋白2过表达对脓毒症大鼠心肌细胞线粒体的保护作用

Protective effect of overexpression of uncoupling protein 2 on mitochondria in cardiomyocytes of rats with sepsis

目的:探讨解偶联蛋白2(UCP2)过表达对脓毒症大鼠心肌细胞线粒体的保护作用,阐明其作用机制。方法:构建UCP2过表达的H9C2心肌细胞。H9C2心肌细胞随机分为正常对照组、脂多糖/聚糖肽(LPS/PepG)刺激组、空病毒组和过表达组,除正常对照组外其余各组予LPS/PepG刺激。采用RT-PCR和Western blotting法检测各组心肌细胞中UCP2mRNA和蛋白表达水平,采用分光光度计法和ELISA法检测脓毒症心肌细胞模型建立指标肌酸激酶(CK)和肿瘤坏死因子α(TNF-α)水平,采用电镜观察各组心肌细胞线粒体形态表现,采用流式细胞术检测各组心肌细胞线粒体膜电位(MMP)水平,采用多功能酶标仪检测各组心肌细胞线粒体活性氧(ROS)和三磷酸腺苷(ATP)水平。结果:与正常对照组比较,LPS/PepG刺激组心肌细胞中CK和TNF-α水平明显升高(P<0.05),且线粒体形态明显损害,MMP水平明显降低(P<0.05),线粒体ROS水平明显升高(P<0.05),ATP水平明显降低(P<0.05);与LPS/PepG刺激组比较,过表达组心肌细胞中CK和TNF-α水平明显降低(P<0.05),且线粒体形态结构明显好转,MMP水平明显升高(P<0.05),线粒体ROS水平明显降低(P<0.05),ATP水平明显升高(P<0.05);与LPS/PepG刺激组比较,空病毒组心肌细胞中CK、TNF-α、MMP、ROS和ATP水平差异均无统计学意义(P>0.05),线粒体肿胀度无明显改变。结论:UCP2过表达对脓毒症大鼠心肌细胞线粒体有保护作用,其机制可能与UCP2通过解偶联作用调控膜电位,进而影响ROS和ATP的合成有关。

Objective：To explore the protective effect of overexpression of uncoupling protein 2（UCP2）on the mitochondrial in cardiomyocytes of the rats with sepsis,and to clarify its mechanisms.Methods：The H9 C2 cardiomyocytes with UCP2 overexpression were reconstructed and randomly devided into normal control group,lipopolysaccharide/peptidoglycan（LPS/PepG）group,PHBLV group and PHBLV-UCP2 group.The H9 C2 cells in various groups except normal control group were stimulated with LPS/PepG.The expression levels of UCP2 mRNA and protein in the cardiomyocytes in various groups were detected with RT-PCR and Western blotting methods.The levels of creatine kinase（CK）and tumor necrosis factorα（TNF-α） were measured by spectrophotometer and enzyme-linked immunosorbent assay（ELISA）. The mitochondrial morphology of cardiomyocytes in various groups was observed by transmission electron microscope（TEM）,the mitochondrial membrane potential（MMP）levels of cardiomyocytes in various groups were detected by flow cytometry（FCM）,and the levels of reactive oxygen species（ROS）and adenosine triphosphate（ATP）in mitochondria of cardiomyocytes were measured by multimode reader.Results：Compared with normal control group,the levels of CK and TNF-αin LPS/PepG group were increased significantly（P〈0.05）,the mitochondrial morphology was injured obviously,the MMP level was decreased（P〈0.05）,the level of ROS was increased（P〈0.05）,and the ATP level was decreased（P〈0.05）.Compared with LPS/PepG group,the CK and TNF-α levels in cardiomyocytes in PHBLV-UCP2 group were decreased（P〈0.05）,the injury of mitochondria was improved,the MMP level was increased（P〈0.05）,the ROS level was decreased signficantly（P〈0.05）,and the ATP level was significantly increased（P〈0.05）.The levels of CK,TNF-α,MMP,ROS,ATP in the cardiomyocytes and the degree of mitochondrial swelling in PHBLV group had no obovionsly changes compared with LPS/PepG group（P〉0.05）.Conclusion：UCP2 overexpression plays a protective role in myocardial mitochondria of the rats with sepsis,which may be related to its regulation on the membrane potential to affect the mitochondrial ROS and ATP synthesis through uncoupling effect under septic condiction.