N-乙酰半胱氨酸改善高脂高糖联合缺氧/复氧致H9C2心肌细胞损伤的实验研究
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摘要
目的探讨抗氧化剂N-乙酰半胱氨酸(NAC)对高脂高糖联合缺氧/复氧所致H9C2心肌细胞损伤及其对时钟基因BMAL1表达的影响。方法将培养的H9C2细胞分为对照组(N组)、高脂高糖联合缺氧/复氧组(HFHG+H/R组)、高脂高糖联合缺氧/复氧+NAC组(HFHG+H/R+NAC组)。CCK-8法检测细胞活性,流式细胞术检测细胞凋亡,DCFH-DA染色观察活性氧(ROS)水平,JC-1染色检测细胞线粒体膜电位(MMP),Western blot测定BMAL1表达水平。结果与N组比较,HFHG+H/R组细胞活性明显降低(P <0.01),凋亡率与ROS水平明显升高(P <0.01),而BMAL1表达水平显著降低(P <0.01)。经NAC处理后,HFHG+H/R+NAC组以上变化均显著减小,差异有高度统计学意义(P <0.01)。HFHG+H/R组MMP较N组明显降低;经NAC处理后,HFHG+H/R+NAC组MMP明显升高。结论 NAC可能通过抑制心肌细胞氧化应激水平,并促进时钟基因BMAL1表达,从而减小高脂高糖与缺氧/复氧所导致的心肌细胞损伤。
Objective To investigate the effect of antioxidant N-acetylcysteine(NAC) on H9C2 cardiomyocyte injury induced by high-fat and high-glucose combined with hypoxia/reoxygenation and its effect on the expression of clock gene BMAL1. Methods H9C2 cells were divided into control group(N group), high-fat and high-glucose combined with hypoxia/reoxygenation group(HFHG+H/R group), high-fat and high-glucose combined with hypoxia/reoxygenation +NAC group(HFHG+H/R+NAC group). Cell viability was detected by CCK-8 method, cell apoptosis was detected by the flow cytometry, reactive oxygen species(ROS) level was observed by DCFH-DA staining, mitochondrial membrane potential(MMP) was detected by JC-1 staining, and the expression level of BMAL1 was determined by Western blot. Results Compared with N group, the cell viability of HFHG+H/R group was significantly lower(P < 0.01), the apoptosis rate and ROS level were significantly increased(P < 0.01), while the expression level of BMAL1 was significantly reduced(P < 0.01). After NAC treatment, the changes of indicators above in the HFHG+H/R+NAC group were significantly reduced, the differences were highly statistically significant(P < 0.01). MMP in the HFHG+H/R group was significantly lower than that in the N group. After NAC treatment, MMP of HFHG+H/R+NAC group was significantly increased. Conclusion NAC may reduce the oxidative stress level of cardiomyocytes and promote the expression level of clock gene BMAL1, thereby reducing cardiomyocyte damage induced by high-fat and high-glucose combined with hypoxia/reoxygenation.
Objective To investigate the effect of antioxidant N-acetylcysteine(NAC) on H9C2 cardiomyocyte injury induced by high-fat and high-glucose combined with hypoxia/reoxygenation and its effect on the expression of clock gene BMAL1. Methods H9C2 cells were divided into control group(N group), high-fat and high-glucose combined with hypoxia/reoxygenation group(HFHG+H/R group), high-fat and high-glucose combined with hypoxia/reoxygenation +NAC group(HFHG+H/R+NAC group). Cell viability was detected by CCK-8 method, cell apoptosis was detected by the flow cytometry, reactive oxygen species(ROS) level was observed by DCFH-DA staining, mitochondrial membrane potential(MMP) was detected by JC-1 staining, and the expression level of BMAL1 was determined by Western blot. Results Compared with N group, the cell viability of HFHG+H/R group was significantly lower(P < 0.01), the apoptosis rate and ROS level were significantly increased(P < 0.01), while the expression level of BMAL1 was significantly reduced(P < 0.01). After NAC treatment, the changes of indicators above in the HFHG+H/R+NAC group were significantly reduced, the differences were highly statistically significant(P < 0.01). MMP in the HFHG+H/R group was significantly lower than that in the N group. After NAC treatment, MMP of HFHG+H/R+NAC group was significantly increased. Conclusion NAC may reduce the oxidative stress level of cardiomyocytes and promote the expression level of clock gene BMAL1, thereby reducing cardiomyocyte damage induced by high-fat and high-glucose combined with hypoxia/reoxygenation.
引文
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[16]Kondratova AA,Dubrovsky YV,Antoch MP,et al.Circadian clock proteins control adaptation to novel environment and,memory formation[J].Aging(Albany NY),2010,2(5):285-297.
[17]Hemmeryckx B,Hohensinner P,Swinnen M,et al.Antioxidant treatment improves cardiac dysfunction in a murine model of premature ageing[J].J Cardiovasc Pharmacol,2016,15(32):6720-6724.
