新型水溶性辅酶Q10对帕金森病线粒体动态平衡和再生的作用

Parkinson's disease （PD）is one of the most common neurodegenerative diseases. The mechanisms responsible for PD are not fully understood. Inhibition of mitochondrial electron transport chain complex I is a cause for the development of PD. Traditional lipid-soluble co-enzyme Q10, although efficacious in reducing PD neuronal death, requires a high dose due to it low distribution in the cells and mitochondria. Comparing to lipid-soluble Q10, the distribution of water-soluble Q10 in the cells and mitochondria increases 60 and 20 times, respectively. Our preliminary results have shown that water-soluble Q10 reduced rotenone-induced cell death in vitro, prevented mitochondrial release of apoptosis-inducing factor, suppressed mitochondrial dynamic imbalance towards fission, and promoted mitochondrial biogenesis, a novel therapeutic target for PD and many other mitochondria related disorders. The objective of the study is to elucidate the roles of mitochondrial dynamics and mitochondrial biogenesis in PD development and water-soluble Q10's neuroprotective mechanism. We anticipate this study will: 1.Establish rotenone-induced PD model in the rat; 2. Use histological methods and immunohistochemistry to evaluate the neuroprotective effects of water-soluble Q10 in PD animals; 3. Employ immunohistochemistry, Western blotting, mitochondrial imaging, and mitochondrial fission inhibitor to study the role of mitochondrial fission in PD and water-soluble Q10 treatment; 4. Elucidate the molecular mechanisms by which the water-soluble Q10 activates mitochondrial biogenesis in PD. The last aim will be achieved by measuring mitochondrial biogenesis related cell signaling pathways and by employing specific inhibitors and siRNA technique to block certain pathway. We hope the above studies will elucidate the role of mitochondrial dynamic imbalance and mitochondrial biogenesis in mediating the development of PD and the neuroprotection of water-soluble Q10. The study will lay basis for subsequent clinical treatment trial in PD. Considering the broad involvement of mitochondrial dysfunction in many clinical disorders, such as cardiac infarction, cerebral stroke, diabetes, myopathies and other mitochondria-related diseases, the application of this research will be broad.

线粒体电子传递链复合酶I功能障碍与帕金森病（PD）关系密切。辅酶Q10有效保护线粒体功能。但传统脂溶性辅酶Q10在细胞和线粒体分布低。新型水溶性辅酶Q10分别在细胞和线粒体内分布提高60和20倍。前期结果显示水溶性辅酶Q10减轻鱼藤酮造成的细胞损伤，抑制促凋亡因子释放和病理分裂，并促进线粒体再生。本课题进一步应用细胞和动物PD模型阐明水溶性辅酶Q10对PD的保护机制。本课题拟完成：1.建立大鼠PD模型；2.应用病理学、免疫组化技术观察辅酶Q10对PD的疗效；3.应用免疫组化、蛋白电泳、线粒体图像分析和特异性抑制剂阐明线粒体分裂在PD发病和辅酶Q10治疗中的作用；4.测定线粒体再生相关调控因子、使用针对激活线粒体再生信号通路的特异性抑制剂和小分子RNA干扰技术确立辅酶Q10激活线粒体再生的分子机制。通过上述研究，力图阐明线粒体分裂和再生在PD发病和辅酶Q10治疗中所发挥的作用。

帕金森病（Parkinson’s disease, PD）是神经系统常见的退行性疾病。发病年龄多在55岁以上，发病率随年龄增加而上升。尽管已知病因为黑质多巴胺能神经元经黑质-纹状体投射纤维释放到基底节的多巴胺减少，其发病分子机制尚未完全阐明。且目前的治疗主要是针对症状的治疗，缺乏针对发病机制的有效治疗。为此，针对由于线粒体功能障碍、线粒体融合裂解动态失衡导致的发病机制开展了一系列的研究。研究结果如下：（1）线粒体复合物I抑制剂鱼藤酮诱导小鼠海马HT22细胞损伤。治疗组水溶性辅酶Q10给予3小时的预处理。鱼藤酮作用24h后Western blotting检测Cytochrome c、Caspase-9及凋亡诱导因子AIF的蛋白表达。鱼藤酮诱导大于50%的细胞死亡，升高ROS水平，AIF的核转移及线粒体膜电位的降低，但未激活Cytochrome c和Caspase-9。水溶性CoQ10预处理改善细胞生存率、降低ROS水平、稳定线粒体膜电位及抑制AIF的核转移。（2）通过鱼藤酮构建HT22细胞PD模型，鱼藤酮提高了线粒体分裂标志物Drp1和Fis1的蛋白表达，通过线粒体染色和形态学分析亦证明其导致了线粒体片段化增加。水溶性CoQ10通过降低Drp1和Fis1蛋白表达降低至鱼藤酮处理前水平，以及减轻鱼藤酮诱导的线粒体片段化而改善线粒体动态失衡。（3）鱼藤酮处理24h后的PC12细胞存活率显著降低并与剂量呈负相关，CoQ10可改善鱼藤酮处理诱导的PC12细胞存活率降低，鱼藤酮引起ROS水平升高，诱导PC12细胞线粒体膜电位降低，导致线粒体片段化，水溶性CoQ10可改善Rot处理诱导的PC12细胞MMP降低和线粒体片段化。Western blotting实验表明CoQ10可降低Rot引起的Caspase-9、active Caspase-3以及Bax的表达增多，上调Bcl-2的表达，阻止AIF向核内转移。（4）辅酶Q10具有抗谷氨酸毒性的作用，该作用与抑制线粒体破碎、维持钙潴留和线粒体膜电位、阻止MPTP形成、AIF释放及DNA片段化有关。（5）在鱼藤酮诱导的大鼠动物模型中，水溶性CoQlO能明显降低Caspase-3、Caspase-9的表达水平，升高survivin的表达水平，但Caspase-8的表达水平无影响．能减少激活性Caspase-3的阳性细胞。