电针对骨癌痛大鼠DRG P2X3受体膜表达的干预及CASK调控机制研究

Cancer pain, especially bone cancer pain, greatly influences quality of life in cancer patients. The mechanism of cancer pain is complicated while cancer pain is usually controlled by opioid analgesics in the clinic due to lack of targeted therapy. Electroacupuncture (EA) has been used clinically to treat cancer pain, however, its mechanism is still unclear. In this study, bone cancer pain were established by intra-tibia injection of MRMT-1 mammary gland carcinoma cells. By measuring the changes of paw withdrawal thresholds as well as paw flinching reflexes induced by intraplantar injection of specific P2X3 receptor antagonist (A-317491) and agonist (α,β-meATP), to explore the role of P2X3 receptor played in the analgesic effect of EA on bone cancer pain. By detecting the expression and ratio of membrane and total P2X3 protein, co-location of P2X3 receptor and plasma membrane marker in DRG and current responses to α,β-meATP in DRG neurons as well as EA’s intervention, to further observe whether changes of P2X3 receptor expression in DRG neuron membrane participated in peripheral sensitization of bone cancer pain and EA’s analgesia. Using intrathecal injection of shRNA lentivirus to slience calcium/calmodulin-dependent serine protein kinase (CASK) gene, the intervention of EA on co-location and co-expression between CASK and P2X3 receptor were investigated. We further clarify EA decreasing membrane expression of P2X3 receptor and its mechanism via regulating CASK. The results of this project may elucidate the novel mechanism of EA on bone cancer pain, which contribute to the enrichment of EA’s analgesic mechanism and widespread application of EA on treating refractory pain.

癌痛是影响癌症患者生活质量的主要原因，以骨癌痛为多见。癌痛发生机制错综复杂，临床治疗主要依赖阿片药物，缺乏针对性治疗。电针已被用于癌痛治疗，其作用机制仍不清楚。本项目采用胫骨内注射MRMT-1乳腺癌细胞建立骨癌痛大鼠模型，检测大鼠缩足阈、足底注射P2X3受体拮抗/激动剂诱发缩足反应及电针干预，观察外周P2X3受体在电针抗骨癌痛中的作用；检测骨癌痛大鼠DRG中P2X3受体膜/总蛋白比、P2X3受体与细胞膜标记物共表达和α,β-meATP诱发神经元电流变化及电针干预，观察DRG P2X3受体膜表达变化在骨癌痛外周敏化和电针镇痛的作用；鞘内注射shRNA慢病毒实现CASK基因沉默，观察骨癌痛大鼠CASK与P2X3受体共存和结合情况及电针干预，探讨电针调节P2X3受体去膜锚定的CASK调控机制。通过本项目研究，从新的角度揭示电针抗骨癌痛的作用机理，为推动电针疗法在难治性疼痛的应用提供科学研究基础。

癌痛是癌症患者的主要并发症，严重影响其生存质量。随着癌症发病率递增和医疗技术手段提高，癌症患者生存期延长的同时癌痛问题日益凸显。电针疗法作为临床常用镇痛手段并NCCN指南推荐应用于癌痛治疗，但其作用机制和靶点仍不清楚。本项目采用胫骨腔内注射MRMT-1乳腺癌细胞诱发骨癌痛大鼠模型，通过检测骨癌痛大鼠患足痛阈变化，观察大鼠骨癌痛造模成功率和电针抗骨癌痛效应。结合足底注射P2X3受体特异性激动剂α,β-me ATP和特异性拮抗剂A-317491等药理手段，检测骨癌痛大鼠局部注射药物诱发的缩足反射及电针干预效应，观察外周P2X3受体在电针抗骨癌痛中的作用。通过检测骨癌痛大鼠患侧L4-6 DRG 中P2X3 受体膜蛋白和总蛋白表达及其比值变化、α,β-meATP诱发内向电流变化以及电针对其的干预，明确DRG 神经元细胞P2X3 受体膜表达变化在骨癌痛外周敏化和电针镇痛中的作用。通过鞘内注射CASK siRNA实现DRG神经元CASK基因沉默，明确CASK在骨癌痛大鼠DRG神经元P2X3受体膜锚定中的作用；通过检测大鼠CASK与P2X3受体共表达和共沉淀情况及电针对其的干预，探讨电针降低P2X3 受体膜锚定的CASK 调控机制。研究结果显示，单侧胫骨腔内注射MRMT-1乳腺癌细胞10d可诱导大鼠骨癌痛，且伴随患侧L4-6 DRG中P2X3R蛋白表达增加。电针可有效对抗大鼠骨癌痛，并抑制其DRG中P2X3R蛋白和免疫阳性细胞表达。电针有效地缓解大鼠骨癌痛，抑制骨癌痛大鼠DRG中P2X3R活性，降低P2X3R阳性细胞和蛋白表达及其膜转运，可能是电针抗大鼠骨癌痛的关键外周机制之一。CASK蛋白可能参与DRG神经元P2X3R膜锚定进而介导骨癌痛的外周敏化，但并不参与电针镇痛。本项目从P2X3受体膜锚定角度研究骨癌痛大鼠DRG神经元细胞膜P2X3受体活化和表达情况，并探究CASK蛋白在调控DRG神经元P2X3受体膜锚定中的作用及电针干预效应，从新角度揭示大鼠骨癌痛发生机制和电针抗大鼠骨癌痛的深层次作用机制，进一步丰富电针治疗癌性痛的作用机制研究，为临床电针治疗癌痛等复杂难治性疼痛提供研究依据。