基于脊髓背角5-羟色胺及其受体调控途径探讨腕踝针治疗神经病理性疼痛的作用机制

ObjectiveTo explore the mechanism of Wrist-Ankle Acupuncture (WAA) in treating neuropathic pain (NP) via 5-hydroxytryptamine (5-HT) and its receptors in the spinal dorsal horn.MethodsA total of 78 clean-grade male Sprague-Dawley rats were randomly assigned into six groups after one week of acclimatization：sham surgery group, model group, WAA group, 5-HT_1A antagonist group, 5-HT_2A antagonist group, and 5-HT₃ antagonist group (18, 18, 18, 8, 8, 8 rats, respectively). Except for the sham group, all other groups underwent chronic constriction injury (CCI) to induce neuropathic pain. From day 7 post-CCI, the WAA group and the three antagonist groups received WAA treatment once daily for 7 days. The sham and model groups were handled similarly without acupuncture. On days 9, 11, and 13 post-surgery, the antagonist groups received respective intraperitoneal injections of WAY-100635, Ketanserin, or Ondansetron 30 minutes before WAA intervention. Pain behavior assessments [paw withdrawal threshold (PWT) and paw withdrawal latency (PWL)] were conducted before surgery and on days 7 and 14 post-surgery. Immunofluorescence was used to detect tryptophan hydroxylase-2 (TPH-2) expression in the dorsal raphe nucleus (DRN). Glial markers in the spinal dorsal horn-ionized calcium-binding adapter molecule 1 (Iba-1) and glial fibrillary acidic protein (GFAP) were observed under a fluorescence microscope. ELISA was used to measure spinal dorsal horn levels of 5-HT and its receptors (5-HT1A, 5-HT2A, 5-HT3), and qRT-PCR was employed to assess mRNA expression levels of inflammatory cytokines interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α).Results(1) Pain Behavior：Compared with the sham group, the model group showed significantly reduced PWT and PWL on days 7 and 14 post-surgery (<italic>P</italic>&lt;0.05). The WAA group showed significantly increased PWT and PWL on day 14 compared to the model group (<italic>P</italic>&lt;0.05). In contrast, the 5-HT_1A and 5-HT₃ antagonist groups showed significantly decreased PWT and PWL compared to the WAA group (<italic>P</italic>&lt;0.05), while the 5-HT_2A antagonist group showed a significant decrease only in PWT (<italic>P</italic>&lt;0.05), with no significant change in PWL (<italic>P</italic>&gt;0.05). (2) TPH-2 and 5-HT Levels：TPH-2 expression in the DRN and 5-HT levels in the spinal dorsal horn were significantly increased in the model group compared to the sham group (<italic>P</italic>&lt;0.05), but significantly decreased in the WAA group compared to the model group (<italic>P</italic>&lt;0.05). (3)5-HT Receptor Expression：The model group showed significantly decreased expression of 5-HT_1A and 5-HT₃ receptors and increased expression of 5-HT_2A receptor compared to the sham group (<italic>P</italic>&lt;0.05). The WAA group showed significantly increased expression of 5-HT_1Aand 5-HT₃ receptors and decreased expression of 5-HT_2A receptor compared to the model group (<italic>P</italic>&lt;0.05). (4) Glial Activation：The model group showed significantly higher Iba-1 and GFAP positivity in the spinal dorsal horn than the sham group (<italic>P</italic>&lt;0.05). The WAA group showed significantly reduced Iba-1 and GFAP positivity compared to the model group (<italic>P</italic>&lt;0.05). These levels were significantly elevated again in the 5-HT_1A and 5-HT₃ antagonist groups compared to the WAA group (<italic>P</italic>&lt;0.05), while no significant changes were observed in the 5-HT_2A antagonist group (<italic>P</italic>&gt;0.05). (5) Inflammatory Cytokines：IL-1β and TNF-α mRNA expression in the spinal dorsal horn were significantly increased in the model group compared to the sham group (<italic>P</italic>&lt;0.05) and significantly reduced in the WAA group compared to the model group (<italic>P</italic>&lt;0.05). These levels were significantly elevated again in the 5-HT_1A and 5-HT₃ antagonist groups compared to the WAA group (<italic>P</italic>&lt;0.05), while the 5-HT_2A antagonist group showed no significant differences (<italic>P</italic>&gt;0.05).ConclusionWAA effectively alleviates hyperalgesia in CCI model rats. The mechanism may involve the suppression of TPH-2 activity in the DRN, modulation of 5-HT and its receptors in the spinal dorsal horn, and inhibition of microglial activation to exert anti-inflammatory effects.