黄芪甲苷调控Nrf2/Bach1/HO-1信号通路抑制缺氧复氧诱导的H9c2心肌细胞损伤
|
摘要
研究黄芪甲苷(astragalosideⅣ,AS-Ⅳ)对缺氧复氧诱导的H9c2心肌细胞损伤的保护作用及其机制。将H9c2心肌细胞缺氧培养12 h再复氧培养8 h复制心肌细胞缺氧复氧损伤(H/R)模型,AS-Ⅳ干预后检测活性氧(ROS)的产生,细胞活力,细胞上清SOD,MDA,IL-6,TNF-α等指标水平,以评估AS-Ⅳ的干预效应;进一步通过蛋白印记试验观察AS-Ⅳ对Nrf2,Bach1,HO-1蛋白表达的影响;最后通过siRNA方法敲低HO-1基因表达观察其对AS-Ⅳ干预效应的逆转作用。结果显示,与H/R模型组比较,H/R+AS-Ⅳ组细胞活力显著提高(P<0.01),细胞内ROS显著减少(P<0.01),细胞上清MDA,hs-CRP,TNF-α含量显著减少(P<0.01),SOD含量显著增加(P<0.01);细胞HO-1蛋白表达显著增加(P<0.01),细胞核蛋白Nrf2表达显著增多(P<0.01),细胞核蛋白Bach1表达显著减少(P<0.01);预先采用脂质体转染HO-1 siRNA至H9c2细胞,敲低HO-1的表达后,可部分逆转AS-Ⅳ的上述效应。该研究结果提示,AS-Ⅳ对H9c2心肌细胞缺氧/复氧损伤具有显著的保护作用,其机制与调控Nrf2/Bach1/HO-1信号通路有关。
Astragaloside Ⅳ(AS-Ⅳ) has protective effects against ischemia-reperfusion injury(IRI), but its mechanism of action has not yet been determined. This study aims to investigate the protective effects and mechanism of AS-Ⅳ on H9c2 cardiomyocyte injury induced by hypoxia-reoxygenation(H/R). The H/R model of myocardial cells was established by hypoxic culture for 12 hours and then reoxygenation culture for 8 hours. After AS-Ⅳ treatment, cell viability, the reactive oxygen species(ROS) levels, as well as the content or activity of superoxide dismutase(SOD), malondialdehyde(MDA), interleukin 6(IL-6), and tumor necrosis factor alpha(TNF-α), were measured to evaluate the effect of AS-Ⅳ treatment. The effect of AS-Ⅳ on HO-1 protein expression and nuclear Nrf2 and Bach1 protein expression was determined by Western blot. Finally, siRNA was used to knock down HO-1 gene expression to observe its reversal effect on AS-Ⅳ intervention. The results showed that as compared with the H/R model group, the cell viability was significantly increased(P<0.01), ROS level in the cells, MDA, hs-CRP and TNF-α in cell supernatant and nuclear protein Bach1 expression in the cells were significantly decreased(P<0.01), while SOD content, HO-1 protein expression in cells and expression of nuclear protein Nrf2 were significantly increased(P<0.01) in H/R+AS-Ⅳ group. However, pre-transfection of HO-1 siRNA into H9c2 cells by liposome could partly reverse the above effects of AS-Ⅳ after knocking down the expression of HO-1. This study suggests that AS-Ⅳ has significant protective effect on H/R injury of H9c2 cardiomyocytes, and Nrf2/Bach1/HO-1 signaling pathway may be a key signaling pathway for the effect.
Astragaloside Ⅳ(AS-Ⅳ) has protective effects against ischemia-reperfusion injury(IRI), but its mechanism of action has not yet been determined. This study aims to investigate the protective effects and mechanism of AS-Ⅳ on H9c2 cardiomyocyte injury induced by hypoxia-reoxygenation(H/R). The H/R model of myocardial cells was established by hypoxic culture for 12 hours and then reoxygenation culture for 8 hours. After AS-Ⅳ treatment, cell viability, the reactive oxygen species(ROS) levels, as well as the content or activity of superoxide dismutase(SOD), malondialdehyde(MDA), interleukin 6(IL-6), and tumor necrosis factor alpha(TNF-α), were measured to evaluate the effect of AS-Ⅳ treatment. The effect of AS-Ⅳ on HO-1 protein expression and nuclear Nrf2 and Bach1 protein expression was determined by Western blot. Finally, siRNA was used to knock down HO-1 gene expression to observe its reversal effect on AS-Ⅳ intervention. The results showed that as compared with the H/R model group, the cell viability was significantly increased(P<0.01), ROS level in the cells, MDA, hs-CRP and TNF-α in cell supernatant and nuclear protein Bach1 expression in the cells were significantly decreased(P<0.01), while SOD content, HO-1 protein expression in cells and expression of nuclear protein Nrf2 were significantly increased(P<0.01) in H/R+AS-Ⅳ group. However, pre-transfection of HO-1 siRNA into H9c2 cells by liposome could partly reverse the above effects of AS-Ⅳ after knocking down the expression of HO-1. This study suggests that AS-Ⅳ has significant protective effect on H/R injury of H9c2 cardiomyocytes, and Nrf2/Bach1/HO-1 signaling pathway may be a key signaling pathway for the effect.
