白脉软膏调控神经活动配体-受体及HIF-1信号通路改善背根神经节慢性压迫大鼠慢性疼痛的作用及机制研究

Baimai Ointment relieves chronic pain induced by chronic compression of dorsal root ganglion in rats by regulating neuroactive ligand-receptor interaction and HIF-1 signaling pathway

具“舒筋活络”核心功效的白脉软膏临床干预疼痛、痉挛、僵硬等症状的白脉病疗效显著,但其药效学特点及机制尚不明确。该研究建立模拟临床的腰椎间盘髓核突出压迫神经的大鼠L4背根神经节慢性压迫(chronic compression of dorsal root ganglion,CCD)模型,采用行为学、副作用评估、网络分析、拮抗剂及分子生物学验证等方法初步探讨白脉软膏治疗CCD的药效及机制。药效学研究表明,白脉软膏能明显改善CCD模型大鼠的痛敏阈值(机械痛敏、热痛敏、冷痛敏),改善步态行为,且无耐受性及明显的毒副作用;靶组织初筛结果显示白脉软膏能抑制小鼠福尔马林实验第二时相伤害性反应,升高正常小鼠的热板阈值,并降低脊髓炎症因子表达;网络分析发现白脉软膏治疗CCD具有协同性且与下行抑制/易化系统及神经炎症相关。实验进一步采用行为学、免疫印迹、免疫荧光等技术发现白脉软膏发挥CCD镇痛作用可能与其调节CHRNA7、ADRA2A、ADRB2等神经活动配体-受体(神经配基)以及降低脊髓小胶质细胞HIF-1信号通路核心调控元件NOS2/p-ERK/PI3K的表达相关。相关研究初步揭示了白脉软膏治疗白脉病的“舒筋活络”机制,并为其临床合理用药及深入研究提供了参考。

The Baimai Ointment with the effect of relaxing sinew and activating collaterals demonstrates a definite effect on Baimai disease with pain,spasm,stiffness and other symptoms,while the pharmacodynamic characteristics and mechanism of this agent remain unclear.In this study,a rat model of chronic compression of L4 dorsal root ganglion(CCD)was established by lumbar disc herniation,and the efficacy and mechanism of Baimai Ointment in the treatment of CCD were preliminarily explored by behavioral tests,side effect evaluation,network analysis,antagonist and molecular biology verification.The pharmacodynamic experiment indicated that Baimai Ointment significantly improved the pain thresholds(mechanical pain,thermal pain,and cold pain)and gait behavior of CCD model rats without causing tolerance or obvious toxic and side effects.Baimai Ointment inhibited the second-phase nociceptive response of mice in the formalin test,increased the hot plate threshold of normal mice,and down-regulated the expression of inflammatory cytokines in the spinal cord.Network analysis showed that Baimai Ointment had synergistic effect in the treatment of CCD and was related to descending inhibition/facilitation system and neuroinflammation.Furthermore,behavioral tests,Western blot,and immunofluorescence assay revealed that the pain-relieving effect of Baimai Ointment on CCD may be related to the regulation of the interaction between neuroactive ligand and receptors(neuroligands)such as CHRNA7,ADRA2A,and ADRB2,and the down-regulation of the expression of NOS2/pERK/PI3K,the core regulatory element of HIF-1 signaling pathway in spinal microglia.The findings preliminarily reveal the mechanism of relaxing sinew and activating collaterals of Baimai Ointment in the treatment of Baimai disease,providing a reference for the rational drug use and further research of this agent.