慢性疼痛模型中枢小胶质细胞活化方式及分子调控机制

Spinal microglial (central macrophages) activation plays a key role in central sensitization that are required for long-term maintenance of neuropathic pain. Macrophages are classified into two distinct polarized phenotypes, classically activated (M1) macrophages releasing proinflammatory mediators and alternatively activated (M2) contributing to the suppression and resolution of inflammation. Recently, the bile acid-responsive G-protein-coupled receptor Tgr5 is expressed in monocytes and macrophages, and its activation mediates potent anti-inflammatory effects. In this proposal, we hypothesized that spinal microglia activated in the neuropathic pain models also have different two groups representing M1/pro-inflammatory and M2/anti-inflammatory phenotypes. The activated microglia with M1/pro-inflammatory phenotypes are dominant and produce typical proinflammatory cytokines and chemokines in the spinal cord following peripheral nerve injury in neuropathic pain model, which drive inflammation and maintain the central sensitization. We also try to rebalance the neuroinflammatory response through Tgr5 activation in spinal microglia, focusing on Tgr5 downstream effector pathways, for example, MAPK/ NF-kB pro-inflammatory and IL-4/Jak/STAT6 anti-inflammatory signaling pathway, the study aims to selectively trigger Tgr5 as a novel therapy to reduce inflammation and attenuate neuronal sensitization both in peripheral and in central.

中枢小胶质细胞被认为是中枢神经系统的巨噬细胞，其在慢性疼痛中枢敏化中的作用得到肯定，并被认为是慢性疼痛持续的必要条件。研究表明，外周巨噬细胞存在促炎M1型和抑炎M2型激活方式，Tgr5信号通路的激活可以调整M1/M2型巨噬细胞的转化，上调抑炎因子的表达，下调致炎因子的表达，起到免疫调节作用。本研究探讨神经病理性疼痛和炎症性疼痛模型中脊髓背角大量活化的小胶质细胞是否也存在类似的活化方式，或至少存在致炎性和抑炎性功能（或细胞表型）的不同类型的活化小胶质细胞，在疾病不同阶段发挥不同的作用；通过Tgr5信号通路的激活在外周抑制致炎性巨噬细胞的激活，在中枢抑制致炎性小胶质细胞的活化（MAPK/ NF-kB通路），并上调抑炎相关的分子信号通路（IL-4/Jak/STAT6通路），调控外周和中枢的炎症免疫反应，达到病变部位致炎和抑炎反应的再平衡，从而减轻外周和中枢敏化，达到改善疼痛的治疗目标。

本课题采用免疫组化、细胞流式分析、Western blot 和Real-time PCR 方法，探讨不同疼痛模型（神经病理性疼痛、骨关节炎和高脂模型）外周损伤部位的巨噬细胞及中枢脊髓小胶质细胞是否存在M1/M2 型活化；明确外周巨噬细胞及中枢小胶质细胞是否表达Tgr5；通过局部和系统给药方式，给予特异性Tgr5 激动剂是否能够抑制模型动物的疼痛行为，是否可以改变活化的巨噬细胞和小胶质细胞的细胞表型；最终探讨Tgr5信号通路调控外周巨噬细胞及中枢小胶质细胞活化方式的分子机制。结果表明，1）外周神经损伤后局部单核巨噬细胞浸润，主要是M1型致炎性巨噬细胞。中枢脊髓小胶质细胞活化，表现为M1型分子表达明显增高。2）神经损伤后，外周组织和中枢脊髓组织NLRP3/caspase-1/IL-1β信号通路激活。3）神经损伤后，外周巨噬细胞和神经纤维均表达Tgr5。4）损伤部位注射、鞘内注射，还是灌胃系统给予特异性Tgr5 激动剂，均能缓解动物的疼痛行为，并可以显著减少M1型巨噬细胞和脊髓小胶质细胞的活化。初步结论，多种因素（损伤、炎症、机械力）均可以导致外周巨噬细胞和中枢小胶质细胞的活化，主要表现为M1型炎症通路的激活，Tgr5信号通路的激活可以抑制致炎性信号通路，如NLRP3炎性小体/caspase-1/IL-1β，调控外周和中枢的炎症免疫反应，达到病变部位致炎和抑炎反应的再平衡，从而减轻外周和中枢敏化，达到改善疼痛的治疗目标。