线粒体解耦联蛋白UCP2介导黑质网状部KCC2功能异常参与肝性脑病诱致运动迟缓的机制研究

Hepatic encephalopathy (HE) is a major neuropsychiatric disorder that occurs in patients with severe liver failure.The mechanisms by which liver failure leads to altered motor function remained unclear.We have focused on the involement of altered mitochondrial funtion and mitochondrial uncoupling protein 2 (UCP2), considering that it has been identified as a critical determinant of fatty acid utilization by adult neurons, within the substantia nigra pars reticulata (SNr) in the toxin thioacetamide (TAA)-induced hepatic encephalopathy using glutamic acid decarboxylase 67-green fluorescent protein knock-in transgenic mice (GAD67-GFP knock-in mice). We have found that, during the first and advanced phase phases of the encephalopathy, the UCP2 expression increases remarkably. Interestingly, the treatment with the targeted suppression of UCP2 within the SNr by lentivirus-mediated RNAi could effectively prevent HE-induced hypolocomotion, while such administration produced a significant effect on the decreased neuron specific chloride transporter KCC2, imbalance of chloride homeostasis and disfunction of mitochondria, considering such changes have been identified to be induced by TAA hepatic encephalopathy. Our data suggest that UCP2 would exert its reverse action on chloride homeostasis regulated by KCC2, and mitochondrial function, at the substantia nigra pars reticulata level, which may be critically involved in the development and maintenance of hepatic encephalopathy. But the exact mechanism associated with hepatic encephalopathy needs to be clarified. Thus we will first use mitochondria isolation and detection methods to investigate the changes of UCP2 expression and four major mitochondrial functions (ATP synthesis, calcium homeostasis, redox reaction and membrane stablity) within the substantia nigra pars reticulata in TAA-induced hepatic encephalopathy model of GAD67-GFP knock-in mice. Second, we will detect the underlying signaling pathways between UCP2 and KCC2 function by molecularand morphological methods in vivo and in vitro.Third, we will use the combinationof patch-clamp recordings and single-cellRT-PCR methods to studythe mechanismof UCP2 and altered mitochondrial function for the pathophysiology of hepaticencephalopathy. Fourth，we will assessthe possiblepharmocological substances on the UCP2 and mitochondrial function, as well as the locomotion behavioral activities during the hepatic encephalopathy model. Thhe aim is to promote the advances in understanding the molecular and cell biology of mitochondria in the hepatic encephalopathy, and to lead to novel approaches for the prevention and treatment of such severe disease.

肝性脑病（HE）机制不清致临床治疗效果不佳。我们前期研究发现：TAA诱导HE小鼠全程可见黑质网状部内线粒体解耦联蛋白UCP2表达增高，下调UCP2能有效缓解HE行为同时，还能纠正HE所致神经元特异性氯离子外向转运体KCC2表达降低、氯稳态失衡以及线粒体功能异常。以上提示UCP2受线粒体介导下调KCC2氯调控功能可能是肝性脑病发生发展的关键事件之一。本课题以此为契机，以GAD67-GFP转基因小鼠为工具，以黑质网状部（SNr）为靶区，以TAA肝性脑病模型为研究对象：①明确HE各时程SNr内UCP2表达水平及线粒体功能改变情况；②找出HE状态下UCP2反向调控KCC2功能的作用机制；③搞清UCP2增多和线粒体功能异常对GABA能神经传递的调控及机制；④最终在整体动物水平探索干预UCP2和线粒体功能对HE行为学影响。本项目旨在从线粒体角度阐明肝性脑病机制，为临床有效治疗肝性脑病提供新思路。

研究结果证实黑质网状部作为运动调控间接通路的重要结构，其神经元功能可塑性变化是肝性脑病诱致整体动物水平的运动迟缓、运动障碍、以及情绪变化的关键事件。研究还证实，黑质网状部作为抑制性GABA能神经元的聚集地，其神经元胞体内部氯离子稳态失衡与GABA能神经元神经传递性质翻转密切相关。发现氧化应激损伤是导致GABA能神经元由抑制性效应向兴奋性效应转变的启动因素。研究结果同时提示，线粒体稳态失衡与神经元稳态失衡不仅同时存在于肝性脑病，而且，线粒体损伤可能早于神经元可塑性变化，并且导致神经元出现不可逆性失代偿损伤，而针对线粒体的干预手段有可能成为恢复GABA能神经元的重要途径。研究验证了一个新策略：调控中枢神经系统氯离子稳态失衡可能成为肝性脑病治疗的潜在靶点。研究提出了一个新见解：利用氯离子成像结合氯离子转运体时、空表达变化检测技术，观察到肝性脑病诱致运动障碍小鼠黑质网状部氯离子稳态失衡现象，提示：中枢神经系统氯离子稳态失衡可能参与肝性脑病的病理过程。研究证明了一种新学说：对比临床肝性脑病和非肝性脑病患者血液内氯离子转运体实时定量RT-PCR检测结果，观察到氯离子转运体表达水平与患者神经功能评分密切相关，提示：检测血液内氯离子转运体的表达失衡可能成为预测肝性脑病的信号。