引文
[1] 曹玉冰.黄芪甲苷的药理作用及其机制的研究进展[J].现代药物与临床,2017,32(5):954.
[2] 王时光,徐雁,陈晓虎.芪甲苷对缺氧/复氧损伤H9c2心肌细胞的影响[J].中药药理与临床,2014,30(3):45.
[3] Zheng Q,Zhu J Z,Bao X Y,et al.A preclinical systematic review and Meta-analysis of astragaloside Ⅳ for myocardial ischemia/reperfusion injury[J].Front Physiol,2018,9:795.
[4] 马可可,鞠营辉,陈清青,等.黄芪甲苷对2型糖尿病肾病大鼠肾组织PI3K/Akt/FoxO1信号调控的影响[J].中国实验方剂学杂志,2019,25(2):74.
[5] 周芹.黄芪甲苷抗凋亡作用机制的研究进展[J].中国现代应用药学,2017,34(5):783.
[6] Lu M,Tang F,Zhang J,et al.Astragaloside Ⅳ attenuates injury caused by myocardial ischemia/reperfusion in rats via regulation of toll-like receptor 4/nuclear factor-κB signaling pathway[J].Phytother Res,2015,29(4):599.
[7] Huang H,Lai S,Wan Q,et al.Astragaloside Ⅳ protects cardiomyocytes from anoxia/reoxygenation injury by upregulating the expression of Hes1 protein[J].Can J Physiol Pharmacol,2016,94(5):542.
[8] 居来提·艾买提,阿布都沙拉木·阿布都热衣木,兰怡.黄芪甲苷对小鼠缺血再灌注心肌细胞自噬及炎症反应的影响[J].中国临床药理学杂志,2018,34(17):2097.
[9] Singh N,Ahmad Z,Baid N,et al.Host heme oxygenase-1:friend or foe in tackling pathogens?[J].IUBMB Life,2018,70(9):869.
[10] Otterbein L E,Foresti R,Motterlini R.Heme oxygenase-1 and carbon monoxide in the heart:the balancing act between danger signaling and pro-survival[J].Circ Res,2016,118(12):1940.
[11] Ndisang J F.Synergistic interaction between heme oxygenase (HO) and nuclear-factor E2-related factor-2 (Nrf2) against oxidative stress in cardiovascular related diseases[J].Curr Pharm Des,2017,23(10):1465.
[12] Omura S,Suzuki H,Toyofuku M,et al.Effects of genetic ablation of bach1 upon smooth muscle cell proliferation and atherosclerosis after cuff injury[J].Genes Cells,2005,10(3):277.
[13] Ibáňez B,Heusch G,Ovize M,et al.Evolving therapies for myocardial ischemia/reperfusion injury[J].J Am Coll Cardiol,2015,65(14):1454.
[14] Cadenas S.ROS and redox signaling in myocardial ischemia-reperfusion injury and cardioprotection [J].Free Radic Biol Med,2018,117:76.
[15] Maulik N,Sharma H S,Das D K.Induction of the heme oxygenase gene-expression during the reperfusion of ischemic rat myocardium[J].J Mol Cell Cardiol,1996,28:1261.
[16] Yet S F,Tian R,Layne M D,et al.Cardiac-specific expression of heme oxygenase-1 protects against ischemia and reperfusion injury in transgenic mice[J].Circ Res,2001,89:168.
[17] Hinkel R,Lange P,Petersen B,et al.Heme oxygenase-1 gene therapy provides cardioprotection via control of post-ischemic inflammation:an experimental study in a pre-clinical pig model[J].J Am Coll Cardiol,2015,66:154.
[18] Liu X,Simpson J A,Brunt K R,et al.Preemptive heme oxygenase-1 gene delivery reveals reduced mortality and preservation of left ventricular function 1 yr after acute myocardial infarction[J].Am J Physiol Heart Circ Physiol,2007,293:H48.
[19] Liu X,Wei J,Peng D H,et al.Absence of heme oxygenase-1 exacerbates myocardial ischemia/reperfusion injury in diabetic mice[J].Diabetes,2005,54:778.
[20] Xue M,Momiji H,Rabbani N,et al.Frequency modulated translocational oscillations of Nrf2 mediate the antioxidant response element cytoprotective transcriptional response[J].Antioxid Redox Signal,2015,23(7):613.
[21] Tanaka Y,Maher J M,Chen C,et al.Hepatic ischemia-reperfusion induces renal heme oxygenase-1 via NF-E2-related factor 2 in rats and mice[J].Mol Pharmacol,2007,71(3):817.
