Cannabinoid type 2 receptor alleviates hyperalgesia in neuropathic pain mice by inhibiting spinal microglia activation / 第二军医大学学报

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.