Nox4/ROS在H2S抗心肌纤维化中的作用研究

NADPH oxidase 4 is a key enzymatic source of mitochondrial reactive oxygen species (ROS), which has been authenticated as a pivotal player in the pathogenesis of myocardial fibrosis. Therefore, Nox4/ROS pathway is likely to provide a novel therapeutic strategy for the treatment of myocardial fibrosis. Hydrogen sulfide (H2S) is endogenously generated by cystathionine γ-lyase (CSE) and has been demonstrated to influence a wide range of physiological and pathological processes in cardiovascular system. Activation of cardiac fibroblasts (CFs) is a key event in the progression of myocardial fibrosis. But our primary study showed that H2S attenuated fibrotic response, Nox4 expression, and ROS production in angiotensin II (Ang II )-activated CFs and in ischemic myocardium. The results provided that H2S might prevent against myocardial fibrosis through modulation of Nox4/ROS pathway. In the current study, we sought to measure proliferation and differentiation of CFs, expression of Nox4, production of mitochondrial ROS and so on to identify H2S attenuated fibrotic response through modulation of Nox4/ROS pathway in Ang II-activated CFs and failure myocardium through lentiviral overexpression/siRNA interference technique. These findings will reveal a novel mechanism of the anti-fibrotic activity of H2S and suggested that modulation of CSE/H2S could offer a therapeutic option for treating myocardial fibrosis.

线粒体氧自由基（ROS）是诱导心肌纤维化的关键因素，NADPH氧化酶4（Nox4）是心肌纤维化过程中ROS的主要来源，因此，Nox4/ROS可能成为治疗心肌纤维化的新策略；硫化氢（H2S）在心血管系统中由胱硫醚-γ-裂解酶（CSE）产生并在心血管系统中发挥重要作用。心肌成纤维细胞（CFs）激活是心肌纤维化的关键环节，我们已发现H2S抑制血管紧张素（AngⅡ）诱导的CFs表达Nox4、ROS以及胶原产生；同时H2S抑制缺血心肌中的Nox4表达、ROS产生以及纤维化，表明H2S可能通过抑制Nox4/ROS发挥抗心肌纤维化作用，但尚无直接证据。本项目通过观察在AngⅡ诱导CFs纤维化反应和心力衰竭模型上，结合慢病毒过表达/siRNA干扰技术，观察CFs增殖、分化、Nox4表达、线粒体ROS等，揭示调控Nox4/ROS是H2S抗纤维化的关键环节。本课题将揭示H2S抗心肌纤维化新机制。

背景 .揭示调控Nox4/ROS是H2S抗纤维化的关键环节，为H2S治疗心肌纤维化过程提供理论依据；另外，Nox4还可能作为其它小分子化合物治疗心肌梗死的靶点；.内容.通过冠状动脉结扎造心肌梗塞模型；采用Ang II激活心肌成纤维细胞；检测缺血心肌和Ang II 刺激的心肌成纤维细胞中的纤维化反应。通过观察硫氢化钠NaHS对Nox4、ROS产生、ERK1/2磷酸化水平、HO-1、CSE、α-SMA、CD34、VEGF、MMP-9、胶原（collagen）的作用来阐明H2S对纤维化反应的保护作用及机制；通过药物筛选发现小分子化合物益母草碱(leonurine)具有抑制Nox4的作用，通过益母草碱对心肌梗死大鼠治疗，观察心肌成纤维细胞激活及心肌梗死情况。.结果.给予NaHS抑制Ang II刺激的成纤维细胞中α-SMA、CTGF和I 型胶原的表达而上调HO-1蛋白表达；NaHS 抑制Ang II诱导的成纤维细胞表达Nox4蛋白表达而伴随着细胞内ROS产生减少、抑制ERK1/2磷酸化水平和 CTGF表达；在心肌梗塞模型中，给予NaHS也可缓解心肌组织中的Nox4 表达和纤维化反应；此外，给予NaHS还可抑制早期心肌炎症反应和上调HO-1和CSE蛋白表达；心肌梗死后42天，外源性H2S显著性降低心肌纤维化面积以及胶原1和胶原3含量、MMP-9表达，并且明显改善心功能相关指标；另外，给予外源性H2S还可显著性提高缺血心肌中的新生血管的密度，包括微血管（CD34）、微动脉（α-SMA）。益母草碱具有显著抑制Nox4的表达及活性，发挥Ang II诱导的成纤维细胞内ROS产生、MMP-2/9表达及活性、α-SMA和胶原表达，并且益母草碱具有改善缺血心肌的心功能、Nox4表达及其介导的炎症反应.科学意义.Nox4是H2S抗心肌纤维化的关键环节并且可作为抗心肌纤维化的靶点。