Department of Neurobiology and Acupuncture Research, The Third School of Clinical Medicine, Key Laboratory of Acupuncture and Neurology of Zhejiang Province, Zhejiang Chinese Medical University, 548 Binwen Road, Binjiang District, Hangzhou, 310053, Zhejiang Province, China.
Department of Acupuncture and Moxibustion, Sanya Traditional Chinese Medicine Hospital, Sanya, 572000, China.
Purinergic Signal. 2023 Mar;19(1):13-27. doi: 10.1007/s11302-022-09861-7. Epub 2022 Apr 28.
Upregulation of P2X3 receptor (P2X3R) has been strongly implicated in nociceptive signaling including bone cancer pain (BCP). The present study, using rat bone cancer model, aimed to explore the role of P2X3R in regulating rat pain behavior under the intervention of electroacupuncture (EA). The BCP model was successfully established by injection with MRMT-1 breast cancer cell into the medullary cavity of left tibia for 3 × 10 cells/3 μL PBS in rats as revealed by obvious bone destruction, decreased paw withdrawal thresholds (PWTs), and reduced paw withdrawal latencies (PWLs). Western blot analyses showed that P2X3R expression was significantly upregulated in ipsilateral lumbar 4-6 (L4-6) dorsal root ganglia (DRG), but the difference not seen in spinal cord dorsal horn (SCDH). With the in-depth study of P2X3R activation, we observed that intrathecal injection of P2X3R agonist α,β-meATP aggravated MRMT-1 induced BCP, while injection of P2X3R inhibitor A-317491 alleviated pain. Subsequently, we demonstrated that BCP induced mechanical allodynia and thermal hyperalgesia were attenuated after EA treatment. Under EA treatment, total P2X3R protein expression in ipsilateral DRGs was decreased, and it is worth mentioning that decreased expression of P2X3R membrane protein, which indicated that both the expression and membrane trafficking of P2X3R were inhibited by EA. The immunofluorescence assay showed that EA stimulation exerted functions by reducing the expression of P2X3R-positive cells in ipsilateral DRGs of BCP rats. Ca imaging analysis revealed that the EA stimulation decreased the percentage of α,β-meATP responsive neurons in DRGs and inhibited calcium influx. Notably, the inhibitory effect of EA on mechanical allodynia and nociceptive flinches was abolished by intrathecal injection of α,β-meATP. These findings demonstrated EA stimulation ameliorated mechanical allodynia and thermal hyperalgesia in rat model of MRMT-1-induced BCP. EA exerts analgesic effect on BCP by reducing the overexpression and functional activity of P2X3R in ipsilateral DRGs of BCP rats. Our work first demonstrates the critical and overall role of P2X3R in EA's analgesia against peripheral sensitization of MRMT-1-induced BCP and further supports EA as a potential therapeutic option for cancer pain in clinic.
嘌呤能受体 P2X3（P2X3R）的上调被强烈认为与伤害性信号转导有关，包括骨癌痛（BCP）。本研究使用大鼠骨癌模型，旨在探讨 P2X3R 在电针（EA）干预下调节大鼠疼痛行为中的作用。通过将 MRMT-1 乳腺癌细胞注入大鼠左侧胫骨骨髓腔，每 3μL PBS 注射 3×10 个细胞，成功建立 BCP 模型，结果显示明显的骨破坏、足底撤回阈值（PWT）降低和足底撤回潜伏期（PWL）缩短。Western blot 分析显示，同侧腰椎 4-6（L4-6）背根神经节（DRG）中 P2X3R 表达明显上调，但脊髓背角（SCDH）中未见差异。随着对 P2X3R 激活的深入研究，我们观察到鞘内注射 P2X3R 激动剂 α，β-meATP 加重了 MRMT-1 诱导的 BCP，而 P2X3R 抑制剂 A-317491 则缓解了疼痛。随后，我们证明 EA 治疗后可减轻 BCP 诱导的机械性痛觉过敏和热痛觉过敏。在 EA 治疗下，同侧 DRG 中总 P2X3R 蛋白表达减少，值得注意的是，P2X3R 膜蛋白表达减少，这表明 EA 抑制了 P2X3R 的表达和膜转运。免疫荧光检测显示，EA 刺激通过降低 BCP 大鼠同侧 DRG 中 P2X3R 阳性细胞的表达发挥作用。钙成像分析显示，EA 刺激降低了 DRG 中对 α，β-meATP 有反应的神经元的百分比，并抑制了钙内流。值得注意的是，鞘内注射 α，β-meATP 可消除 EA 对机械性痛觉过敏和伤害性退缩的抑制作用。这些发现表明，EA 刺激通过降低 BCP 大鼠同侧 DRG 中 P2X3R 的过度表达和功能活性，改善了 MRMT-1 诱导的 BCP 大鼠的机械性痛觉过敏和热痛觉过敏。EA 通过降低 BCP 大鼠同侧 DRG 中 P2X3R 的过度表达和功能活性，对 MRMT-1 诱导的 BCP 外周敏化发挥镇痛作用。我们的工作首次证明了 P2X3R 在 EA 对抗 MRMT-1 诱导的 BCP 外周敏化的镇痛作用中的关键和整体作用，并进一步支持 EA 作为临床癌症疼痛的潜在治疗选择。
