脊髓水平BDNF/TrkB信号级联调控线粒体分裂和功能介导突触囊泡释放参与骨癌痛的机制研究

Bone metastasis of tumor cells induces increased spinal dorsal horn neuronal excitability which promoting the transmission of pain information. The process depends on mitochondria-mediated synaptic vesicle release. As a pain modulator, brain-derived neurotrophic factor (BDNF) is involved in the transmission of pain information, but the mechanism remains unclear. Our previous studies found that increased pain sensitivity in bone cancer pain (BCP) rats was accompanied by the increased BDNF expression and abnormalities of mitochondrial fission and function; TrkB receptor antagonist treatment significantly improved the pain behavior of BCP animals. Accordingly, we proposed the hypothesis: BDNF/TrkB singnaling mediated PI3K/Akt/GSK-3β modulates mitochondrial dynamics by regulating Drp1 phosphorylation; PLCγ/IP3 regulates mitochondrial function through affecting intracellular Ca2+ level; the above two pathways regulate synaptic vesicle release and transmission of pain information which plays a key role in bone cancer pain. The current study intends to employ BCP animals and SH-SY5Y cell; to analyze the correlation between the activity of BDNF/TrkB signaling pathway and the animal pain behavior; to detect mitochondrial dynamics and function, and changes of synaptic vesicle protein interactions; to clarify the molecular mechanisms of BDNF/TrkB signaling cascade regulates transmission of pain information and affects pain sensitivity in animals.

肿瘤细胞骨转移诱导脊髓背角神经元兴奋性增加，促进痛觉信息传递；该过程依赖于线粒体介导的突触囊泡释放。脑源性神经营养因子（BDNF）作为痛觉调制因子参与痛觉信息传递，但作用机制尚未明确。本课题组前期研究表明，骨癌痛（BCP）大鼠痛敏增加伴随BDNF上调及线粒体分裂和功能异常；TrkB受体拮抗剂显著改善BCP动物痛觉行为。据此提出假说：BDNF/TrkB信号介导PI3K/Akt/GSK-3β调节Drp1磷酸化影响线粒体动力学；PLCγ/IP3调节胞内Ca2+水平影响线粒体功能；从而调控突触囊泡释放，影响痛觉信息传递并在骨癌痛中发挥关键作用。本项目拟以BCP动物和SH-SY5Y细胞为载体，靶向调控TrkB活性，分析BDNF/TrkB信号途径的活性与动物痛觉行为的相关性；检测线粒体动力学和功能以及突触囊泡蛋白相互作用的变化；阐明BDNF/TrkB信号级联调节痛觉信息传递及影响动物痛敏的分子机制。

骨癌痛（BCP）是由骨肉瘤或实体瘤转移而产生的一种慢性疼痛。骨癌痛的产生和维持与多种调节因子相关，脑源性神经营养因子（BDNF）作为痛觉调制因子参与痛觉信息传递，BDNF/TrkB信号在脊髓中枢敏化中的作用是痛觉维持的基础。本课题组以胫骨骨髓腔内注入乳腺癌细胞的方法建立骨癌痛大鼠模型，痛觉行为学检测表明BCP大鼠痛敏增加、运动能力分析表明BCP大鼠运动能力下降。影像学及形态学分析显示骨组织损伤、正电子发射断层扫描（PET）确定BCP脊髓组织的活性增强。进一步分析显示BCP大鼠脊髓背角BDNF/TrkB信号显著激活，脊髓神经元活性上调，线粒体结构和功能损伤，突触蛋白、Ca2+信号相关蛋白及线粒体相关蛋白表达异常。运用ANA-12和LY294002靶向抑制BDNF/TrkB及其下游信号可显著改善BCP大鼠的痛觉行为及运动能力；免疫印迹分析表明突触蛋白、Ca2+信号相关蛋白及线粒体相关蛋白表达水平显著恢复。上述研究结果为阐明BDNF/TrkB信号级联调节痛觉信息传递及影响动物痛敏的分子机制提供了研究基础。