黄芪总苷对H_2O_2诱导的衰老心肌细胞氧化应激及PI3K/AKT通路的影响
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摘要
【目的】观察黄芪总苷对H_2O_2诱导的衰老心肌细胞氧化应激及磷脂酰肌醇-3-激酶/蛋白激酶B(PI3K/AKT)通路的影响,探讨黄芪总苷对心肌细胞的保护作用。【方法】通过H_2O_2诱导大鼠心肌H9C2细胞,建立衰老心肌细胞模型。将造模后的细胞随机分成模型对照组,黄芪总苷低、中、高剂量组(剂量分别为10、20、40 mg/L),另设空白对照组。黄芪总苷低、中、高剂量组细胞分别给予对应质量浓度处理48 h,空白对照组和模型对照组不加黄芪总苷处理。采用四甲基偶氮唑盐(MTT)法检测细胞增殖能力,羟胺法检测超氧化物歧化酶(SOD)活性,硫代巴比妥酸(TBA)检测丙二醛(MDA)含量,化学荧光法检测活性氧(ROS)含量,蛋白免疫印迹(Western blot)法检测PI3K/AKT通路蛋白PI3K、AKT磷酸化水平。【结果】与空白对照组比较,模型对照组细胞存活率、SOD活性、p-PI3K/PI3K比值和p-AKT/AKT比值显著降低,MDA、ROS含量显著升高(P<0.05);黄芪总苷低、中、高剂量组对上述指标有均显著改善作用,并呈剂量依赖性。【结论】黄芪总苷可减缓H_2O_2诱导的心肌细胞衰老,其机制可能与减轻氧化应激反应、激活PI3K/AKT通路有关。
Objective To observe the effects of astragalosides on oxidative stress and phosphatidylinositol 3-kinase(PI3K)/protein kinase B(AKT)signaling pathway in H2O2-induced aging cardiomyocytes, and to explore itsprotective effects on cardiomyocytes. Methods The aging rat cardiomyocyte H9C2 model was established by H2O2 induction. The modeled cells were randomly divided into model group, and low-, middle-and high-doseastragalosides groups(at the dosage of 10,20,40 mg/L,respectively). Meanwhile,blank control group wasalso set up. The H9C2 cells in the low-,middle-and high-dose astragalosides groups were treated withcorresponding mass concentrations of astragalosides for 48 h,while the blank control group and the model grouphad no astragalosides treatment. The cell proliferative viability was tested by methyl thiazolyltetrazolium(MTT)method. The superoxide dismutase(SOD)activity was measured by hydroxylamine method, malondialdehyde(MDA) content was detected with thiobarbituric acid(TBA)method,and reactive oxygen species(ROS)contentwas examined by chemiluminescence analysis. The phosphorylated levels of PI3 K and AKT proteins in PI3K/AKTsignaling pathway were detected with Western blotting method. Results Compared with the blank control group,the cell survival rate,SOD activity,p-PI3K/PI3 K ratio,and p-AKT/AKT ratio were decreased,and MDA and ROS contents were increased in the model group(P < 0.05). The low-,middle-and high-dose astragalosideshad significant effects on improving the above indicators, and the effects were dose-dependent. Conclusion Astragalosides have certain effects on delaying the aging of myocardial cells induced by H2O2,and the mechanismmay be related with alleviating oxidative stress and activating the PI3K/AKT pathway.
Objective To observe the effects of astragalosides on oxidative stress and phosphatidylinositol 3-kinase(PI3K)/protein kinase B(AKT)signaling pathway in H2O2-induced aging cardiomyocytes, and to explore itsprotective effects on cardiomyocytes. Methods The aging rat cardiomyocyte H9C2 model was established by H2O2 induction. The modeled cells were randomly divided into model group, and low-, middle-and high-doseastragalosides groups(at the dosage of 10,20,40 mg/L,respectively). Meanwhile,blank control group wasalso set up. The H9C2 cells in the low-,middle-and high-dose astragalosides groups were treated withcorresponding mass concentrations of astragalosides for 48 h,while the blank control group and the model grouphad no astragalosides treatment. The cell proliferative viability was tested by methyl thiazolyltetrazolium(MTT)method. The superoxide dismutase(SOD)activity was measured by hydroxylamine method, malondialdehyde(MDA) content was detected with thiobarbituric acid(TBA)method,and reactive oxygen species(ROS)contentwas examined by chemiluminescence analysis. The phosphorylated levels of PI3 K and AKT proteins in PI3K/AKTsignaling pathway were detected with Western blotting method. Results Compared with the blank control group,the cell survival rate,SOD activity,p-PI3K/PI3 K ratio,and p-AKT/AKT ratio were decreased,and MDA and ROS contents were increased in the model group(P < 0.05). The low-,middle-and high-dose astragalosideshad significant effects on improving the above indicators, and the effects were dose-dependent. Conclusion Astragalosides have certain effects on delaying the aging of myocardial cells induced by H2O2,and the mechanismmay be related with alleviating oxidative stress and activating the PI3K/AKT pathway.
引文
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[2]夏世金.前言——重视炎性衰老的研究[J].实用老年医学,2014,28(2):91.
[3]张磊,聂磊,王唯红.黄芪注射液色谱指纹图谱与抗氧化作用的相关分析[J].中药材,2009,32(11):1757.
[4]裴志萍,牛文革,柴静波,等.黄芪预处理对大鼠肠系膜缺血再灌注损伤后氧化应激和炎症反应的抑制作用[J].中国中医急症,2016,25(6):967.
[5]刘晋华,陈静然,尤光甫.黄芪对心肌保护作用的药理学研究进展[J].中成药,2002,24(8):623.
[6] Harman D. The free radical theory of aging[J]. Antioxid Redox Signal,2003,5(5):557.
[7]张欣文,徐思红,厉曙光.黑腹果蝇体内SOD活力和MDA含量随龄变化趋势[J].中国食品卫生杂志,2000,12(2):3.
[8]王春花,刘克明,张明月,等. SOD基因表达与衰老的相关性[J].中国公共卫生,2004,20(8):953.
[9]李振洁,彭丽倩,高爱莉,等.枸杞多糖对紫外线致人皮肤成纤维细胞脂质损伤的防御作用[J].中国激光医学杂志,2017,26(3):127.
[10] Latunde-Dada G O. Ferroptosis:Role of lipid peroxidation,iron and ferritinophagy[J]. Biochim Biophys Acta,2017,1861(8):1893.
[11] Miyaguchi C,Muranaka S,Kanno T,et al. 17 beta-estradiol suppresses ROS-induced apoptosis of CHO cells through inhibition of lipid peroxidation-coupled membrane permeability transition[J]. Physiol Chem Phys Med NMR, 2004, 36(1):21.
[12]赵宏,刘国良,丁丽颖,等.酰基化ghrelin通过PI3K/Akt通路抑制高糖诱导的内皮细胞凋亡[J].中国糖尿病杂志,2009,17(3):225.
[13] Kobayashi N, Mita S, Yoshida K, et al. Celiprolol activates eNOS through the PI3K-Akt pathway and inhibits VCAM-1 via NF-kappaB induced by oxidative stress[J]. Hypertension,2003,42(5):1004.
[14] Qi S, Xin Y, Guo Y, et al. Ampelopsin reduces endotoxic inflammation via repressing ROS-mediated activation of PI3K/Akt/NF-κB signaling pathways[J]. Int Immunopharmacol,2012,12(1):278.