基于TLR信号通路的泛素E3连接酶Pellino1参与神经病理性疼痛研究

There are still no specific treatment strategies for neuropathic pain. Although activation of microglia has been demonstrated as playing a pivotal role in the pathogenesis of neuropathic pain, the intracellular mechanisms involved in microglia activation remain unclear. Toll-like receptor-mediated signaling contributes to microglia activation. Moreover, E3 ubiquitin ligase regulates microglia activation via ubiquitination in signaling by TLR. The pellino1 E3 ubiquitin ligase is newly found to associate with microglia activation. However, the role of pellino1 and its underlying mechanisms in neuropathic pain has still uncovered. We found that pellino1 levels are significantly upregulated in ipsilateral dorsal horn after CCI (chronic constriction injury). Immunofluorescence staining shows that pellino1 is colocalized with microglial marker. More importantly, shRNA-mediated inhibition of pellino1 in the spinal cord persistently attenuated CCI-induced mechanical allodynia and thermal hyperalgesia. Moreover, inhibition of pellino1 by shRNA lentivirus attenuates TLR signaling-mediated MAPKs signaling and NF-κB activation. Therefore, in the current study, we will examine the role of the E3 ubiquitin ligase Pellino1 in neuropathic pain in vivo and in vitro. Moreover, we will elucidate whether pellino1 mediates microglia activation via modulation of molecules ubiquitination in TLR signaling, leading to regulate neuronal excitability in neuropathic pain. This study will provide the new and novel therapeutic strategy for the treatment and management of neuropathic pain patients.

神经病理性疼痛临床上仍缺乏特异性治疗措施。小胶质细胞活化已成为神经病理性疼痛(NP)发生发展的重要病理学基础，而其发挥效应的胞内机制尚不明确。Toll样受体(TLR)信号通路是介导小胶质细胞活化的重要因素，且该过程与泛素E3连接酶泛素化修饰TLR信号通路有关。Pellino1是新近发现参与小胶质细胞活化的E3连接酶，但Pellino1对NP的调控作用及机制尚不清楚。预实验证实，NP状态下，脊髓背角Pellino1过度激活，且Pellino1高表达于活化的小胶质细胞，而抑制Pellino1表达能减轻痛觉过敏，同时降低TLR信号介导的MAPKs表达及NF-kB活性。因此，本项目拟以NP模型和小胶质细胞为研究对象，从整体、细胞及分子水平探讨pellino1通过泛素化修饰TLR信号通路下游分子，介导小胶质细胞活化，调节神经元兴奋性，参与神经病理性疼痛。本研究将会为神经病理性疼痛治疗提供新思路。

脊髓小胶质细胞在神经病理性疼痛的发生发展中起着重要的作用。但在神经病理性疼痛过程中，脊髓小胶质细胞激活的具体机制尚不完全清楚。本研究探讨在神经病理性疼痛中，脊髓Peli1所起的作用及其与脊髓小胶质细胞活化的相互作用。本研究中，我们建立了神经病理性疼痛模型（CCI），观察脊髓Peli1对痛觉敏化和脊髓小胶质细胞的作用。通过利用免疫印迹、免疫荧光、定量聚合酶链反应和酶联免疫吸附实验探讨Peli1介导的痛觉过敏的分子机制。利用免疫共沉淀实验检测CCI后肿瘤坏死因子受体相关因子6的泛素化。此外，我们还探讨了Peli1对脂多糖诱导的BV2小胶质细胞炎症反应的影响及其作用机制。我们发现在神经病理性疼痛状态下，脊髓Peli1表达显著增加，且主要表达于脊髓背角中的小胶质细胞。脊髓Peli1不仅参与神经病性疼痛的形成阶段还参与其维持阶段。我们的体内实验结果表明CCI诱导的脊髓Peli1显著增加促分裂原激活的蛋白激酶（MAPK）磷酸化，核因子κB (NF-κB)活化及促炎细胞因子的产生，并伴随着脊髓小胶质细胞活化。脊髓Peli1还能够促使CCI后损伤侧脊髓中TRAF6-K63位泛素化。此外，我们体外实验结果还表明Peli1能明显增加脂多糖诱导的BV2小胶质细胞的炎症反应。综上所述，我们的结果表明，脊髓Peli1通过活化MAPK/NF-κB通路，激活脊髓小胶质细胞，促进炎症因子释放，最终促使神经病理性疼痛的发生发展。因此，调节脊髓Peli1有可能成为治疗神经病理性疼痛的新策略。