辛伐他汀抑制大鼠心肌细胞TNF-α的表达及其与活性氧的关系

摘要/Abstract

摘要： 目的：探讨辛伐他汀对脂多糖( LPS) 诱导大鼠心肌细胞TNF-α表达的影响及其分子机制。方法：以培养的新生SD 大鼠心肌细胞为实验模型, 应用RT-PCR 和ELISA 检测TNF-αmRNA 和蛋白表达水平, 采用荧光探针二氯荧光素二乙酸(DCF-DA) 测定细胞内活性氧( ROS) 水平, 通过细胞色素C 还原试验测定还原型辅酶Ⅱ( NADPH)氧化酶活性。结果：LPS 组心肌细胞TNF-αmRNA和蛋白表达水平分别为( 0.29 ±0.07) 、( 83.6 ±14.5) pg/mL, 与对照组( 0.07 ±0.02) 、( 22.1 ±7.1) pg/mL 比较显著升高( P <0.01) 。1 μmol/L和10 μmol/L 辛伐他汀干预下TNF-αmRNA 水平为0.18 ±0.05 、0.15 ±0.02, 蛋白含量为( 43.1 ±8.3) 、( 40.1 ±7.3) pg/mL, 与LPS 组比较显著降低( P <0.01) 。在抗氧化剂氮乙酰半胱氨酸和二亚苯基碘金翁预处理后, TNF-α蛋白含量分别为( 39.1 ±10.2) 、( 37.9 ±8.9) pg/mL, 与LPS 组比较均显著降低( P <0.01) 。LPS 组DCF 荧光强度为83 ±15, NADPH 氧化酶活性为( 246 ±43) %, 均明显高于对照组( P <0.01) 。辛伐他汀干预下DCF荧光强度为41 ±8, 氧化酶活性为( 120 ±17) %, 与LPS 组比较均显著下降( P <0.01) 。在外源性H₂O₂ 刺激下, TNF-α 蛋白含量为( 65.0 ±17.2) pg/mL, 与对照组比较显著升高( P <0.01) 。辛伐他汀干预下TNF-α蛋白含量为( 58.8 ±15.8) pg/mL, 与H₂O₂ 组比较变化相近( P >0.05) 。在甲羟戊酸和辛伐他汀共同干预作用下,心肌细胞TNF-αmRNA 和蛋白表达水平、ROS 水平及NADPH 氧化酶活性与辛伐他汀干预组比较均显著升高( P <0.01), 与LPS 组比较变化相近( P >0.05) 。结论：辛伐他汀能够抑制LPS 诱导心肌细胞TNF-α表达的作用, 其机制可能与降低NADPH 氧化酶活性从而减少ROS 的生成有关。

关键词: 他汀类药物, 脂多糖, 炎症细胞因子, 心肌细胞, 活性氧

Abstract: AIM: To investigate the effects of simvastatin (Sim) on the expression of tumor necrosis factor- α( TNF-α) in neonatal rat cardiomyocytes stimulated by lipopolysaccharide ( LPS) and approach the underlying molecular mechanisms.METHODS: The cadiocyte, as experimental model, was isolated and cultured from neonatal Sprague-Dawley rats.The expressions of TNF-αmRNA and protein level were detected by RT-PCR and ELISA.The reactive oxygen species ( ROS) levels in cadiocyte were detected by peroxide specific probe 2 ', 7 '-dichlorofluorescin diacetate (DCF-DA).The relative activity of NADPH oxidase was measured by cytochrome C reduction assay.RESULTS: The TNF-αmRNA of cadiocyte in cardiomyocytes stimulated with LPS, the expressions of TNF-α mRNA and protein level [ ( 0.29 ±0.07), ( 83.6 ± 14.5) pg/mL] were higher than those in control group [ ( 0.07 ±0.02), ( 22.1 ±7.1) pg/mL] ( P <0.01). The levels of TNF-αmRNA and protein in 1 μmol/L Sim +LPS and 10 μmol/L Sim +LPS groups were (0.18 ±0.05), ( 0.15 ±0.02) and ( 43.1 ±8.3), ( 40.1 ±7.3) pg/mL, respectively.Compared with LPS group, which significantly were decreased( P <0.01). The TNF-αprotein contents were ( 39.1 ±10.2), ( 37.9 ±8.9) pg/mL pretreatment with antioxidants Nacetylcysteine and diphenyleneiodonium, compared with LPS group, which significantly were decreased ( P < 0.01).Compared with control group, the DCF-fluorescence intensity ( 83 ±15) and the NADPH oxidase activity( 246 ±43) % in LPS group were significantly increased ( P <0.01), which were decreased by pretreatment with Sim[ ( 41 ±8), ( 120 ±17) %, P < 0.01].Compared with control group, the TNF-αprotein content was increased( 65.0 ±17.2) pg/mL, stimulated by exogenous hydrogen peroxide.The TNF-α protein content was ( 58.8 ±15.8) pg/mL pretreatment with Sim, compared with H₂O₂ group, there was no siginificant difference.Compared with Sim group, the expression of TNF-αmRNA and protein level, ROS level, NADPH oxidase activity were increased pretreatment with mevalonate and Sim.CONCLUSION: Sim probably inhibits the LPS-induced expression of TNF-αin cultured cadiocyte via modulating the activity of NADPH oxidase and subsequent production of ROS.

