脊髓P2X4R-BDNF-TrkB-KCC2信号通路参与尼古丁戒断大鼠痛觉过敏形成的研究

Clinical findings demonstrated high incidence of chronic pain and increased postoperative opioid requirement in patients with long-term smokers.But the precise molecular mechanisms are still unclear. In the early our group established modified rat model of nicotine dependence / withdrawal and found that pain sensitivity and activity of spinal microglia increased in rats with nicotine withdrawal. Research has shown that activation of spinal microglia, and disinhibition of GABA system, and activation of P2X4R-BDNF-TrkB-KCC2 signaling pathways may play an important role in pain sensitization.But whether the above-mentioned factors are related to increased pain sensitivity after nicotine withdrawal has yet to be confirmed. Applying molecular biology technology and scientific and reliable rat model of nicotine dependence/withdrawal, this research intends to observe the expression of activation of microglia in vitro and in vivo, and P2X4R- BDNF-TrkB-KCC2 signal pathway when nicotine withdrawal induced hyperalgesia, and effects of intervention related links on overall pain behavior, in order to provide clear evidence that activation of microglial cells, nAChR and P2X4R-BDNF signaling pathways involved in the developement of pain sensitization after nicotine withdrawal. Expected results contribute to reveal the mechanism of nicotine withdrawal induced hyperalgesia, and provide new interventions for pain control in smoker.

临床发现长期吸烟患者慢性疼痛发生率高，手术后阿片药物需要量增加，但其确切分子机制尚不清楚。本课题组前期通过自行改良尼古丁依赖/戒断大鼠模型研究发现，尼古丁戒断后大鼠疼痛敏感性增高，脊髓小胶质细胞的活性增强。有研究表明脊髓小胶质细胞活化，GABA系统去抑制和P2X4R-BDNF-TrkB-KCC2信号通路的激活可能在痛觉敏化中发挥重要作用，但尼古丁戒断所致疼痛敏感性增高是否与上述因素有关尚待证实。本研究拟利用分子生物学技术和科学可靠的尼古丁依赖/戒断大鼠模型，从离体和整体观测小胶质细胞活化，P2X4R-BDNF-TrkB-KCC2信号通路在尼古丁戒断所致痛敏时的表达和干预相关环节对整体痛行为的影响，以期为小胶质细胞活化，激活nAChR及P2X4R-BDNF信号通路参与尼古丁戒断后痛觉敏化形成提供明确的证据。预期结果有助于揭示尼古丁戒断所致痛敏的形成机制，并为吸烟患者疼痛防治提供新的干预思路。

临床发现长期吸烟患者慢性疼痛发生率高，手术后阿片药物需要量增加，但其确切分子机制尚不清楚。本课题组前期通过自行改良的尼古丁依赖/戒断大鼠模型研究发现，尼古丁戒断后大鼠疼痛敏感性增高，脊髓小胶质细胞的活性增强。有研究表明脊髓小胶质细胞活化，GABA系统去抑制和P2X4R-BDNF-TrkB-KCC2信号通路的激活可能在痛觉敏化中发挥重要作用，但尼古丁戒断所致疼痛敏感性增高是否与上述因素有关尚待证实。本研究应用稳定可靠的尼古丁依赖/戒断大鼠模型及相关分子生物学技术，从离体和整体观测小胶质细胞活化，P2X4R-BDNF-TrkB-KCC2信号通路及GABA系统在尼古丁戒断所致疼痛过敏时的表达和干预相关环节对整体痛行为的影响。通过离体培养BV-2小胶质细胞，发现尼古丁作用BV-2小胶质细胞后尼古丁受体α7nAChR的表达显著上调，并进一步引起BV-2小胶质细胞P2X4R的表达上调。通过在体实验发现尼古丁戒断后大鼠疼痛敏感性增高且在尼古丁戒断后第4天最为显著，脊髓P2X4R、BDNF的表达增多，KCC2的表达减少，且与上述行为学结果相一致，鞘内应用小胶质细胞抑制剂米诺环素及TrkB抑制剂K252a可以抑制上述分子的表达,同时降低尼古丁戒断所导致的疼痛敏感性增高，从而在体验证了脊髓P2X4R-BDNF-TrkB-KCC2信号通路在尼古丁戒断大鼠痛觉过敏形成中的重要作用；在体实验发现大鼠尼古丁戒断后脊髓GABA、GAD67的表达减少，说明脊髓GABA系统去抑制有可能参与尼古丁戒断大鼠痛觉过敏的形成；另外在完成原研究计划的基础上在体条件下证实了脊髓小胶质细胞CX3CR1-p38MAPK信号通路参与尼古丁戒断大鼠痛觉过敏的形成。这些研究结果不仅有助于揭示一般痛觉敏化及尼古丁戒断后痛觉敏化的形成机制，而且为长期吸烟患者的疼痛防治提供新思路、新启示、新靶点。