ROS反向增强脊髓背角神经元线粒体生物活性参与胰腺癌性痛的机制研究

Pancreatic cancer causes severe pain in 50 to 70% of patients and is is often difficult to treat. Mitochondria are involved in the most common neurological disorders, however, its role in the the pathogenesis of chronic pain remains uncertain. We have focused on the the involvement of altered mitochondrial function and reactive oxygen species (ROS), considering that mitochondria compartment is presumed as the main source and susceptible target of intracellular ROS, within the spinal dorsal horn in the 7,12-dimethyl-1,2-benz[a]anthracene (DMBA)-induced pancreatic cancer pain 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 phases of the cancer pain, the ROS generation increase remarkably. Moreover, DMBA induced loss of mitochondrial membrane potential, reduction of tissue Na/K, Na and Ca ATPase activities, and decreases of expression of mitochondrial uncoupling protein 2 (UCP2), during the first phase of the pain model. Interestingly, during the advanced phase, DMBA reduced the loss of mitochondrial membrane potential, increased tissue ATPase activities, and upregulated UCP2 expression in the spinal dorsal horn. Preemptive treatment with TEMPOL, a superoxide selective scavenger, is effective in preventing the first development of visceral pain behavior, while such administration produced a much smaller effect in the advanced phase. Our data suggest that ROS production in the advanced phase of cancer pain would exert its positive action on mitochondrial function mainly at spinal cord level, which may be critically involved in the development and maintenance of visceral pain. But the exact mechanisms associated with cancer pain needs to be clarified. Thus we will first use mitochondria isolation and detection methods to investigate the changes of ROS generation and four major mitochondrial functions (ATP synthesis, redox reaction, membrane stablity and activation of mitochondrial protective system) within the spinal dorsal horn in DMBA-induced pancreactive cancer pain model of GAD-67-GFP knock-in mice. Second, we will detect the underlying signaling pathways between ROS and mitochondrial function by molecular and morphological methods in vivo and in vitro. Third, we will use the combination of patch-clamp recordings and single-cell RT-PCR methhods to study the mechanisms of ROS and altered mitochondrial function for the pathophysiology of chronic pain. Fourth, we will assess the possible pharmacological substances on the mitochondrial function, as well as the visceral pain behavioral activities during the advanced phase of the cancer pain models. The aim is to promote the advances in understanding the molecular and cell biology of mitochondria in chronic cancer pain and to lead to novel approaches for the prevention and treatment of such severe pain.

癌性痛机制亟需阐明。最新研究表明，线粒体功能改变参与多种病理性痛，但在癌性痛中作用不清。我们前期研究发现：DMBA胰腺癌性痛小鼠全程见活性氧分子ROS生成增多，但ROS在初始和发展两阶段对脊髓背角线粒体之膜电位水平、ATP酶活性以及解偶联蛋白合成等作用相反，提示ROS反向增强线粒体活性可能是胰腺癌疼痛慢性化关键事件之一。本项目拟以此为契机，以GAD67-GFP转基因小鼠为工具，以脊髓背角为靶区，以DMBA胰腺癌性痛为研究对象：①利用线粒体检测技术，明确癌性痛小鼠脊髓背角ROS水平及线粒体功能改变情况；②利用在体和离体方法，找出癌性痛状态下ROS反向调控线粒体功能的作用机制；③利用膜片钳技术，搞清ROS和线粒体对脊髓背角GABA能传递的调控；④利用行为药理学方法，探索调控ROS及线粒体不同环节对胰腺癌小鼠痛行为学的改变。本项目旨在从线粒体角度阐明癌性痛分子机制，为临床癌性痛治疗提供新思路。

慢性痛机制不明致临床镇痛效果不佳。最新研究揭示线粒体参与慢性痛发生发展过程，但具体机制不清。本研究发现：第一，慢性痛状态下线粒体发生可塑性改变：通过鞘内注射线粒体示踪剂探针（Mito-Red），结合基于共聚焦显微镜的线粒体成像技术，检测脊髓背角线粒体分布和亚细胞形态改变，发现持续性炎性痛和神经病理性痛等慢性痛状态下，脊髓背角浅层（I – II 层）和深层（III – V层）内神经元线粒体发生形态可塑性改变，即散在、颗粒状转变为聚合、簇状；通过流式细胞技术及多种生化手段，检测脊髓背角线粒体氧化应激产物（丙二醛、活性氧和过氧化氢）水平、线粒体膜电位变化及线粒体解偶联蛋白表达，发现在慢性痛不同时相，线粒体功能呈现可塑性改变，即急性期线粒体减低，而在慢性期恢复。第二，线粒体解偶联蛋白（UCP）表达变化引起痛行为改变及痛相关分子变化：利用腺相关病毒表达载体干预脊髓背角神经元UCP4表达，通过行为学检测发现：痛模型小鼠过表达UCP4后可缓解触诱发痛行为，而正常小鼠下调UCP4表达则能够明显引起小鼠痛行为；利用蛋白质谱技术，检测表达病毒干预后脊髓背角线粒体蛋白表达变化，发现UCP4表达干预后44种痛相关分子表达发生相应改变。第三，ROS引起大脑前额叶皮层神经元发生突触可塑性改变：采用全细胞电压膜片钳记录方法，利用ROS类似物t-BOOH（tert-butyl hydroperoxide）及ROS清除剂PBN（N-tert-Butyl-α-phenylnitrone）调节ROS水平，检测微小兴奋性突触后电流（mEPSCs）、微小抑制性突触后电流（mIPSCs），发现低浓度ROS会引起抑制性神经元突触前谷氨酸释放减少，GABA释放增多；兴奋性神经元突触前谷氨酸释放无明显影响，GABA释放减少。高浓度ROS则会引起抑制性神经元突触前谷氨酸释放增多，GABA释放减少；兴奋性神经元突触前谷氨酸释放减少，GABA释放减少。提示线粒体功能紊乱引起ROS增多会引起突触间递质释放紊乱。以上研究从线粒体可塑性、神经元突触可塑性等角度深入探究线粒体功能障碍与疼痛慢性化的关系，并找到线粒体开启自身保护系统关键蛋白UCP4，为镇痛新药的开发提供新的靶点。