大鼠脊髓小胶质细胞CX3CR1/ERK5信号通路在神经病理性疼痛中的作用

Role of CX3CR1/ERK5 pathway in spinal microglia for the development of neuropathic pain

目的 探讨脊髓小胶质细胞CX3CR1/ERK5信号通路在神经病理性疼痛中的作用.方法 建立脊神经结扎（SNL）疼痛模型,检测大鼠脊髓p-ERK5表达的改变及其表达的细胞类型.鞘内注射ERK5反义寡核苷酸,观察抑制脊髓ERK5表达对SNL大鼠PWT与TWL的影响,以确定ERK5在神经病理性疼痛中的作用.SNL大鼠鞘内注射CX3CR1中和抗体,观察阻断该受体对ERK5活化的影响；鞘内注射CX3CR1的配体CX3CL1,观察CX3CL1能否激活脊髓小胶质细胞和ERK5,以及预先抑制ERK5表达能否逆转CX3CL1的作用,以判定神经病理性疼痛条件下CX3CR1对ERK5的调节作用.结果 与假手术组相比,SNL大鼠术后脊髓p-ERK5免疫反应阳性细胞表达增加（61.75±11.52比2.2±0.12; 58.01 ±10.45比1.1 ±0.11）（P＜0.05）,荧光双标结果表明p-ERK5主要表达于小胶质细胞.抑制脊髓ERK5表达有效缓解SNL所致的热（13.48±2.0）s比（18.0±3.7）s;（11.6 ±2.3）s比（17.7 ±1.4）s（P＜0.05）与机械（15.42 ±3.46）g比（22.7±3.2）g；（13.6±2.9）g比（21.4±4.1）g痛敏（P＜0.05）.与对照组相比,阻断CX3CR1可有效减少SNL大鼠脊髓ERK5的活化（30±9）比（58±12）；（50±12）比（36±4）（P＜0.05）.抑制脊髓ERK5的表达缓解鞘内注射CX3CL1所致痛觉过敏与小胶质细胞活化.结论 在神经病理性疼痛条件下,CX3CL1/CX3CR1通过激活脊髓小胶质细胞ERK5,调节脊髓小胶质细胞的活化,该通路参与了脊髓疼痛信号的转导过程.

Objective To explore the role of spinal microglial CX3CR1/ERK5 pathway in the development of neuropathic pain.Methods The model of spinal nerve ligation （SNL） was established by ligating the L5 spinal nerve with 6-0 silk thread in male Sprague Dawley rats.The expression of activated ERK5 （p-ERK5） was examined by immunohistochemistry test.To detect the role of ERK5 in neuropathic pain,PWT and PWL were measured after an intrathecal knockdown of ERK5.For determining the regulating effect of CX3CL1/CX3CR1 on the activity of microglial ERK5,CX3CR1 was blocked by an intrathecal injection of anti-rat CX3CR1 antibody and the activity of spinal ERK5 tested.Then whether an intrathecal knockdown of ERK5 could reverse the effect of CX3CL1 on pain hypersensitivity and microglia activation was investigated.Results ERK5 was activated in spinal microglia after SNL compared to the sham group （61.75 ± 11.52 vs 2.2 ±0.12; 58.01 ± 10.45 vs 1.1 ±0.11）.The knockdown of ERK5 by an intrathecal injection of antisense oligonucleotides suppressed the mechanical （15.42 ± 3.46 vs 22.7 ± 3.2g; 13.6± 2.9 vs 21.4 ± 4.1 g） and thermal hyperalgesia （13.48 ± 2.0） vs （18.0 ± 3.7） s ; （1 l.6 ± 2.3） vs （17.7 ± 1.4） s induced by nerve injury.The blockage of CX3CR1,a receptor of CX3CL1,significantly reduced the level of ERK5 activation followingSNL （30±9） vs （58±12）;（50±12） vs （36±4） （P〈0.05）.In addition,the antisense knockdown of ERK5 reversed the CX3CL1-induced hyperalgesia and spinal microglia activation.Conclusion CX3CL1/CX3CR1 regulates nerve injury-induced pain hypersensitivity through ERK5.