槟榔醇提物对H9C2心肌细胞的抗缺氧保护作用
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摘要
目的研究槟榔无水醇提物对低氧H9C2心肌细胞的作用及其保护机制。方法建立H9C2心肌细胞低氧模型,CCK-8法检测槟榔无水醇提物对H9C2细胞存活率的影响;通过检测细胞内丙二醛(MDA)含量,过氧化物歧化酶(SOD)、还原型谷胱甘肽(GSH)活力,研究槟榔无水醇提物对H9C2细胞的保护作用;RT-PCR法检测细胞内Nrf2、Caspase-3 mRNA水平的表达,从分子水平研究槟榔无水醇提物对低氧H9C2细胞的保护机制。结果与常氧对照组相比,低氧24 h时细胞存活为28.46%(P<0.01),细胞内MDA含量升高44.33%(P<0.05),SOD、GSH活力分别下降16.18%、30.64%(P<0.05或P<0.01)。RT-PCR实验结果表明,Nrf2被激活,其mRNA表达上升1.74倍(P<0.05)。与低氧模型组相比,槟榔无水醇提物处理组H9C2细胞存活率明显升高(P<0.01),且呈现剂量依赖性,槟榔无水醇提物处理组细胞内SOD、GSH活力分别增加14.90%、28.94%(P<0.05),Nrf2基因mRNA相对表达下降66%(P<0.05)。结论槟榔无水醇提物对低氧H9C2细胞有显著的保护作用,其保护机制可能与提高细胞内抗氧化物酶活力,减轻细胞氧化应激损伤,提高细胞耐低氧能力有关。
Objective To study the protective effect and mechanism of anhydrous ethanol extract of areca catechu linn against hypoxia in H9C2 cells.Methods Myocardial cell hypoxic mold was established by H9C2 cell line.The effect of areca catechu linn anhydrous ethanol extract on cells livability was studied by CCK-8.The intracellular content of MDA,activities of SOD and GSH were measured to determine the protective effect of the extract.The mRNA of Nrf-2 and caspase-3 were detected by real-time PCR to explore the protective mechanism.Results Compared to the normoxic control group,the cell survival rate in hypoxic group was 28.46%(P<0.01) after 24 hour hypoxia.The intracellular content of MDA increased 44.33%(P<0.05).The activities of SOD and GSH decreased by 16.18% and 30.64%,respectively(P<0.05 or P<0.01).The result of RT-PCR showed the activation of Nrf2 with a 1.74 times increase of mRNA expression(P<0.05).Compared with hypoxic group,there was an dose-dependent increase of cell survival rate in H9C2 cells(P<0.01) when treated with areca catechu linn anhydrous ethanol extract during hypoxia.The intracellular activities of SOD and GSH also increased by 14.90% and 28.94%(P<0.05).The relative expression of Nrf2 mRNA decreased significantly by 66%(P<0.05).Conclusion The anhydrous alcohol extract of areca catechu linn has a significant protective effect on H9C2 cells against hypoxia.Its protective mechanism may relate to the improvement of intracellular antioxidant enzyme activity,the reduction of oxidative stress damage and the improvement of cell hypoxic tolerance.
Objective To study the protective effect and mechanism of anhydrous ethanol extract of areca catechu linn against hypoxia in H9C2 cells.Methods Myocardial cell hypoxic mold was established by H9C2 cell line.The effect of areca catechu linn anhydrous ethanol extract on cells livability was studied by CCK-8.The intracellular content of MDA,activities of SOD and GSH were measured to determine the protective effect of the extract.The mRNA of Nrf-2 and caspase-3 were detected by real-time PCR to explore the protective mechanism.Results Compared to the normoxic control group,the cell survival rate in hypoxic group was 28.46%(P<0.01) after 24 hour hypoxia.The intracellular content of MDA increased 44.33%(P<0.05).The activities of SOD and GSH decreased by 16.18% and 30.64%,respectively(P<0.05 or P<0.01).The result of RT-PCR showed the activation of Nrf2 with a 1.74 times increase of mRNA expression(P<0.05).Compared with hypoxic group,there was an dose-dependent increase of cell survival rate in H9C2 cells(P<0.01) when treated with areca catechu linn anhydrous ethanol extract during hypoxia.The intracellular activities of SOD and GSH also increased by 14.90% and 28.94%(P<0.05).The relative expression of Nrf2 mRNA decreased significantly by 66%(P<0.05).Conclusion The anhydrous alcohol extract of areca catechu linn has a significant protective effect on H9C2 cells against hypoxia.Its protective mechanism may relate to the improvement of intracellular antioxidant enzyme activity,the reduction of oxidative stress damage and the improvement of cell hypoxic tolerance.
引文
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[12] EARNSHAW W C,MARTINS L M,KAUFMANN S H.Mammalian caspases:structure,activation,substrates,and functions during apoptosis[J].Annu Rev Biochem,1999,68(1):383-424.
[2] PARK E S,KANG D H,KANG J C,et al.Cardioprotective effect of KR-33889,a novel PARP inhibitor,against oxidative stress-induced apoptosis in H9C2 cells and isolated rat hearts[J].Arch Pharm Res,2017,40(5):640-654.
[3] CHAVAN Y V,SINGHAL R S.Separation of polyphenols and arecoline from areca nut (Areca catechu L.) by solvent extraction,its antioxidant activity,and identification of polyphenols[J].J Sci Food Agric,2013,93(10):2580-2589.
[4] LIN E S,LI C C.Evaluation of superoxide radical scavenging capacity and reducing power of areca flower extracts [J].J Med Plant Res,2010,4(10):975-981.
[5] LEE KK,CHO J J,PARK E J,et al.Anti-elastase and anti-hyaluronidase of phenolic substance from Areca catechu as a new anti-ageing agent[J].Int J Cosmet Sci,2001,23(6):341-346.
[6] 靳婷.槟榔提取物抗高原缺氧药效学及其机制研究[D].兰州:兰州大学,2018.
[7] 普义鑫.槟榔多酚提取、纯化及组分分析[D].长沙:中南林业科技大学,2012.
[8] 吴秋生,宋菲,黄玉林,等.大孔树脂分离纯化槟榔花多酚研究[J].广东农业科学,2015,42(23):122-126.
[9] TKACHEV V O,MENSHCHIKOVA E B,ZENKOV N K.Mechanism of the Nrf2/Keap1/ARE signaling system[J].Biochemistry Mosc,2011,76(4):407-422.
[10] ABDO S,ZHANG S L,CHAN J S.Reactive oxygen species and nuclear factor erythroid 2-related factor 2 activation in diabetic nephropathy:A hidden target[J].J Diabetes Metab,2015,6(6):1375-1379.
[11] KANNAN K,JAIN S K.Oxidative stress and apoptosis [J].Pathophysiology,2000,7(3):153-163.
[12] EARNSHAW W C,MARTINS L M,KAUFMANN S H.Mammalian caspases:structure,activation,substrates,and functions during apoptosis[J].Annu Rev Biochem,1999,68(1):383-424.