【摘要】 目的 从线粒体动力相关蛋白1(Drp1)介导线粒体分裂平衡角度,解读黄芪多糖(APS)对感染性心肌病小鼠的心功能保护作用及机制。方法 按随机数字表法将18只成年雄性C57BL/6J小鼠分为对照组、脂多糖(LPS)组和LPS+APS组,每组6只。采用LPS诱导制备感染性心肌病模型。各组均于术后6h经尾静脉取外周血,采用酶联免疫吸附试验(ELISA)检测小鼠血清炎性因子水平;LPS处理后24 h行超声心动图检查心室功能,包括左室射血分数(LVEF)、左室短轴缩短率(LVFS)、左室舒张期末内径(LVEDD)和左室收缩期末内径(LVESD);检查后即刻处死小鼠取心肌组织,采用蛋白质印迹试验(Western blotting)检测肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)、C3a、Drp1、线粒体分裂蛋白1(Fis1)、线粒体融合蛋白2(Mfn2)、视神经萎缩蛋白1 (Opa1)蛋白表达。将购买的小鼠心房肌细胞株HL-1分为对照组、LPS组、LPS+APS组和LPS+APS+线粒体氧化磷酸化解偶联剂(FCCP)组。采用活性氧(ROS)检测试剂盒检测分离线粒体及HL-1细胞内的ROS含量;采用线粒体膜电位检测试剂盒(JC-1)检测线粒体膜电位(MMP);采用三磷酸腺苷(ATP)检测试剂盒检测HL-1细胞内ATP含量。结果 与对照组比较,LPS组小鼠心脏的LVEF、LVFS、LVEDD和LVESD均明显降低,线粒体内ROS含量明显升高,MMP和ATP含量均明显下降;而APS可改善感染性心肌病小鼠的心功能指标[LVEF:0.571±0.026比0.287±0.045,LVFS(%):33.13±2.11比21.22±0.99,LVEDD(mm):3.74±0.10比2.77±0.05,LVESD(mm):2.74±0.09比1.99±0.04,均P<0.05],明显降低心肌内ROS含量[ROS(%):138.39±9.28比260.97±19.22,P<0.05],明显抑制MMP和ATP含量下降[MMP(红色/绿色荧光强度比值):0.91±0.05比0.55±0.01,ATP(%):94.22±10.11比58.74±5.39,均P<0.05],且APS可明显抑制LPS处理后的Drp1和Fis1蛋白过表达以及Mfn2和Opa1蛋白低表达[Drpl/GAPDH(%):126.33±11.70比218.75±19.44,Fis1/GAPDH (%):105.71±15.77比194.39±5.27,Mfn2/GAPDH (%):92.75±10.44比41.88±3.95,Opal/GAPDH(%):98.14±3.71比55.94±7.30,均P<0.05]。故进一步采用HL-1细胞构建感染性心肌病模型,HL-1细胞经FCCP处理后可明显抑制APS的保护效应。结论 APS通过抑制Drp1过表达,抑制线粒体的过度分裂,减轻感染性心肌细胞的氧化应激和线粒体损伤,最终改善心脏功能,该机制的发现可为感染性心肌病的治疗提供新的理论依据和临床参考。更多还原

【Abstract】 Objective From the point of view of mitochondrial fissure balance mediated by mitochondrial dynamic related protein 1(Drpl) to probe the protective effect and mechanism of Astragalus polysaccharide(APS) on cardiac function of mice with septic cardiomyopathy.Methods Eighteen adult male C57 BL/6 J mice were randomly divided into 3 groups:control,lipopolysaccharide(LPS) and LPS+APS groups,with 6 mice in each group.LPS was used to induce the mice model of septic cardiomyopathy.After 6 hours,the peripheral blood was taken from the mouse caudal vein of each group,and the levels of serum inflammatory factors were detected by enzyme-linked immunosorbent assay(ELISA);24 hours after LPS treatment,ventricular function was examined by echocardiography,including left ventricular ejection fraction(LVEF),left ventricular fractional shortening(LVFS),left ventricular end diastolic diameter(LVEDD) and left ventricular end systolic diameter(LVESD);the mice were sacrificed immediately after examinations to obtain cardiac tissue.The protein expression levels of tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),C3 a,Drp1,mitochondrial mitotic protein 1(Fisl),mitochondrial fusion protein 2(Mfn2) and optic atrophy protein 1(Opal)were detected by Western blotting.The purchased mouse atrial muscle cell line HL-1 was divided into 4 groups:control,LPS,LPS+APS and LPS+APS+carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone(FCCP) groups.Reactive oxygen species(ROS) assay kit was used to detect the content of ROS in isolated mitochondria and HL-1 cells;mitochondrial membrane potential(MMP) was detected by mitochondrial membrane potential detection kit(JC-1);the content of ATP in HL-1 cells was detected by adenosine triphosphate(ATP) detection kit.Results Compared with those of the control group,the levels of LVEF,LVFS,LVEDD and LVESD were significantly decreased,the content of ROS in mitochondria was significantly increased,and the contents of MMP and ATP were significantly decreased in the hearts of LPS mice group;while APS could improve the indexes of cardiac function in mice with infectious cardiomyopathy [LVEF:0.571 ±0.026 vs.0.287±0.045,LVFS(%):33.13±2.11 vs.21.22±0.99,LVEDD(mm):3.74±0.10 vs.2.77±0.05,LVESD(mm):2.74±0.09 vs.1.99±0.04,all P <0.05],and obviously reduce the content of ROS in myocardium [ROS(%):138.39±9.28 vs.260.97±19.22,P <0.05],significantly inhibit the decrease of MMP and ATP contents [MMP(red/green fluorescence intensity ratio):0.91±0.05 vs.0.55±0.01,ATP(%):94.22±10.11 vs.58.74±5.39,all P <0.05],and APS could significantly inhibit the over expression of Drp1 and Fisl protein and the lower expression of Mfn2 and Opal protein after LPS treatment [Drpl/GAPDH(%):126.33±11.70 vs.218.75±19.44,Fisl/GAPDH(%):105.71± 15.77 vs.194.39±5.27,Mfn2/GAPDH(%):92.75±10.44 vs.41.88±3.95,Opal/GAPDH(%):98.14±3.71 vs.55.94±7.30,all P <0.05].Therefore,HL-1 cells were further used to construct the model of infectious cardiomyopathy,and after the HL-1 cells were treated with FCCP could significantly inhibit the protective effect of APS on the model.Conclusions APS can inhibit the overexpression of Drpl± inhibit the excessive division of mitochondria,reduce the oxidative stress and mitochondrial damage of infectious cardiomyocytes,and finally improve cardiac function.The discovery of this mechanism can provide a new theoretical basis and clinical reference for the treatment of patients with infectious cardiomyopathy.更多还原