荭草花提取物对H9c2心肌细胞缺氧复氧损伤的改善作用
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篇名: 荭草花提取物对H9c2心肌细胞缺氧复氧损伤的改善作用
TITLE: Study on Improvement Effects of Polygonum orientale Flower Extract on Hypoxia-reoxygenation Injury of H 9c2 Cardiomyocytes
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/LCoCl2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(Cytoc)/线粒体中Cytoc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+CoCl2组(15μmol/LLY294002+800μmol/LCoCl2)、LY294002+荭草花提取物组(15μmol/LLY294002+80μg/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(25μmol/LPD98059+800μmol/LCoCl2)、PD98059+荭草花提取物组(25μmol/LPD98059+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Cytoc/线粒体中Cytoc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。
ABSTRACT: OBJECTIVE:To study the improvement effects and mechanism of Polygonum orientale flower extract on hypoxia- reoxygenation injury of H 9c2 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 Ca 2+(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 pathwayrelated protein [protein kinase B (Akt)and extracellular signal regulated kinase 1/2(ERK1/2)] as well as protein expression of HIF- 1 α were detected respectively. In addition,the cells were divided into normal control group ,model group and P. orientale flower extract group (80 μ g/mL),PI3K inhibitor LY294002+CoCl2 group(15 μmol/L LY294002+80 μmol/L ,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 ERK 1/2 protein phosphorylation level were significantly increased (P<0.01). After treated with PI 3K inhibitor LY 294002 and MEK inhibitor PD 98059,Akt protein and ERK 1/2 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 H 9c2 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.
期刊: 2021年第32卷第11期
作者: 李月婷,刘亭,吴琼,陆定艳,王明金,王永林,李勇军,周孟,刘春花
AUTHORS: LI Yueting, LIU Ting,WU Qiong,LU Dingyan,WANG Mingjin,WANG Yonglin,LI Yongjun,ZHOU Meng,LIU Chunhua
关键字: 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
KEYWORDS: Polygonum orientale flower extract ;H9c2 cardiomyocytes;Hypoxia-reoxygenation injury ;RISK signaling
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