脊髓水平GR-NPAS4信号通路对GABA能抑制性突触的调节在术前焦虑加重术后疼痛中的作用

Preoperative anxiety is a clinically significant problem for many patients undergoing surgery, and is associated with the prolonged and exacerbated postoperative pain. However, the molecular mechanisms are still unclear. Our previous studies demonstrated that preoperative anxiety resulted in exacerbated and prolonged mechanical allodynia that lasted up to 30 postoperative days in a rat model of incisional pain. The decrease in inhibitory neurotransmitter GABA release and the down-regulation of GABAA receptor γ2 subunit in spinal cord may participate in the mechanism of this phenomenon. Studies in vitro indicated that transcription factor Neuronal Per Arnt Sim (PAS) domain protein 4 (NPAS4) palys an important role in the activity-dependent regulation of inhibitory synapse. Stress can down-regulate the expression of NPAS4 via glucocorticoid receptor (GR) in hippocampus, inhibit the development and function of GABAergic inhibitory synapse, and contribute to the stress-induced brain dysfunction. As mentioned above, we hypothesize that preoperative anxiety can derectly inhibit the release of GABA and the development of GABAergic inhibitory synapse via the neuron GR-NPAS4 signaling pathway in spinal cord. The imbalance of inhibitory synapse function and excitatory synapse function results in the exacerbated and prolonged postoperative pain. The spinal neuron cultivation, pain behaviors measurement, microdialysis, western blot, chromatin immunoprecipitation assay and immunofluorescence technique will be applied to investigate the release of GABA and the expression of the inhibitory synaptic proteins, and to investigate the spinal mechanisms of preoperative anxiety-induced exacerbated and prolonged postoperative pain in vivo and in vitro. The implementation of this project provides novel intervention targets for the prophylaxis and treatment of preoperative anxiety-related postoperative pain.

术前焦虑加重术后疼痛是临床常见现象，其机制尚未明确。我们前期实验发现，术前焦虑可使切口痛大鼠术后疼痛明显加重，且持续时间延长，该现象同时伴随脊髓水平抑制性神经递质GABA释放减少及GABAA受体γ2亚基表达下调。研究提示，转录因子NPAS4在抑制性突触形成和维持的功能依赖性调节中具有关键作用。应激可通过激活糖皮质激素受体（GR）而显著下调海马NPAS4的表达水平，从而抑制GABA能抑制性突触的形成和功能，参与应激诱导的神经功能失调。我们提出假说：术前焦虑通过脊髓神经元GR-NPAS4信号通路，抑制GABA能抑制性突触的形成及突触囊泡释放GABA，最终导致术后疼痛的加重和延长。我们拟通过干预上述信号通路中的关键靶点，检测GABA囊泡释放量及GABA能抑制性突触相关蛋白的表达，在离体和在体水平探讨术前焦虑加重术后疼痛的分子机制，为临床防治与焦虑相关的术后疼痛提供新的策略。

术前焦虑加重术后疼痛是临床常见现象，严重影响患者生活质量。我们前期研究发现，术前焦虑可使切口痛大鼠术后疼痛明显加重，且持续时间延长，该现象同时伴随脊髓水平抑制性神经递质GABA释放减少及GABAA受体γ2亚基表达下调。文献提示，转录因子NPAS4在抑制性突触形成和维持的功能依赖性调节中具有关键作用。应激可通过激活糖皮质激素受体（GR）而显著下调海马NPAS4的表达水平，从而抑制GABA能抑制性突触的形成和功能，参与应激诱导的神经功能失调。本项目首先建立了术前单次延长应激（SPS）诱发术后慢性疼痛的大鼠模型，在离体和在体水平深入探讨了术前焦虑与术后慢性疼痛之间的机制关联。研究结果显示：①术前焦虑应激引起大鼠切口手术后血皮质酮（GCs）水平升高，通过脊髓神经元GR-NPAS4信号通路，下调GABA能抑制性突触相关蛋白及其mRNA的水平（GABARα1，GABARγ2，GAD65，GAD67，VGAT），并抑制神经递质GABA的释放，导致术后疼痛的加重和延长。②术前焦虑应激引起的GCs升高对脊髓小胶质细胞具有敏化作用，敏化的小胶质细胞在切口手术作用下进一步被激活，在术后慢性疼痛的起始阶段发挥关键作用；α7nAchR的活化通过调控小胶质细胞介导的促炎性反应，预防术后慢性疼痛的发生。③术前焦虑应激引起的GCs升高通过作用于星形胶质细胞上SGK1激活星形胶质细胞，并促进细胞内ATP释放至细胞外，在术后慢性疼痛的维持阶段发挥关键作用。本项目揭示了术前焦虑应激对脊髓神经元与胶质细胞的影响，及其在术后慢性疼痛中的作用机制，为临床防治与焦虑相关的术后疼痛提供了新的策略与理论依据。