Key words: statins, lipopolysaccharide, inflammatory cytokines, cadiocyte, reactive oxygen species

中图分类号:

R965.2

尚福军, 王捷频, 郑强荪, 刘雄涛, 薛玉生, 何勇, 张录兴, 刘慧, 王彬, 赵连友. 辛伐他汀抑制大鼠心肌细胞TNF-α的表达及其与活性氧的关系[J]. 中国临床药理学与治疗学, 2009, 14(12): 1379-1385.

SHANG Fu-jun, WANG Jie-pin, ZHENG Qiang-sun, LIU Xiong-tao, XUE Yu-sheng, HE Yong, ZHANG Lu-xing, LIU Hui, WANG Bin, ZHAO Lian-you. Simvastatin inhibit the expression of tumor necrosis factor-αin rat cadiocyte and the relation with reactive oxygen species[J]. Chinese Journal of Clinical Pharmacology and Therapeutics, 2009, 14(12): 1379-1385.

参考文献

[1] Schillaci G, Verdecchia P, Porcellati C, et al.Continuous relation between left ventricular mass and cardiovascular risk in essential hypertension[J].Hypertension, 2000, 35( 2):580-586.
[2] Wilson EM, Diwan A, Spinale FG, et al.Duality of innate stress responses in cardiac injury, repair, and remodeling[J].J Mol Cell Cardiol, 2004, 37( 4):801-811.
[3] Mann DL.Inflammatory mediators and the failing heart: past, present, and the foreseeable future[J].Circ Res, 2002, 91( 11):988-998.
[4] Werner N, Nickenig G, Laufs U.Pleiotropic effects of HMG-CoA reductase inhibitors[J].Basic Res Cardiol, 2002, 97( 3):105-116.
[5] 田建伟, 赵连友, 王士雯, 等.阿伐他汀对幼年大鼠心肌成纤维细胞增殖和胶原合成的影响及其意义[J]. 中华医学杂志, 2003, 83( 2):118-122.
[6] Zhang J, Cheng X, Liao YH, et al.Simvastatin regulates myocardial cytokine expression and improves ventricular remodeling in rats after acute myocardial infarction[J]. Cardiovasc Drugs Ther, 2005, 19( 1):13-21.
[7] Wallace CK, Stetson SJ, Kucuker SA, et al.Simvastatin decreases myocardial tumor necrosis factor alpha content in heart transplant recipients[J].J Heart Lung Transplant, 2005, 24( 1):46-51.
[8] Li JM, Gall NP, Grieve DJ, et al.Activation of NADPH oxidase during progression of cardiac hypertrophy to failure[J].Hypertension, 2002, 40( 4):477-484.
[9] Wright G, Singh IS, Hasday JD, et al.Endotoxin stressresponse in cardiomyocytes:NF-κB activation and tumor necrosis factor-αexpression[J].Am J Physiol Heart Circ Physiol, 2002, 282( 3):H872-879.
[10] 唐惠芳, 吴书林, 邓春玉, 等.辛伐他汀减轻舒张功能不全大鼠心肌线粒体损伤[J].中国临床药理学与治疗学, 2009, 14( 7):780-784.
[11] 吴旭斌, 周胜华, 林英忠, 等.罗格列酮联合阿托伐他汀对高胆固醇血症兔主动脉斑块面积和TNF-α的影响[J].中国临床药理学与治疗学, 2009, 14( 3): 255-260.
[12] 赵连友, 陈永清, 郑强荪, 等.辛伐他汀对自发性高血压大鼠左心室肥厚影响与蛋白激酶B 表达的关系[J].中华医学杂志, 2005, 85( 19):1344-1347.
[13] 王彦珍, 罗建东.活性氧介导内皮素-1 诱导的培养新生大鼠心肌细胞肥大[J].生理学报, 2004, 56( 3): 403-406.
[14] Delbosc S, Cristol JP, Descomps B, et al.Simvastatin prevents angiotensin II-induced cardiac alteration and oxidative stress[J].Hypertension, 2002, 40( 2):142-147.
[15] Wassmann S, Laufs U, Muller K, et al.Cellular antioxidant effects of atorvastatin in vitro and in vivo[J].Arterioscler Thromb Vasc Biol, 2002, 22( 2):300-305.
[16] Touyz RM, Schiffrin EL.Reactive oxygen species in vascular biology:implications in hypertension[J].Histochem Cell Biol, 2004, 122( 4):339-352.
[17] Ndengele MM, Muscoli C, Wang ZQ, et al.Superoxide potentiates NF-kappaB activation and modulates endotoxininduced cytokine production in alveolar macrophages[J]. Shock, 2005, 23( 2):186-193.
[18] Ali MH, Schlidt SA, Chandel NS, et al.Endothelial permeability and IL-6 production during hypoxia:role of ROS in signal transduction[J].Am J Phy siol, 1999, 277( 5 Pt 1):L1057-1065.
[19] Khadour FH, Panas D, Ferdinandy P, et al.Enhanced NO and superoxide generation in dysfunctional hearts from endotoxemic rats[J].Am J Physiol Heart Circ Physiol, 2002, 283( 3):H1108-H1115.
[20] Laufs U, Kilter H, Konkol C, et al.Impact of HMG CoA reductase inhibition on small GTPases in the heart[J]. Cardiovasc Res, 2002, 53( 4):911-920.
[21] Maack C, Kartes T, Kilter H, et al.Oxygen free radical release in human failing myocardium is associated with increased activity of rac1-GTPase and represents a target for statin treatment[J].Circulation, 2003, 108( 13):1567-1574.