基于VAMP7介导的细胞自噬探讨突触结构重塑对神经病理性疼痛的影响

The synaptic structural remodeling was one of important pathological fundament for neuropathic pain. However, the detailed pathogenesis of synaptic structural remodeling in neuropathic pain is still unclear. Autophagy was supposed to play an important role during such remodeling process. Meanwhile, it was suggested neuron autophagy is closely related with the synaptic structural remodeling. But it is not clear the relationship between autophagy and tissue structural remodeling in neuropathic pain. VAMP7 is an important member in SNAREs protein family, and played an important role in the membrane fusion during autophagy. Meanwhile, VAMP7 is essential for neuronal morphogenesis. Besides, our bioinformatics and molecular biology analyses demonstrated the decreased expression of VAMP7 in neuropathic pain, and the up-regulation of VAMP7 expression could relieve pain syndrome by activating autophagy process. Therefore, we speculate that autophagy process mediated by the VAMP7 may be related with the synaptic structural remodeling in neuropathic pain. In this study we plan to discuss whether the VAMP7 expression can influence the synaptic structural remodeling and neuropathic pain by regulation the autophagy process on both cell (spinal cord neurons) and animal (SNL model on GFP-LC3 transgenic rats) levels. Besides, we plan to verify the difference on treatment effect for neuropathic pain between the up-regulation of the VAMP7 expression and the application of gabapentin. This study would provide new theoretical basis for the research of neuropathic pain mechanism and treatment.

突触结构重塑是导致神经病理性疼痛反复发作的重要病理基础，其具体机制尚未阐明，并可能影响疼痛长期疗效。研究表明自噬与许多疾病组织结构重塑密切相关，可能是极有前景的治疗靶点；VAMP7是调控自噬体膜融合关键蛋白SNAREs成员，我们前期证明VAMP7在疼痛大鼠脊髓表达下调，并伴突触结构重塑，而上调其表达能激活脊髓内自噬并改善疼痛症状，据此我们提出假说：“VAMP7介导的细胞自噬参与突触结构重塑，在神经病理疼痛发生和迁延不愈中起着关键作用”。本项目拟采用GFP-LC3转基因大鼠和原代脊髓神经元，分别建立疼痛动物模型和细胞剌激模型，应用自噬抑制剂和激动剂、基因沉默和过表达等手段干预自噬及VAMP7表达，通过观察自噬荧光、自噬蛋白表达、突触结构及疼痛行为等指标，探讨VAMP7介导的自噬在突触结构重塑及疼痛中的作用。本项目有望揭示VAMP7介导的自噬在神经病理性疼痛中的机制，为新镇痛靶点确立提供依据。

神经病理性疼痛是由躯体感觉系统损害或疾病导致的疼痛，发病率高，治疗难度大，并严重影响患者生活质量。突触结构重塑是导致神经病理性疼痛反复发作的重要病理基础，其具体机制尚未阐明，并可能影响疼痛长期疗效。研究表明自噬与许多疾病组织结构重塑密切相关，因此其可能是极有前景的治疗靶点。本项目主要通过建立脊神经结扎（SNL）神经病理性疼痛大鼠模型及原代培养大鼠脊髓神经元，动态观察及评价神经病理性疼痛发生过程中大鼠脊髓及神经元自噬水平及突触结构重塑情况，并在动物及细胞层面明确干预 VAMP7 表达是否调控细胞自噬，并影响突触结构重塑及动物疼痛行为。通过相关分子生物学实验，我们发现（1）SNL大鼠脊髓内自噬水平出现紊乱，并主要发生在脊髓背角区域；（2）通过动物及细胞实验证实，采用慢病毒过表达载体能够上调大鼠脊髓或原代培养神经元VAMP7的蛋白表达，并增加自噬标记物LC3表达水平；（3）通过动物模型鞘内注射慢病毒过表达载体及SiRNA，证明VAMP7表达增加能够有效缓解神经病理性疼痛大鼠导致的脊髓突触结构重塑，显著增加SNL大鼠脊髓突触后膜厚度及活性区长度，同时降低突触脊宽度，而这一作用依赖细胞自噬激活；（4）通过上调VAMP7表达能够显著缓解SNL大鼠的机械痛敏及热痛敏；（5）此外，通过与加巴喷丁对比，我们发现通过上调VAMP7能够获得更好地镇痛效果。在项目执行期间，共发表期刊论文6篇，其中SCI收录论文5篇，培养在读硕士生3名；项目负责人以本项目思路为主要研究结果作为申报基础获得了2020年度高校基本科研业务费-中山大学青年教师培育项目资助。