呼吸链复合体Ⅲ电子漏在电离辐射诱导肺成纤维细胞分化中的作用及其发生机制研究

Myofibroblasts，mainly differentiated from lung fibroblasts, play a pivotal role in the pathogenesis of pulmonary fibrosis.Recently,it was suggested that radiation-induced fibroblast differentiation into myofibroblasts was mediated by reactive oxygen species (ROS). It has been shown that the electron leak from mitochondrial respiratory chain is the primary source of ROS generation in nonphagocytic cells. However, the precise roles of the electron leak in radiation-related lung fibroblasts differentiation have yet to be determined. Sponsored by the Natural Science Foundation of China, our previous studies have shown that the increasing of electron leak from the respiratory chain play an important role in the fibroblast differentiation into myofibroblast induced by ionizing irradiation.Morever, the results of our study indicated that the electron leak may come from complex III of the respiratory chain. That improving of the electron leak may become the new strategy of inhibiting fibroblast differentiation induced by radiation. In this project, by using cellular bioenergetics analysis, blue native-PAGE, RNAi and other technological methods, the mitochondrial dysfunction,such as basal oxygen consumption,glycolysis rates, ATP production, and respiratory capacity induced by radiation will be explored. Also, the the changes of the components in complex Ⅲ and the construuction of supermolecular complex will be stuided. Based on the results of the study, the key role that the electron leak from complex Ⅲof the respiratory chain may contribute to driving fibroblasts differentiation will be elucidated. And the molecular mechanisms responsible for the electron leak in response to radiation will be clarified. Thus new strategies for the prevention and treatment of radiation-induced lung fibrosis will be provided from the experiments.

主要由肺成纤维细胞（LF）分化而来的肌成纤维细胞（MFB）在肺纤维化发生、发展过程中发挥着关键作用。新近发现，电离辐射诱导LF分化依赖于细胞活性氧（ROS）水平，而线粒体电子漏程度是决定细胞ROS水平的关键因素。以电子漏为切入点，探讨电离辐射诱导LF分化机制的相关研究，尚未见报道。基于前一自然基金项目发现呼吸链电子漏在电离辐射诱导LF分化中发挥了关键作用，且电子漏可能主要发生于呼吸链复合体Ⅲ，本项目拟应用细胞能量代谢实时测定、Blue native-page、RNAi等先进技术手段，结合动物和细胞模型，系统研究辐射后LF线粒体功能状态、复合体Ⅲ结构功能及超分子复合物稳定性、构成比的变化等科学问题，旨在明确呼吸链复合体Ⅲ底物端电子漏在电离辐射致LF分化中的重要地位，阐明复合体Ⅲ电子漏发生的生化及分子机制，为探寻放射性肺纤维化的防治新措施提供新思路。

电离辐射诱导肺成纤维细胞（LF）分化为肌成纤维细胞依赖于细胞活性氧（ROS）水平，而线粒体电子漏程度是决定细胞ROS水平的关键因素。本课题以电子漏为切入点，进行了电离辐射诱导LF分化机制的相关研究。研究发现，辐射后细胞内ROS显著升高，ATP含量增加、线粒体O2-•含量显著增加。辐射后细胞呼吸功能增加，电子漏显著增加。同时，辐照导致细胞线粒体膜电位显著降低、结构受损、线粒体动力学失衡。电离辐射后细胞糖酵解增加。NOX4在辐射引起肺成纤维细胞分化过程中的发挥了关键作用，肺成纤维细胞分化与p38MAPK/Akt信号通路有关。进一步的研究证实，电离辐射导致呼吸链复合体I和复合体Ⅲ关键组分表达和功能变化是线粒体发生电子漏，最后诱导肺成纤维细胞分化的重要原因。采用线粒体电子漏抑制剂MitoQ以及中药单体---原青花素均能显著降低线粒体电子漏，防止电离辐射导致呼吸链复合体I和复合体Ⅲ关键组分变化，抑制辐射后细胞内ROS升高和细胞ATP上升，降低线粒体O2-•含量，改善线粒体功能，改善线粒体动力学失衡，纠正细胞糖酵解增加的状态，抑制NOX4/ p38MAPK/Akt信号通路, 阻止电离辐射所诱导的肺成纤维细胞分化。本研究从线粒体呼吸链底物端电子漏这一全新角度研究电离辐射诱导肺成纤维细胞分化、从而促进放射性肺纤维化的机制，研究结果将为放射性肺损伤的防治提供新思路。