I型大麻素受体去苏素化调节脊髓损伤后小胶质细胞向M1型极化在引起脊髓损伤后慢性疼痛中的作用及机制研究

Chronic pain has always been a catastrophic complication after spinal cord injury (SCI) that affects quality of life of those patients. The imbalance between M1- and M2- microglia polarization is widely accepted as a critical mechanism accounts for this complex process. Activation of cannabinoid CB1 receptor (CB1R) may offer therapeutic strategies in the suppression of microglia polarization to M1 phenotype, but the efficiency of CB1R agonists may be impaired by the CB1R desensitization and internalization. Posttranslational modifications, such as protein phosphorylation, are required for CB1R desensitization. Small ubiquitin-like modifier conjugation (SUMOylation) plays a critical role in regulating inflammation as well as pain-like behaviors, and that is also an important posttranslational modification for CB1R. Our preliminary results showed that the expression of CB1R and CB1R-SUMO1 conjugated proteins is decreased in SCI rats, while SENP1 is increased. However, whether Small ubiquitin-like modifier (SUMO)-specific protease (SENP1) mediates CB1R downregulation，and how SENP1 affects microglia polarization remain unclear. The main aim of present study are as follows: to investigate the effects of SENP1 expression on the microglia polarization, CB1R, inflammatory cytokines expression, as well as pain-like behaviors; and to identify the critical target of CB1R SUMOylation. Our work will uncover a novel mechanism that accounts for CB1R desensitization and microglia polarization to M1 phenotype, and may provide a new insight for the treatment of chronic pain.

慢性疼痛灾难性地影响着脊髓损伤（SCI）患者的生活质量，机制涉及脊髓背角的小胶质细胞极化失衡。尽管激活I型大麻素受体（CB1R）在抑制小胶质细胞向M1型极化有重要价值，但其疗效受限于大麻素减敏，机制涉及CB1R的翻译后修饰过程—磷酸化。苏素化（SUMOylation）也是CB1R重要的翻译后修饰形式，参与炎症及痛行为学调控。我们发现脊髓损伤后CB1R-SUMO1表达减少，而SUMO1特异性蛋白酶（SENP1）表达增多。然而，SENP1是否介导CB1R减敏机制及如何调节SCI后小胶质细胞极化至今不明。本项目主要目的为：阐明SENP1表达变化对CB1R、炎性因子表达、小胶极化、以及痛行为学的影响，并明确CB1R的苏素化关键位点。本研究将揭示CB1R去苏素化介导CB1R减敏及小胶向M1型极化的分子机制，为缓解SCI后慢性疼痛的治疗提供新思路。

脊髓损伤引起的慢性疼痛依然是棘手的临床问题。持续的小胶质细胞激活介导的过度炎症反应是脊髓损伤发生后的显著特征。小胶质细胞激活后分化为M1促炎型和M2促炎型。小胶质细胞介导的促炎反应关键性促进了慢性疼痛的发生发展。诸多研究提示I型大麻素受体（CB1R）是调控小胶质细胞/巨噬细胞极化的关键靶点，但选择性激活CB1R极易产生耐受并增加不良反应。同其他G蛋白偶联受体类似，CB1R的内化可能涉及蛋白翻译后修饰调控。静息状态下的大鼠神经元中CB1R 处于苏素化状态，机制涉及 SUMO-1 对 CB1R 酪氨酸 43 位点（K43）的苏素化，而SENP1 可特异性参与反转这一过程。我们前期研究发现脊髓损伤后脊髓水平小胶M1型表达相对增加，伴随着去苏素化蛋白酶SENP1的上调、CB1R表达的下调；CB1R可表达于脊髓背角小胶质细胞上。因此，我们推测SENP1介导的CB1R下调可能是调节脊髓损伤后小胶M1极化及促进慢性疼痛发生的关键。本研究应用LPS刺激离体BV2细胞系实验及大鼠脊髓损伤动物模型，主要揭示了体外LPS刺激或脊髓损伤后，随着小胶质细胞向M1型极化，SENP1上调，CB1R下调，苏素化形式CB1R下调，炎症因子表达增加；CB1R在核周表达增加而在胞膜表达减少，提示LPS刺激后或脊髓损伤后CB1R发生内化。应用IBA1-siSENP1-AAV选择性敲低小胶质细胞的CB1R，可显著降低则阻止了LPS刺激或脊髓损伤引起的SENP1上调，CB1R下调，苏素化形式CB1R下调，M1极化、炎症因子表达增加，以及CB1R的内化；而应用IBA1-AdSENP1-AAV过表达SENP1则促进上述过程。在行为学上，低表达SENP1时可出现痛行为学的显著缓解；过表达SENP1时则进一步加剧痛敏的发生。综合以上，脊髓损伤后SENP1表达上调通过介导小胶质细胞的CB1R内化，加重了小胶M1型极化失衡及炎性反应的发生，从而促进了脊髓损伤后慢性疼痛发生发展。本项目揭示了脊髓水平SENP1介导小胶质细胞极化在脊髓损伤后慢性疼痛中的作用机制，为临床防治慢性疼痛提供了新的策略与理论依据。