目的 探讨抗增殖蛋白2（PHB2）脓毒症心肌损伤线粒体功能的调控机制。方法 体外培养大鼠心肌细胞株（H9C2）,分为对照组、脂多糖（LPS）组、LPS+PHB2 siRNA（si-PHB2）组。检测氧化应激指标细胞内丙二醛（MDA）水平、荧光探针检测细胞内活性氧（ROS）水平;线粒体指标：三磷酸腺苷（ATP）水平、线粒体膜电位、线粒体电镜、线粒体半定量评分;免疫印迹法检测PHB2、PTEN诱导激酶1（PTNKI）、帕金蛋白（Parkin）、线粒体转录因子（TFAM）的表达。结果 LPS刺激后MDA水平和ROS水平升高、ATP水平低,LPS+si-PHB2组MDA（6.21±0.39 vs 3.59±0.33, P<0.05）、细胞内的ROS（15 131.37±88.72 vs 8 628.67±71.95, P<0.05）的水平较LPS组升高,ATP（3.46±0.34 vs 4.52±0.25, P<0.05）和线粒体膜电位水平（0.33±0.04 vs 0.55±0.09, P<0.05）进一步降低;电镜观察显示与正常组相比,LPS组、LPS+si-PHB2组出现不同程度线粒体损伤,线粒体损伤半定量评分显示LPS+si-PHB2组的损伤较LPS组更为明显（1.42±0.10 vs 0.81±0.04, P<0.05）; 免疫印迹法结果显示LPS处理后PHB2、PINK1、Parkin 表达上调,TFAM表达下调,LPS+si-PHB2组的线粒体自噬相关蛋白PINK1（1.33±0.06 vs 1.79±0.21, P<0.05）、Parkin（1.43±0.08 vs 1.86±0.09, P<0.05）和线粒体生物发生关键蛋白TFAM（0.29±0.01 vs 0.74±0.06, P<0.05）表达均较LPS组降低。结论 LPS可促进大鼠心肌细胞PHB2表达,si-PHB2干扰后线粒体自噬蛋白和生物发生蛋白表达抑制,心肌细胞氧化应激损害和线粒体功能障碍加重,提示PHB2表达上调可能恢复线粒体稳态改善脓毒症心肌损伤的线粒体功能。

Objective To explore the regulatory mechanism of septic myocardial injury by prohibitin 2（PHB2）. Methods Rat myocardial cell lines（H9C2）were cultured in vitro and divided into control group,LPS group,LPS + PHB2 siRNA（si-PHB2） group. The indicators for detecting oxidative stress include the levels of intracellular malondialdehyde（MDA）and reactive oxygen species（ROS）. The indicators for mitochondrial detection include adenosine triphosphate（ATP）levels,mitochondrial membrane potential,mitochondrial electron microscopy,and semi-quantitative mitochondrial scoring. Western blotting was used to detect the expression of PHB2,PTEN induced putative kinase（PINK1）,Parkin,mitochondrial transcription factor A（TFAM）. Results After LPS stimulation,MDA level and intracellular ROS level increased,ATP level decreased. Compared with LPS group,MDA（6. 21±0. 39 vs 3. 59±0. 33, P<0. 05）level and intracellular ROS level（15 131. 37±88. 72 vs 8 628. 67±71. 95, P<0. 05）in LPS + si-PHB2 group increased significantly,while ATP（3. 46±0. 34 vs 4. 52±0. 25, P<0. 05）and MMP（0. 33±0. 04 vs 0. 55±0. 09, P<0. 05）level further decreased. Compared with the normal group,the structure of mitochondria in LPS group and LPS + si-PHB2 group was damaged in different degree. The semi-quantitative score of mitochondrial damage showed that the damage in LPS + si-PHB2 group was more obvious than that in LPS group（1. 42±0. 10 vs 0. 81±0. 04, P<0. 05）. Western blotting showed that the expression of PHB2,PINK1 and Parkin were up-regulated and the expression of TFAM was down-regulated after LPS treatment,mitohagy-related proteins PINK1（1. 33±0. 06 vs 1. 79±0. 21, P<0. 05）,Parkin（1. 43±0. 08 vs 1. 86±0. 09, P<0. 05）and mitochondrial biogenetic protein TFAM（0. 29±0. 01 vs 0. 74±0. 06, P<0. 05）in LPS+si-PHB2 group were lower than those in LPS group. Conclusions LPS can promote the expression of PHB2 in rat cardiomyocytes. After interfering with PHB2 expression,we found that mitochondrial autophagy and biogenesis are inhibited,and mitochondrial dysfunction,oxidative stress exacerbated,suggesting that the up-regulation of PHB2 expression may restore mitochondrial homeostasis and improve mitochondrial function in septic myocardial injury.