李月婷 刘亭 吴琼 陆定艳 王明金 王永林 李勇军 周孟 刘春花

摘 要 目的：研究葒草花提取物对H9c2心肌细胞缺氧复氧损伤的的改善作用及机制。方法：将H9c2心肌细胞为正常对照组、模型组和荭草花提取物低、中、高浓度组（20、40、80 μg/mL），除正常对照组外，其余各组细胞以800 μmol/L CoCl2复制缺氧复氧损伤模型，然后观察细胞凋亡情况，并分别检测细胞中ROS水平、钙离子水平（细胞质中）、线粒体膜电位（MMP）水平、ATP酶（Na+-K+-ATP酶、Ca2+-Mg2+-ATP酶）活性、细胞质中细胞色素c（Cyto c）/线粒体中Cyto c比值、再灌注补救激酶（RISK）信号通路相关蛋白[蛋白激酶B（Akt）、细胞外信号调节激酶1/2（ERK1/2）]的磷酸化水平以及缺氧诱导因子1α（HIF-1α）的蛋白表达水平。另取细胞分为正常对照组、模型组、荭草花提取物组（80 μg/mL）、磷脂酰肌醇3激酶（PI3K）抑制剂LY294002+CoCl2组（15 μmol/L LY294002+800 μmol/L CoCl2）、LY294002+荭草花提取物组（15 μmol/L LY294002+80 μg/mL荭草花提取物）、丝裂原活化蛋白激酶激酶（MEK）抑制剂PD98059+CoCl2组（25 μmol/L PD98059+800 μmol/L CoCl2）、PD98059+荭草花提取物组（25 μmol/L PD98059+80 μg/mL荭草花提取物），同法培养后，测定细胞中Akt蛋白和ERK1/2蛋白的磷酸化水平，以验证荭草花提取物对RISK信号通路的激活作用。结果：与模型组比较，荭草花提取物各浓度组细胞的细胞核固缩现象减轻，凋亡小体数量减少;细胞中ROS水平、钙离子水平（低浓度组除外）、MMP、细胞质中Cyto c/线粒体中Cyto c比值和HIF-1α的蛋白表达水平均显著降低（P<0.05或P<0.01）;ATP酶活性（低浓度组除外）、Akt蛋白和ERK1/2蛋白磷酸化水平均显著升高（P<0.01）。加入抑制剂LY294002、PD98059后，细胞中Akt蛋白和ERK1/2蛋白磷酸化水平均显著降低（P<0.05或P<0.01）。结论：荭草花提取物可改善H9c2心肌细胞缺氧复氧损伤，其作用机制可能与抑制心肌细胞凋亡、提高ATP酶活性、保护线粒体、调节RISK信号通路相关蛋白和HIF-1α蛋白表达有关。

关键词 荭草花提取物;H9c2心肌细胞;缺氧复氧损伤;补救激酶信号通路;缺氧诱导因子1α

ABSTRACT   OBJECTIVE： To study the improvement effects and mechanism of Polygonum orientale flower extract on hypoxia- reoxygenation injury of H9c2 cardiomyocytes. METHODS： H9c2 cardiomyocytes were divided into normal control group， model group and low-， medium- and high-concentrations groups of P. orientale flower extract （20， 40， 80 μg/mL）. Except for normal control group， other groups were given 800 μmol/L CoCl2 to induce hypoxia-reoxygenation injury model. Cell apoptosis was observed. The levels of Ca2+ （in cytoplasm）， mitochondrial membrane potential （MMP）， ATP enzyme （Na+-K+-ATP enzyme， Ca2+-Mg2+-ATP enzyme） activities， the ratio of cytochrome c（Cyto c）   protein in cytosol to mitochondria， phosphorylation levels of reperfusion injury salvage kinase （RISK） signaling pathway related protein [protein kinase B （Akt） and extracellular signal regulated kinase 1/2 （ERK1/2）] as well as protein expression of HIF-1α were detected respectively. In addition， the cells were divided into normal control group， model group and P. orientale flower extract group （80 μg/mL）， PI3K inhibitor LY294002+CoCl2 group （15 μmol/L LY294002+80 μmol/L CoCl2），  LY294002+P. orientale flower extract group （15 μmol/L LY294002+80 μg/mL P. orientale flower extract）， MEK inhibitor PD98059+CoCl2 group （25 μmol/L PD98059+800 μmol/L CoCl2）， PD98059+P. orientale flower extract group （25 μmol/L PD98059+80 μg/mL P. orientale flower extract）. After cultured by the same method， the phosphorylation levels of Akt protein and ERK1/2 protein in the cells were measured to verify the activation of P. orientale flower extract to RISK signaling pathway. RESULTS： Compared with model group， nuclear pyknosis and the number of apoptotic bodies were reduced in different concentrations groups of P. orientale flower extract. ROS level， Ca2+ level （except for low-concentration group）， MMP， ratio of Cyto c in cytoplasm to Cyto c in mitochondria， protein expression of HIF-1α were decreased significantly （P<0.05 or P<0.01）; the activity of ATP enzyme （except for the low-concentration group）， Akt protein and ERK1/2 protein phosphorylation level were significantly increased （P<0.01）. After treated with PI3K inhibitor LY294002 and MEK inhibitor PD98059， Akt protein and ERK1/2 protein phosphorylation level in cadiomyocyte were decreased significantly （P<0.05 or P<0.01）. CONCLUSIONS： P. orientale flower extract can improve hypoxia-reoxygenation injury of H9c2 cardiomyocytes， the mechanism of which may be associated with inhibiting cardiomyocyte apoptosis， improving ATPase activity， protecting mitochondria， regulating RISK signaling pathway related proteins and HIF-1α protein expression.

KEYWORDS   Polygonum orientale flower extract; H9c2 cardiomyocytes; Hypoxia-reoxygenation injury; RISK signaling pathway; HIF-1α

荭草为蓼科植物荭草Polygonum orientale L.的干燥果穗及带叶茎枝，其味辛，性凉，有小毒，归心、肝、胃、大肠经，具有清热解毒、祛风除湿、活血消肿的功效，常用于治疗风湿性关节炎、冠心病、心胃气痛、疝气、脚气、疮肿[1]。……