The antinociceptive effects of spirocyclopiperazinium salt compound DXL-A-24 on neuropathic pain and chemical-stimulated pain were investigated in this study. After the administration of DXL-A-24, the paw withdrawal latency (PWL) and mechanical withdrawal threshold (MWT) were increased in rats suffering from neuropathic pain (chronic constriction injury, CCI) on days 1, 3, 5, 7 and 14 after surgery, and pain responses were inhibited in mice stimulated with chemicals (formalin or acetic acid). In the analysis of antinociceptive targets, the effect of DXL-A-24 was blocked by a peripheral nicotinic acetylcholine receptor (nAChR) antagonist (hexamethonium, Hex) or α7 nAChR antagonist (methyllycaconitine, MLA) in the formalin test. Meanwhile, the effect of DXL-A-24 was also blocked by a peripheral muscarinic acetylcholine receptor (mAChR) antagonist (atropine methylnitrate, Amn) or M4 mAChR antagonist (tropicamide, TRO). The antinociceptive signalling pathway was explored using molecular biology methods in ipsilateral dorsal root ganglions (DRGs) of CCI rats after the administration of DXL-A-24 for 7 days. Western blot analyses showed that the increased levels of phosphorylation of calcium/calmodulin-dependent protein kinase II alpha (CaMKIIα) and cAMP response element-binding protein (CREB) were eliminated, and the qRT-PCR assay showed that the increase in the expression of Tumor necrosis factor alpha (TNF-α) mRNA was reduced. Meanwhile, immunofluorescence staining revealed that the increase in calcitonin gene related peptide (CGRP) expression was inhibited by the administration of DXL-A-24, and the effect was blocked by MLA or TRO. In conclusion, DXL-A-24 exerts significant antinociceptive effects on neuropathic pain and chemical-stimulated pain. The antinociceptive effect of DXL-A-24 is probably attributed to the activation of peripheral α7 nAChR and M4 mAChR, the subsequent inhibition of the CaMKIIα/CREB signalling pathway, and finally the inhibition of TNF-α and CGRP expression.

本研究调查了螺环哌嗪盐化合物 DXL-A-24 对神经性疼痛和化学刺激性疼痛的镇痛作用。给予 DXL-A-24 后，第 1、3、5、7 和 14 天患有神经性疼痛（慢性压迫性损伤，CCI）的大鼠的缩爪潜伏期 (PWL) 和机械缩回阈值 (MWT) 增加手术后，用化学物质（福尔马林或乙酸）刺激的小鼠的疼痛反应受到抑制。在抗伤害靶点分析中，在福尔马林试验中，DXL-A-24 的作用被外周烟碱乙酰胆碱受体 (nAChR) 拮抗剂（六甲铵，Hex）或 α7 nAChR 拮抗剂（甲基lycaconitine，MLA）阻断。同时，DXL-A-24的作用也被外周毒蕈碱乙酰胆碱受体（mAChR）拮抗剂（硝酸甲酯阿托品，Amn）或M4 mAChR拮抗剂（托吡卡胺，TRO）阻断。在给予 DXL-A-24 7 天后，使用分子生物学方法探索 CCI 大鼠同侧背根神经节 (DRG) 的镇痛信号通路。 Western blot 分析显示，钙/钙调蛋白依赖性蛋白激酶 II α (CaMKIIα) 和 cAMP 反应元件结合蛋白 (CREB) 磷酸化水平的增加被消除，qRT-PCR 检测显示，肿瘤坏死因子 α (TNF-α) mRNA 减少。同时，免疫荧光染色显示，降钙素基因相关肽（CGRP）表达的增加受到DXL-A-24给药的抑制，并且该作用被MLA或TRO阻断。总之，DXL-A-24 对神经性疼痛和化学刺激性疼痛具有显着的镇痛作用。 DXL-A-24 的镇痛作用可能归因于外周 α7 nAChR 和 M4 mAChR 的激活，随后抑制 CaMKIIα/CREB ​​信号通路，最后抑制 TNF-α 和 CGRP 表达。