线粒体异常可打破mitophagy和凋亡的平衡，诱发疾病。我们发现抑制蛛网膜下腔出血(subarachnoid hemorrhage，SAH)早期脑损伤（early brain injury，EBI）阶段的p38活性通过DJ-1保护线粒体，上调自噬，抑制细胞凋亡。故推测“SAH后调控p38/DJ-1介导mitophagy对线粒体的保护途径可能成为SAH防治的新靶点”。本课题拟：1）探讨p38/DJ-1通路和线粒体的动态变化，及p38和DJ-1的互作；2）观察p38/DJ-1通路对线粒体功能、mitophagy及凋亡的影响；3）分析mitophagy在EBI中的作用，筛选调控mitophagy和凋亡转换的关键蛋白；4）明确以干预p38/DJ-1/mitophagy途径为靶点的疗效，最终阐明SAH后EBI时期p38/DJ-1信号通路对mitophagy调控的分子机制，为SAH的治疗提供新思路。

Recent studies showed that mitochondrial dysfunction might destroy the imbalance of mitophagy and apoptosis, which may contribute to the pathogenesis. Therefore, targeting mitochondria has long been regarded as a potential and reasonable therapeutic method, which has been widely used in therapy many central nerves system disease. In our previous works, we found that inhibition of p38 expression might maintain normal mitochondrial funtion by incearsing autophagy and inhibiting apoptosis in early brain injury (EBI) after subarachnoid hemorrhage (SAH). So, we hypothesized that modulation of the p38/DJ-1 mediated mitophagy signaling pathway would protect mitochondria integrity, which may be a new insight in SAH therapy..Therefore, some studies should be done to varify this propose: 1) to elucidate the dynamic states of p38, DJ-1, and mitochondrial funcion and shape in EBI stage after SAH by using cell biology technologies, meanwhile, identification the interaction of p38 and DJ-1 by using coimmunoprecipitation and mass spectrometric technique; 2) to observe the effect of p38/DJ-1 signaling pathway on mitochondrial function, mitophagy, and apoptosis by using RNAi and gene knockout techniques; 3) to clerify the role of mitophagy in EBI development, so that some key proteins, which modulate the open and/or close eihter mitophagy or apoptosis pathway, can be scrrened by using molecular-cellbiologic techinology; 4) to identify the therapeutic effect by targeting interferring p38/DJ-1/mitophagy pathway. .The aim of these researches was to elucidate the interation and roles of p38 and DJ-1 signaling pathway, by which the molecular signlaling mechamisms have been uncover to understand how p38/DJ-1 regulates mitophagy. These findings may be helpful for us by providing new insights into prevention and therapy SAH.

既往研究认为阻断SAH早期脑损伤的发展可能是行之有效的治疗方法之一。大脑是需能较多的组织，线粒体为细胞提供了主要的能量，此外还其介导了细胞死亡通路，包括凋亡和自噬等。本研究针对以p38/DJ-1通路介导的mitophagy开展了以下研究：（1）p38和DJ-1在SAH发生后的3h表达即升高，且持续至72h。利用免疫共沉淀技术发现p38可以结合DJ-1，提示二者可以互作。进一步研究发现SAH后DJ-1主要定位至线粒体，利用p38抑制剂处理后发现p-p38的表达活性受抑制后，DJ-1的线粒体定位也减少，提示SAH后p38作用于DJ-1后，通过DJ-1的线粒体转位发挥生物学活性，而抑制p38表达可保护细胞免受SAH后的损伤。（2）由于SAH后DJ-1靶向线粒体，故线粒体可能在SAH后受到损伤。本项目发现SAH后线粒体膜电位发生去极化，细胞内ROS生产增多，提示线粒体受损。同时我们发现SAH后线粒体的再生失调，线粒体分裂能力显著提高，线粒体拷贝数的增加进一步证明了细胞中线粒体数量在SAH后增多，但细胞中线粒体形态却由正常的棒状变为弥散装分布，提示SAH后出现的这种线粒体形态与功能异常极有可能作为SAH治疗的潜在靶点。我们利用SB203580发现抑制p38的表达可保护SAH后线粒体功能和形态。（3）SAH后线粒体膜电位去极化、ROS升高及线粒体形态的变化均可激活细胞自噬途径以清除受损的线粒体。然而过度自噬后会激活细胞凋亡途径诱导细胞死亡，故自噬具有双重作用。研究发现SAH后线粒体自噬途径过度激活，而SB203580可抑制细胞的过度自噬发生，进而下调Bcl-2/Bax在线粒体上形成的异源二聚体，进而降低凋亡的发生。（4）利用p38抑制剂在动物模型上进行了初步的治疗实验，发现抑制p38的表达可在SAH后的早期脑损伤阶段通过保护线粒体途径降低细胞损伤，提示p38抑制可能为潜在的SAH治疗药物。这些结果初步揭示了p38和DJ-1干预SAH发生、发展机制，为后续深入开展靶点线粒体的p38/DJ-1通路的SAH治疗机制的研究奠定了重要的理论基础。