荭草花提取物对H9c2心肌细胞缺氧复氧损伤的改善作用  

Study on Improvement Effects of Polygonum orientale Flower Extract on Hypoxia-reoxygenation Injury of H9c2 Cardiomyocytes

作　　者：李月婷[1] 刘亭[1] 吴琼[2] 陆定艳 王明金 王永林[1] 李勇军[2] 周孟[2] 刘春花[2] LI Yueting;LIU Ting;WU Qiong;LU Dingyan;WANG Mingjin;WANG Yonglin;LI Yongjun;ZHOU Meng;LIU Chunhua(Guizhou Provincial Key Laboratory of Pharmaceutics/State Key Laboratory of Functions and Applications of Medicinal Plants,Guizhou Medical University,Guiyang 550004,China;Engineering Research Center of the Ministry of Education for the Development and Application of Ethnic Medicine and TCM,Guizhou Medical University,Guiyang 550004,China;School of Pharmacy,Guizhou Medical University,Guiyang 550004,China)

机构地区：[1]贵州医科大学贵州省药物制剂重点实验室/省部共建药用植物功效与利用国家重点实验室,贵阳550004 [2]贵州医科大学民族药与中药开发应用教育部工程研究中心,贵阳550004 [3]贵州医科大学药学院,贵阳550004

出　　处：《中国药房》2021年第11期1304-1312,共9页China Pharmacy

基　　金：国家自然科学基金资助项目(No.81803700,No.81560630,No.81660570,No.U1812403);中央引导地方科技发展专项(No.黔科中引地〔2018〕4006);贵州省科技计划项目(No.黔科合基础〔2020〕1Z068号,No.黔科合基础〔2019〕1439号,No.黔科合基础〔2019〕1280号)。

摘　　要：目的:研究荭草花提取物对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、80μg/mL),除正常对照组外,其余各组细胞以800μmol/L CoCl_(2)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、Ca^(2+)-Mg^(2+)-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(80μg/mL)、磷脂酰肌醇3激酶(PI3K)抑制剂LY294002+CoCl_(2)组(15μmol/L LY294002+800μmol/L CoCl_(2))、LY294002+荭草花提取物组(15μmol/L LY294002+80μg/mL荭草花提取物)、丝裂原活化蛋白激酶激酶(MEK)抑制剂PD98059+CoCl_(2)组(25μmol/L PD98059+800μmol/L CoCl_(2))、PD98059+荭草花提取物组(25μmol/L PD98059+80μg/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HIOBJECTIVE:To study the improvement effects and mechanism of Polygonum orientale flower extract on hypoxiareoxygenation 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(20,40,80μg/mL).Except for normal control group,other groups were given 800μmol/L CoCl_(2) to induce hypoxia-reoxygenation injury model.Cell apoptosis was observed.The levels of Ca^(2+)(in cytoplasm),mitochondrial membrane potential(MMP),ATP enzyme(Na^(+)-K^(+)-ATP enzyme,Ca^(2+)-Mg^(2+)-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-1αwere detected respectively.In addition,the cells were divided into normal control group,model group and P.orientale flower extract group(80μg/mL),PI3K inhibitor LY29400^(2+)CoCl_(2) group(15μmol/L LY29400^(2+)80μmol/L CoCl_(2)),LY29400^(2+)P.orientale flower extract group(15μmol/L LY29400^(2+)80μg/mL P.orientale flower extract),MEK inhibitor PD98059+CoCl_(2) group(25μmol/L PD98059+800μmol/L CoCl_(2)),PD98059+P.orientale flower extract group(25μmol/L PD98059+80μg/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,Ca^(2+)level(except for low-concentration group),MMP,ratio of Cyto c in cytoplasm to Cyto c in mitochondria,protein expression of HIF-1αwere decreased significantly(P<0.05 or P<0.01);the activity of ATP enzyme(except for the low-concentration group),Akt protein an

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 

分 类 号：R965[医药卫生—药理学]