[18]Koektuerk B,Aksoy M,Horlitz M,et al.Role of diabetes in heart rhythm disorders[J].World J Diabetes,2016,7(3):45-49.
[19]Wu T,Yang L,Jiang J,et al.Chronic glucocorticoid treatment induced circadian clock disorder leads to lipid metabolism and gut microbiota alterations in rats[J].Life Sci,2018,192:173-182.
[20]Zhao R,Xu G,Zhang T,et al.Effects of Lycium barbarum.polysaccharide on type 2 diabetes mellitus rats by regulating biological rhythms[J].Iran J Basic Med Sci,2016,19(9):1024-1030.
[2]Bhattacharya S,Manna P,Gachhui R,et al.D-saccharic acid 1,4-lactone protects diabetic rat kidney by ameliorating hyperglycemia-mediated oxidative stress and renal inflammatory cytokines via NF-κB and PKC signaling[J].Toxicol Appl Pharmacol,2013,267(1):16-29.
[3]Cheng YC,Sheen JM,Hu WL,et al.Polyphenols and Oxidative Stress in Atherosclerosis-Related Ischemic Heart Disease and Stroke[J].Oxid Med Cell Longev,2017,2017(5):1-16.
[4]Peliciari-Garcia RA,Goel M,Aristorenas JA,et al.Altered myocardial metabolic adaptation to increased fatty acid availability in cardiomyocyte-specific CLOCK mutant mice[J].Biochim Biophys Acta,2016,1861(10):1579-1595.
[5]Rakshit K,Thomas AP,Matveyenko AV.Does disruption of circadian rhythms contribute to beta-cell failure in type2 diabetes?[J].Curr Diab Rep,2014,14(4):474.
[6]Lee J,Moulik M,Fang Z,et al.Bmal1 andβ-cell clock are required for adaptation to circadian disruption,and their loss of function leads to oxidative stress-inducedβ-cell failure in mice[J].Mol Cell Biol,2013,33(11):2327-2338.
[7]Lee TI,Bai KJ,Chen YC,et al.Histone deacetylase inhibition of cardiac autophagy in rats on a high-fat diet with low-dose streptozotocin-induced type 2 diabetes mellitus[J].Mol Med Rep,2017,17(1):594-601
[8]Casellini CM,Parson HK,Hodges K,et al.Bariatric Surgery Restores Cardiac and Sudomotor Autonomic C-Fiber Dysfunction towards Normal in Obese Subjects with Type 2Diabetes[J].PLoS One,2016,11(5):e0154211.
[9]Boudina S,Abel ED.Diabetic cardiomyopathy,causes and effects[J].Rev Endocr Metab Disord,2010,11(1):31-39.
[10]Nabeebaccus A,Zhang M,Shah AM.NADPH oxidases and cardiac remodelling[J].Heart Fail Rev,2011,16(1):5-12.
[11]崔雅忠,杨丹,张倩,等.STZ诱导的2型与1型糖尿病大鼠模型的对比研究[J].医学研究杂志,2018,47(5):36-38.
[12]Chowdhry MF,Vohra HA,Gali觡anes M.Diabetes increases apoptosis and necrosis in both ischemic and nonischemic human myocardium:role of caspases and polyadenosine diphosphate-ribose polymerase[J].J Thorac Cardiovasc Surg,2007,134(1):124-131.
[13]Ricci C,Jong CJ,Schaffer SW.Proapoptotic and antiapoptotic effects of hyperglycemia:role of insulin signaling[J].Can J Physiol Pharmacol,2008,86(4):166-172.
[14]Buja LM,Entman ML.Modes of Myocardial Cell Injury and Cell Death in Ischemic Heart Disease[J].Circulation,1998,98(14):1355-1357.
[15]Tungjai M,Phathakanon N,Rithidech KN.Effects of Medical Diagnostic Low-dose X Rays on Human Lymphocytes:Mitochondrial Membrane Potential,Apoptosis and Cell Cycle[J].Health Phys,2017,112(5):458-464.
[16]Kondratova AA,Dubrovsky YV,Antoch MP,et al.Circadian clock proteins control adaptation to novel environment and,memory formation[J].Aging(Albany NY),2010,2(5):285-297.
[17]Hemmeryckx B,Hohensinner P,Swinnen M,et al.Antioxidant treatment improves cardiac dysfunction in a murine model of premature ageing[J].J Cardiovasc Pharmacol,2016,15(32):6720-6724.
[18]Koektuerk B,Aksoy M,Horlitz M,et al.Role of diabetes in heart rhythm disorders[J].World J Diabetes,2016,7(3):45-49.
[19]Wu T,Yang L,Jiang J,et al.Chronic glucocorticoid treatment induced circadian clock disorder leads to lipid metabolism and gut microbiota alterations in rats[J].Life Sci,2018,192:173-182.
[20]Zhao R,Xu G,Zhang T,et al.Effects of Lycium barbarum.polysaccharide on type 2 diabetes mellitus rats by regulating biological rhythms[J].Iran J Basic Med Sci,2016,19(9):1024-1030.