[22] Baird L,Dinkova-Kostova A T.The cytoprotective role of the Keap1-Nrf2 pathway[J].Arch Toxicol,2011,85(4):241.
[2] 王时光,徐雁,陈晓虎.芪甲苷对缺氧/复氧损伤H9c2心肌细胞的影响[J].中药药理与临床,2014,30(3):45.
[3] Zheng Q,Zhu J Z,Bao X Y,et al.A preclinical systematic review and Meta-analysis of astragaloside Ⅳ for myocardial ischemia/reperfusion injury[J].Front Physiol,2018,9:795.
[4] 马可可,鞠营辉,陈清青,等.黄芪甲苷对2型糖尿病肾病大鼠肾组织PI3K/Akt/FoxO1信号调控的影响[J].中国实验方剂学杂志,2019,25(2):74.
[5] 周芹.黄芪甲苷抗凋亡作用机制的研究进展[J].中国现代应用药学,2017,34(5):783.
[6] Lu M,Tang F,Zhang J,et al.Astragaloside Ⅳ attenuates injury caused by myocardial ischemia/reperfusion in rats via regulation of toll-like receptor 4/nuclear factor-κB signaling pathway[J].Phytother Res,2015,29(4):599.
[7] Huang H,Lai S,Wan Q,et al.Astragaloside Ⅳ protects cardiomyocytes from anoxia/reoxygenation injury by upregulating the expression of Hes1 protein[J].Can J Physiol Pharmacol,2016,94(5):542.
[8] 居来提·艾买提,阿布都沙拉木·阿布都热衣木,兰怡.黄芪甲苷对小鼠缺血再灌注心肌细胞自噬及炎症反应的影响[J].中国临床药理学杂志,2018,34(17):2097.
[9] Singh N,Ahmad Z,Baid N,et al.Host heme oxygenase-1:friend or foe in tackling pathogens?[J].IUBMB Life,2018,70(9):869.
[10] Otterbein L E,Foresti R,Motterlini R.Heme oxygenase-1 and carbon monoxide in the heart:the balancing act between danger signaling and pro-survival[J].Circ Res,2016,118(12):1940.
[11] Ndisang J F.Synergistic interaction between heme oxygenase (HO) and nuclear-factor E2-related factor-2 (Nrf2) against oxidative stress in cardiovascular related diseases[J].Curr Pharm Des,2017,23(10):1465.
[12] Omura S,Suzuki H,Toyofuku M,et al.Effects of genetic ablation of bach1 upon smooth muscle cell proliferation and atherosclerosis after cuff injury[J].Genes Cells,2005,10(3):277.
[13] Ibáňez B,Heusch G,Ovize M,et al.Evolving therapies for myocardial ischemia/reperfusion injury[J].J Am Coll Cardiol,2015,65(14):1454.
[14] Cadenas S.ROS and redox signaling in myocardial ischemia-reperfusion injury and cardioprotection [J].Free Radic Biol Med,2018,117:76.
[15] Maulik N,Sharma H S,Das D K.Induction of the heme oxygenase gene-expression during the reperfusion of ischemic rat myocardium[J].J Mol Cell Cardiol,1996,28:1261.
[16] Yet S F,Tian R,Layne M D,et al.Cardiac-specific expression of heme oxygenase-1 protects against ischemia and reperfusion injury in transgenic mice[J].Circ Res,2001,89:168.
[17] Hinkel R,Lange P,Petersen B,et al.Heme oxygenase-1 gene therapy provides cardioprotection via control of post-ischemic inflammation:an experimental study in a pre-clinical pig model[J].J Am Coll Cardiol,2015,66:154.
[18] Liu X,Simpson J A,Brunt K R,et al.Preemptive heme oxygenase-1 gene delivery reveals reduced mortality and preservation of left ventricular function 1 yr after acute myocardial infarction[J].Am J Physiol Heart Circ Physiol,2007,293:H48.
[19] Liu X,Wei J,Peng D H,et al.Absence of heme oxygenase-1 exacerbates myocardial ischemia/reperfusion injury in diabetic mice[J].Diabetes,2005,54:778.
[20] Xue M,Momiji H,Rabbani N,et al.Frequency modulated translocational oscillations of Nrf2 mediate the antioxidant response element cytoprotective transcriptional response[J].Antioxid Redox Signal,2015,23(7):613.
[21] Tanaka Y,Maher J M,Chen C,et al.Hepatic ischemia-reperfusion induces renal heme oxygenase-1 via NF-E2-related factor 2 in rats and mice[J].Mol Pharmacol,2007,71(3):817.
[22] Baird L,Dinkova-Kostova A T.The cytoprotective role of the Keap1-Nrf2 pathway[J].Arch Toxicol,2011,85(4):241.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |