染料木素抑制心肌细胞内IL-6表达及其与ROS的关系

Genistein inhibits expression of IL-6 in cultured cardiomyocytes and implication of ROS

目的探讨染料木素(Gen)对体外培养的心肌细胞内炎性细胞因子白介素(IL)-6表达的影响及活性氧(ROS)在此过程中的作用。方法以培养的新生SD大鼠心肌细胞为模型,随机分为对照(Control)组、Ang II组、Ang II+Gen组和Gen组,通过RT-PCR和ELISA检测IL-6 mRNA和蛋白表达水平;ROS敏感的荧光探针二氯荧光素二乙酸(DCF-DA)测定细胞内ROS水平;细胞色素C还原实验测定NADPH氧化酶活性。结果在血管紧张素(Ang)II作用下,心肌细胞中IL-6 mRNA和蛋白表达水平均较对照组显著升高(P<0.05)。在Ang II作用前给予1×10-7mol/L Gen进行干预,IL-6 mRNA水平和蛋白含量均降低(P<0.05),与单独Ang II刺激组比较均有显著性差异(P<0.05)。与对照组比较,Ang II作用下心肌细胞内DCF荧光强度显著增加(P<0.05),NADPH氧化酶活性明显升高,在Ang II作用前给予Gen干预后DCF荧光强度和NADPH氧化酶活性明显下降,与单独Ang II刺激组比较有显著性差异(P<0.05)。结论 Gen可抑制Ang II诱导的心肌细胞内炎性细胞因子IL-6的表达,降低心肌细胞内ROS水平可能是Gen抑制IL-6表达的重要机制之一。

AIM To investigate the effect of genistein （Gen） on the expression of interleukin 6 （ IL-6）, an important inflammatory cytokine, in cultured cardiomyoeytes and the role of reactive oxygen species （ROS） in signal transduction mechanisms. METHODS Cardiomyocytes were isolated and cultured from neonatal Sprague Dawley rats, which were divided into four groups： control group, Ang II group, AngII ＋ Gen group and Gen group. Expression of IL-6 mRNA and protein level were determined by RT-PCR and ELISA. ROS generation in cardiomyocytes was determined by peroxide-specific probe 2＇ ,7＇- dichlorofluorescein diacetate （DCF-DA）. Activity of NADPH oxidase in cardiomyocytes was measured by superoxide dismutase inhibitable cytochrome C reduction assay. RESULTS In cardiomyocytes stimulated with angiotensin II （Ang Ⅱ）, expression of IL-6 mRNA was higher than in untreated cells （P 〈 0. 05）, which was markedly attenuated by pretreatment with 1 × 10-7 mol/L Gen （P 〈0. 05）. Similarly, compared with blank group, IL-6 content in culture medium significantly increased in LPS group （P 〈 0. 05 ）. Pretreatment with Gen decreased IL-6 content induced by Ang II （P 〈 0. 05 ）. Furthermore, AngII increased ROS generation and activity of NADPH oxidase in myocytes in comparison with that in blank control group, which was also attenuated by pretreatment with Gen （ P 〈 0. 05 ）. CONCLUSION Genistein inhibits expression of IL-6 in cultured cardiomyocytes induced by Ang II. ROS is probably an important downstream signaling molecule in the molecular mechanism.