毛冬青皂苷E对H9C2心肌细胞缺氧/复氧损伤的影响
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摘要
目的考察毛冬青皂苷E对H9C2心肌细胞缺氧/复氧损伤的保护作用,并探讨其作用机制。方法将H9C2心肌细胞随机分为正常对照组,模型组,毛冬青皂苷E低、中、高剂量对照组(10,50,250μg·m L-1)、毛冬青皂苷E低、中、高剂量治疗组(10,50,250μg·m L-1)。造模前24 h给予相应浓度的药物预处理,缺氧4 h,复氧4 h后,采用CCK-8法检测细胞存活率,DCFH-DA荧光探针检测细胞内活性氧(ROS)含量,并采用蛋白印迹法(Western Blot)检测Caspase-3和Cleaved caspase-3凋亡蛋白的表达。结果缺氧/复氧后,与正常对照组比较,模型组细胞存活率显著降低(P<0.05),细胞内ROS含量显著升高(P<0.01),毛冬青皂苷E各剂量治疗组预处理后,细胞存活率增加,ROS含量降低。缺氧/复氧损伤可使Caspase-3和Cleaved caspase-3蛋白表达增加。毛冬青皂苷E各剂量治疗组预处理后,Caspase-3和Cleaved caspase-3蛋白表达均下降。结论毛冬青皂苷E对H9C2心肌细胞缺氧/复氧损伤具有保护作用,其机制可能与减少ROS生成,抑制凋亡相关蛋白Caspase-3和Cleaved caspase-3的表达有关。
Objective To study the protective effects of ilexoside E on H9C2 myocardial cells after hypoxia/reoxygenation and to explore its mechanism.Methods H9C2 myocardial cells were randomly divided into normal control group,hypoxia/reoxygenation model group,and low-,middle-and high-dose ilexoside E(10,50,250 μg/m L)treatment groups.H9C2 myocardial cells were pretreated with ilexoside E for 24 h before hypoxia.After hypoxia for 4h and reoxygenation for 4h,cell viability was measured by CCK-8 method,intracellular reactive oxygen species(ROS) was detected by DCFH-DA fluorescent probe,and the expression levels of Caspase-3 and cleaved Caspase-3 were detected by Western blot.Results Compared with the normal control group,the viability of H9C2 myocardial cells decreased significantly(P <0.01),and intracellular ROS level was elevated significantly(P <0.01) after hypoxia /reoxygenation.After pretreatment with 10,50,250 μg/m L of ilexoside E,cell viability was significantly increased,the intracelluar ROS level was significantly decreased.Moreover,the expression levels of Caspase-3 and cleaved Caspase-3 were all increased in myocardial cells injured by hypoxia /reoxygenation.After pretreated with 10,50,250 μg·m L-1 of Ilexoside E,the expression levels of Caspase-3 and cleaved Caspase-3 were all reduced.C onclusion Ilexoside E has protective effects on H9C2 myocardial cells injured by H/R.And the mechanism may be related with the reduction of intracellular ROS level,with the inhibition of the expression of apoptosis-related protein Caspase-3 and cleaved Caspase-3.
Objective To study the protective effects of ilexoside E on H9C2 myocardial cells after hypoxia/reoxygenation and to explore its mechanism.Methods H9C2 myocardial cells were randomly divided into normal control group,hypoxia/reoxygenation model group,and low-,middle-and high-dose ilexoside E(10,50,250 μg/m L)treatment groups.H9C2 myocardial cells were pretreated with ilexoside E for 24 h before hypoxia.After hypoxia for 4h and reoxygenation for 4h,cell viability was measured by CCK-8 method,intracellular reactive oxygen species(ROS) was detected by DCFH-DA fluorescent probe,and the expression levels of Caspase-3 and cleaved Caspase-3 were detected by Western blot.Results Compared with the normal control group,the viability of H9C2 myocardial cells decreased significantly(P <0.01),and intracellular ROS level was elevated significantly(P <0.01) after hypoxia /reoxygenation.After pretreatment with 10,50,250 μg/m L of ilexoside E,cell viability was significantly increased,the intracelluar ROS level was significantly decreased.Moreover,the expression levels of Caspase-3 and cleaved Caspase-3 were all increased in myocardial cells injured by hypoxia /reoxygenation.After pretreated with 10,50,250 μg·m L-1 of Ilexoside E,the expression levels of Caspase-3 and cleaved Caspase-3 were all reduced.C onclusion Ilexoside E has protective effects on H9C2 myocardial cells injured by H/R.And the mechanism may be related with the reduction of intracellular ROS level,with the inhibition of the expression of apoptosis-related protein Caspase-3 and cleaved Caspase-3.
引文
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[14]Mocanu MM1,Baxter GF,Yellon DM.Caspase inhibition and limitation of myocardial infarct size:protection against lethal reperfusion injury[J].Br J Pharmacol,2000,130(2):197-200.
[2]周园,熊天琴,林朝展,等.毛冬青皂苷ilexsaponin A1的制备及其药理活性研究[J].中药材,2011,34(5):765-767.
[3]鲍涵.毛冬青药材不同部位与成分对大鼠乳鼠心肌细胞缺氧/复氧损伤的保护作用[D].广州:广州中医药大学,2012:21-51.
[4]强皎.毛冬青皂苷E对缺氧/复氧诱导的心肌细胞凋亡的影响及机制研究[D].广州:广州中医药大学,2014:29-42.
[5]Mc Milin JB,Dowhan W.Cardiolipin and apoptosis[J].Biochim Biophs Acta,2002,1585(2-3):97-107.
[6]Shirito K,Otani H,Yamamoto F,et al.MK2-/-gene knockout mouse hearts carry anti-apoptotic signal and are resistant to ischemia reperfusion injure[J].JMol Cell Cardiaol,2005,38(1):93-97.
[7]杨天潇.促红细胞生成素对缺氧复氧心肌细胞的保护作用及其机制[D].广州:南方医科大学,2011.
[8]曹雪明.黄连素预处理对H9C2心肌细胞缺氧/复氧损伤保护作用的机制研究[D].南方医科大学,2014.
[9]王艳冰,任素萍,王庆军,等.新型组蛋白去乙酰化酶抑制剂对缺氧损伤心肌细胞的保护作用研究[J].军事医学,2015,39(1):30-35,70.
[10]Gao C,Liu Y,Yu Q,et al.TNF-αantagonism ameliorates myocardial ischemia-reperfusion injury in mice by upregulating adiponectin[J].Am J Physiol Heart Circ Physiol,2015,308(12):H1583-91.
[11]Zhang Y,Zhao J,Li R,et al.Adipo Ron,the First Orally Active Adiponectin Receptor Activator,Attenuates Post-Ischemic Myocardial Apoptosis[J].Am J Physiol Endocrinol Metab,2015,309(3):275-282.
[12]Pisarenko O,Shulzhenko V,Studneva I,et al.Structural apelin analogues:mitochondrial ROS inhibition and cardiometabolic protection in myocardial ischaemia reperfusion injury[J].Br J Pharmacol,2015,172(12):2933-2945.
[13]陈建伟,钟玲.细胞色素C与caspase家族在缺血/再灌注所致心肌细胞凋亡中的作用[J].医学综述,2009,15(2):173-176.
[14]Mocanu MM1,Baxter GF,Yellon DM.Caspase inhibition and limitation of myocardial infarct size:protection against lethal reperfusion injury[J].Br J Pharmacol,2000,130(2):197-200.