神经损伤诱导前扣带皮层趋化因子-致炎细胞因子级联反应的痛调制作用及机制

Anterior cingulate cortex (ACC) has been regarded as necessary in the encoding of the sensory discriminative and affective consequences of nociceptor stimulation. However, the immune mechanisms underlying the pain-related regulation of ACC is still unknown. In our preliminary experiment, we used the stimulatory designer receptors exclusively activated by designer drugs (DREADD-hM3Dq or hM4Di, where hM3Dq indicates Gq-coupled human M3 muscarinic receptor and hM4Di indicates Gi-coupled human M4 muscarinic receptor), which couples through a Gq or Gi pathway to depolarize or hyperpolarize neuronal membrane potential upon the application of synthetic ligand clozapine-N-oxide (CNO).We found that inhibition of ACC pyramidal neurons with use of Gi-DREADD reversed the mechanical allodynia induced by spared sciatic nerve injury (SNI). On the contrary, administration of Gq-DREADD into the unilateral ACC layer Ⅴ/Ⅵ without any nerve injury decreased the bilateral paw withdrawal thresholds to von Frey hairs through activating ACC. Furthermore, the protein levels of chemokine CX3CL1 and tumor necrosis factor-alpha (TNF-α) and interleukin-6 were increased in ACC following SNI surgery. The increased TNF-α was co-localized with c-Fos (neuronal excited marker) and GFAP (astrocyte marker). Pre-treatment with anti-TNF-α antibody into ACC significantly blocked c-Fos expression, increased the animal paw withdrawal threshold and attenuated the pain-related negative emotion, suggesting the cascade of chemokines-proinflammatory cytokines in ACC might be responsible for the development of chronic pain. Based on our present works, we will further investigate the ACC mechanisms underlying different models of neuropathic pain, reveal the role of chemokine-proinflammatory cytokine cascade in the glial-neuronal interactions as well as the regulations of NF-κB, p38 MAPK and JNK signals on the cytokines secretions in ACC following nerve injury. The present project aims to reveal the immune mechanism of ACC in neuropathic pain, and to provide a new target for clinical therapy.

前扣带皮层（ACC）在痛分辨、痛情绪反应中扮演重要角色，但其免疫机制尚不十分清楚。本项目预实验将携带有人M3/M4毒蕈碱受体（hM3Dq或hM4Di）基因的AAV病毒转染ACC，在给予配体氯氮平-N-氧化物后可通过Gq或Gi通路兴奋或抑制ACC锥体神经元，行为学结果表明ACC参与痛调制；分子生物学实验显示神经损伤后ACC内趋化因子CX3CL1、致炎细胞因子TNF-α上调，并与神经元兴奋标志物c-Fos共表达；ACC内预处理TNF-α中和抗体抑制神经损伤诱导的c-Fos表达、缓解触诱发痛和痛情绪。本项目拟用多种神经科学手段进一步揭示不同表型神经病理性痛模型ACC的痛调制差异；ACC神经元↔胶质细胞交互激活网络中趋化因子-致炎细胞因子级联反应在脊髓下行易化中的作用；NF-κB、p38 MAPK、JNK通路介导ACC兴奋、细胞因子微环境改变的分子机制。本项目旨在为缓解慢性痛和痛情绪提供新靶点。

前扣带皮层(anterior cingulate cortex，ACC)是痛信息处理的重要中枢，在痛的感觉分辨、痛情绪反应等信息处理中扮演重要角色，但目前关于ACC痛调制的神经免疫机制仍不十分清楚。在本项目基金资助支持下，我们首先发现，外周坐骨神经损伤后ACC内神经元与小胶质细胞交互作用诱发肿瘤坏死因子α（tumor necrosis factor α, TNF-α）表达升高以及神经元异常兴奋，是外周神经损伤后痛过敏和痛情绪产生的重要原因，揭示了ACC内神经免疫调控紊乱与痛信息处理之间的紧密联系，为后续工作的开展给予了重要的理论和实验室数据支持。随后研究发现ACC内趋化因子CX3CL1介导的下游级联免疫反应在下行易化脊髓痛信息传递中发挥关键作用，恶化脊髓背角神经炎症，导致痛敏增强，甚至镜像痛。首次从神经免疫学角度揭示ACC与脊髓背角炎症反应之间的直接联系，为临床慢性痛及镜像痛治疗提供新靶点。在项目执行过程中，我们还意外发现在2021年诺贝尔奖舞台上大放光彩的“明星人物”——机械敏感性通道Piezo1在ACC内也可能参与了痛信息处理。在离体的细胞培养实验中，外源性TNFα可刺激细胞NLRP3炎症小体相关蛋白表达升高以及Piezo1表达增强；在体实验中发现，外周坐骨神经损伤可通过激活NLRP3炎症小体-Piezo1通路介导小胶质细胞对受损抑制性PV中间神经元吞噬，这可能是ACC兴奋/抑制失平衡及慢性痛形成的重要原因，首次揭示机械性通道Piezo1在高位中枢痛处理中的可能作用及机制。此外，我们还用聚类分析的方法分析了TNFα诱导坏死性凋亡在痛研究领域的前景，为慢性痛的基础研究和临床治疗提供新思路。通过本项目的研究，我们已经对外周神经损伤后ACC痛信息处理的中枢免疫调控机制有了较充分的认识，可为临床慢性痛和镜像痛的治疗提供新思路和药物作用靶点。