| 摘要: |
| 目的 探讨脊髓2型大麻素受体（CB2R）与小胶质细胞活化对神经病理性疼痛小鼠痛觉过敏的影响。方法 若干健康雄性C57/BL小鼠随机分为6组：假手术组、脊神经结扎（SNL）组、SNL+CB2R激动剂（AM1241）组、SNL+小胶质细胞抑制剂（米诺环素）组、SNL+CB2R小干扰RNA（siRNA）组（SNL+siRNA组）、SNL+CB2R小干扰RNA+小胶质细胞抑制剂组（SNL+siRNA+米诺环素组）。采用SNL方法建立神经病理性疼痛小鼠模型。采用蛋白质印迹法检测小鼠脊髓组织中CB2R和小胶质细胞活化特异蛋白钙离子结合调节因子1（IBA-1）的蛋白表达，Von Frey纤维丝测定小鼠机械性痛阈，免疫荧光观察脊髓背角IBA-1荧光定量表达，qRT-PCR测定小鼠脊髓透析液中炎性因子释放水平，电生理技术观察CB2R激动剂对脊髓背角自发性抑制性突触后电流（sIPSC）的影响。结果 与假手术组相比，SNL组小鼠脊髓组织中CB2R表达减少（P<0.012 5），痛阈降低（P<0.016 7），IBA-1荧光定量和蛋白表达均增高（P均<0.008 3），炎性因子TNF-α、IL-1β、IL-6 mRNA表达均增加（P均<0.008 3）。鞘内注射CB2R激动剂AM1241或小胶质细胞抑制剂米诺环素后，与SNL组相比，SNL+AM1241、SNL+米诺环素组小鼠的痛阈均升高（P均<0.008 3），IBA-1荧光定量和蛋白表达均降低（P均<0.008 3），TNF-α、IL-1β、IL-6 mRNA表达均减少（P均<0.008 3）。siRNA靶向干扰CB2R表达后，与SNL组相比，SNL+siRNA组小鼠的痛阈降低（P<0.008 3），IBA-1荧光定量和蛋白表达均增加（P均<0.008 3），TNF-α、IL-1β、IL-6 mRNA表达均增加（P均<0.008 3）；而鞘内注射米诺环素逆转了siRNA靶向干扰CB2R表达导致的上述变化（P均<0.008 3）。AM1241体外干预能够增强小鼠脊髓背角sIPSC的频率和振幅，与干预前相比差异均有统计学意义（P均<0.05），而米诺环素持续处理抑制了AM1241对sIPSC的增强效应。结论 CB2R通过抑制脊髓小胶质细胞活化减轻神经炎症反应、增强抑制性电活动，减轻小鼠神经病理性疼痛的痛觉过敏。 |
| 关键词: 大麻酚受体CB2 小神经胶质细胞 神经病理性疼痛 炎症 脊髓 痛觉过敏 |
| DOI：10.16781/j.0258-879x.2020.10.1115 |
| 投稿时间：2019-11-30修订日期：2020-01-06 |
| 基金项目:国家自然科学基金（81671082）． |
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| Cannabinoid type 2 receptor alleviates hyperalgesia in neuropathic pain mice by inhibiting spinal microglia activation |
| KONG Er-liang1,WU Fei-xiang2,BAI Yun-hu3,ZHANG Yang1,ZHONG Cheng-yue1,FENG Xu-dong1* |
(1. Department of Anesthesiology, No. 988 Hospital of Logistic Support Forces of PLA, Zhengzhou 450042, Henan, China;
2. Department of Anesthesiology, Eastern Hepatobiliary Surgery Hospital, Naval Medical University(Second Military Medical University), Shanghai 200438, China;
3. Department of General Surgery, No. 988 Hospital of Logistic Support Forces of PLA, Zhengzhou 450042, Henan, China
*Corresponding author) |
| Abstract: |
| Objective To investigate the effects of spinal cannabinoid type 2 receptor (CB2R) and microglia activation on hyperalgesia in neuropathic pain mice. Methods Male C57/BL mice were randomly divided into six groups:sham, spinal nerve ligation (SNL), SNL+CB2R agonist AM1241 (SNL+AM1241), SNL+microglia inhibitor minocycline (SNL+ minocycline), SNL+small interfering RNA (siRNA) targeting CB2R (SNL+siRNA), and SNL+siRNA+minocycline groups. A neuropathic pain mouse model was established by SNL. The expression levels of spinal CB2R and microglia-specific protein ionized calcium-binding adapter molecule 1 (IBA-1) were determined by Western blotting, mechanical pain thresholds were measured by Von Frey, spinal microglia activation was observed by IBA-1 immunofluorescence, and the expression levels of inflammatory factors in spinal cord dialysate were determined by quantitative real-time polymerase chain reaction (qRT-PCR). Electrophysiology was applied to observe the effect of CB2R agonist on spontaneous inhibitory postsynaptic current (sIPSC) in the spinal dorsal horn. Results Compared with the sham group, the expression of CB2R in spinal cord was significantly decreased in the SNL group (P<0.012 5), the pain threshold was significantly reduced (P<0.016 7), the fluorescence quantification and protein expression of IBA-1 were significantly increased (both P<0.008 3), and the mRNA expression levels of tumor necrosis factor α (TNF-α), interleukin (IL)-1β and IL-6 were significantly increased (all P<0.008 3). After intrathecal injection of CB2R agonist AM1241 or microglial inhibitor minocycline, compared with the SNL group, the pain thresholds of mice were significantly increased in the SNL+AM1241 and SNL+minocycline groups (both P<0.008 3), the fluorescence quantification and protein expression of IBA-1 were significantly decreased (both P<0.008 3), and the mRNA expression levels of TNF-α, IL-1β and IL-6 were significantly decreased (all P<0.008 3). After targeted interfering CB2R expression by siRNA, compared with the SNL group, the pain threshold was significantly decreased in the SNL+siRNA group (P<0.008 3), the fluorescence quantification and protein expression of IBA-1 were significantly increased (both P<0.008 3), and the mRNA expression levels of TNF-α, IL-1β and IL-6 were significantly increased (all P<0.008 3); while intrathecal injection of minocycline significantly reversed the above changes (all P<0.008 3). Intervention in vitro of AM1241 could significantly enhance the frequency and amplitude of sIPSC in the spinal dorsal horn (both P<0.05), while continuous treatment with minocycline inhibited the enhancement effects of AM1241 on sIPSC. Conclusion CB2R can reduce the neuroinflammatory responses and enhance the inhibitory electrical activity in the spinal cord by inhibiting spinal microglia activation, thereby alleviating hyperalgesia of neuropathic pain in mice. |
| Key words: cannabinoid receptor CB2 microglia neuropathic pain inflammation spinal cord hyperalgesia |
