摘 要 目的：觀察参附益心方对缺氧原代心肌细胞活性氧（ROS）和能量代谢的影响。方法：新生SD大鼠原代心肌细胞经分离、培养、鉴定后，随机分为正常组、模型组、阳性对照组（辅酶Q10，0.1 mmol/L）和参附益心方低、高剂量组（0.25、0.5 mg/mL）。除正常组外，其余各组细胞均于5%O2、5%CO2、90%N2条件下培养6 h以复制缺氧损伤模型。缺氧6 h后，采用ROS探针和流式细胞术分别检测各组细胞及其线粒体中ROS的含量，采用荧光素酶发光法和Western blotting法分别检测各组细胞中腺苷三磷酸（ATP）的含量以及肌酸激酶（CK）蛋白的表达水平，并使用透射电子显微镜观察各组细胞的超微结构。结果：与正常组比较，模型组缺氧原代心肌细胞及其线粒体中ROS的表达均明显增加，其ROS含量均显著升高，ATP的含量以及CK蛋白的表达水平均显著降低（P<0.05）；细胞内质网、线粒体肿胀，线粒体嵴溶解甚至消失，损伤明显。与模型组比较，各给药组缺氧原代心肌细胞及其线粒体中ROS的表达均有所减少，阳性对照组和参附益心方高剂量组缺氧原代心肌细胞中ROS的含量以及各给药组缺氧原代心肌细胞线粒体中ROS的含量均显著降低，阳性对照组和参附益心方高剂量组缺氧原代心肌细胞中ATP的含量以及各给药组缺氧原代心肌细胞中CK蛋白的表达水平均显著升高（P<0.05）；阳性对照组和参附益心方高剂量组缺氧原代心肌细胞损伤明显减轻。
结论：参附益心方对缺氧原代心肌细胞具有一定的改善作用，可下调细胞及线粒体中ROS的表达，并可改善其能量代谢。
关键词 参附益心方；缺氧损伤； 原代心肌细胞；活性氧；能量代谢
Effects of Shenfu Yixin Decoction on Reactive Oxygen Species and Energy Metabolism of Primary Hypoxic Cardiomyocytes
LI Bin1，2，WANG Xinlu2，XIE Shiyang3，GAO Yuan3，WANG Youping3，CUI Lin3，WANG Yongxia2，ZHU Mingjun2（1. Graduate Division， the First Affiliated Hospital of Henan University of TCM， Zhengzhou 450099， China； 2. Center of Cardiology， the First Affiliated Hospital of Henan University of TCM， Zhengzhou 450099， China； 3. Central Laboratory， the First Affiliated Hospital of Henan University of TCM， Zhengzhou 450099， China）
ABSTRACT OBJECTIVE： To observe the effects of Shenfu yixin decoction on reactive oxygen species （ROS） and energy metabolism in primary hypoxic cardiomyocytes. METHODS： After isolation， culture and identification， primary cardiomyocytes of neonatal SD rats were randomly divided into normal group， model group， positive control group （coenzyme Q10， 0.1 mmol/L） and Shenfu yixin decoction low-dose and high-dose groups （0.25， 0.5 mg/mL）. Except for normal group， other groups were cultured with 5%O2， 5%CO2 and 90%N2 for 6 h to induce hypoxic injury model. After 6 hours of hypoxia， ROS contents in cardiomyocytes and mitochondria of each group were detected by ROS probe and flow cytometry. Luciferase luminescence and Western blotting were used to detect ATP content and CK protein expression of each group. Transmission electron microscope was used to observe ultrastructure of cardiomyocytes in each group. RESULTS： Compared with normal group， the expression of ROS in primary hypoxic cardiomyocytes and mitochondria as well as the content of ROS were increased significantly， while the content of ATP and expression levels of CK protein were decreased significantly （P<0.05）； there were swelling of endoplasmic reticulum and mitochondria， dissolution or even disappearance of mitochondrial ridge， obvious cardiomyocytes injury. Compared with model group， the expression of ROS in primary hypoxic cardiomyocytes and mitochondria of administration groups， the contents of ROS in primary hypoxic cardiomyocytes of positive control group and Shenfu yixin decoction high-dose group as well as the content of ROS in primary hypoxic cardiomyocytes mitochondria of administration groups were all decreased significantly， while ATP contents in primary hypoxic cardiomyocytes of positive control group and Shenfu yixin decoction high-dose group as well as expression levels of CK protein in primary hypoxic cardiomyocytes of administration groups were all increased significantly （P<0.05）. The primary hypoxic cardiomyocytes injury was relieved significantly in positive control group and Shenfu yixin decoction high-dose group. CONCLUSIONS： Shenfu yixin decoction can improve primary hypoxic cardiomyocytes， down-regulate the expression of ROS in cardiomyocytes and mitochondria and also improve its energy metabolism.