缺氧诱导慢性肾衰大鼠肾脏线粒体自噬与凋亡的机制及肾衰II方的干预作用

Hypoxia is the pathological key point of renal fibrosis.Latest research has proved that mitophagy induced by hypoxia is related to signal pathway by HIF-1α/BNIP3/Beclin-1.Mitophagy and apoptosis are inseparable. The projet is based on preliminary study on improvement oxygen consumption and down-regulation expressions of HIF-1αin renal cortex and medulla. The study investigates deeply the new mechanism which "Meliorated Renal Faiure Decotion,MRFD "can regulate the expressions of Beclin-1,LC3-Ⅱ, Bcl-2、Bax and Caspase-3 for treatment and prevention of renal fibrosis. It takes CRF rats induced by 5/6 ablation/infarction(A/I) model as the study objects and selects AngII inhibiting agents as the contrast by means of measurement matabolism of oxygen consumption,cell-molecular levels,biochemistry,electronic speculum and pathology. The research proves the scientic hypothesis if MRFD can regulate the signal pathway of mitophagy and apoptosis relationship between hypoxia and mitophagy and apoptosis as a point of penetration and clarifies the new mechanism of MRFD against renal fibrosis.It supplies microscopic basis in cellulla level and new methodology of research on treatment and prevention of renal fibrosis caused by hypoxia in TCM.

缺氧是肾纤维化的关键病理环节，最新研究证实，缺氧诱导肾脏线粒体自噬与HIF-1α/BNIP3/Beclin-1信号通路有关，线粒体自噬与凋亡密不可分。本课题基于我们前期研究肾衰II号方改善肾氧耗效应，下调肾皮质和髓质HIF-1α过表达的基础上，深入研究该方影响肾氧耗调控肾脏线粒体自噬标志性Beclin-1和LC3-Ⅱ以及凋亡标志性Bcl-2、Bax和 Caspase-3表达新靶点防治肾纤维化的作用机制，拟以5/6（A/I）慢性肾衰模型大鼠为研究对象，AngII受体阻滞剂为对照，采用肾氧耗代谢测定、细胞与分子水平、生化、电镜及病理等手段，从缺氧与线粒体自噬和凋亡的相关性为切入点，验证肾衰II号方通过改善肾氧耗调控肾脏线粒体自噬和凋亡信号通路实现抗肾纤维化作用的科学假说，阐明肾衰II号方抗肾纤维化的新机制，为中医药防治缺氧性肾损伤导致肾纤维化理论发展提供细胞层面的微观依据和新的研究思路与方法。

肾内慢性缺氧是肾纤维化的介导因素与关键病理环节，缺氧最易影响线粒体功能，造成线粒体损伤，研究证实，缺氧诱导肾脏线粒体自噬与HIF-1α/BNIP3/Beclin-1信号通路有关，线粒体自噬与凋亡密不可分,线粒体自噬与凋亡异常是其介导肾纤维化进展的重要一环。基于前期研究肾衰II号方改善肾氧耗效应，下调HIF-1α过表达的基础上，拟以5/6(ablation /infarction，A/I)慢性肾衰模型大鼠为研究对象，AngII受体阻滞剂为对照，研究肾衰Ⅱ号方对慢性肾衰肾内缺氧的调控效应和对线粒体自噬与凋亡通路的影响及作用机制。. 本研究以65只SD雄性大鼠按5/6(A/I)法制备慢性肾衰模型，30日后,将造模成功的大鼠随机分为三组：模型组、中药组（肾衰Ⅱ号方）和西药组（氯沙坦钾），每组15只，另取15只大鼠为假手术组。干预60天后，采用生化、肾氧耗代谢测定、细胞与分子水平、电镜及病理等手段，观察各组大鼠凋亡相关因子Bax、Bcl2和Caspase3的表达，自噬相关因子Beclin1和Lc3的表达以及残余肾组织的病理和线粒体形态结构的变化。. 研究发现，肾衰Ⅱ号方可改善5/6(A/I)大鼠肾血流和肾内氧耗，抑制促凋亡蛋白Bax在线粒体的聚积，上调线粒体上抗凋亡蛋白Bcl2的水平，线粒体Bax/Bcl2的比值降低，上调自噬关键蛋白Beclin1的表达及Lc3-Ⅱ/Lc3-Ⅰ的比值，可以减轻肾小球硬化及肾小管间质病理变化，减轻线粒体形态结构的变化。.研究阐明了肾衰Ⅱ号方通过改善慢性肾衰大鼠肾内缺氧影响线粒体自噬与凋亡途径而发挥抗肾纤维化作用的新机制，验证了“脾肾气虚，湿瘀内蕴”的中医病机与“缺氧-线粒体自噬与凋亡途径-肾纤维化”之间的病理特征相关，为中医药防治缺氧性肾损伤导致肾纤维化理论发展提供细胞层面的微观依据和创新的思路与方法，对延缓慢性肾衰向终末期肾病的进展具有重要的临床指导意义。