Cobratoxin Alleviates Cancer-Induced Bone Pain in Rats via Inhibiting CaMKII Signaling Pathway after Acting on M4 Muscarinic Cholinergic Receptors
Cobratoxin Alleviates Cancer-Induced Bone Pain in Rats via Inhibiting CaMKII Signaling Pathway after Acting on M4 Muscarinic Cholinergic Receptors
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DOI：
10.1021/acschemneuro.2c00048
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发表时间：
2022-05-04
影响因子：
5
通讯作者：
Yao, Ming
中科院分区：
文献类型：
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作者：
Xu, Longsheng;Wang, Shizhen;Yao, Ming
癌性骨痛(CIBP)是临床常见的疼痛，可降低患者的生活质量，增加患者的死亡率，但CIBP的治疗有限。本研究旨在探讨α-眼镜蛇毒素对CIBP的镇痛作用，并进一步探讨其分子靶点和潜在的信号通路。 CIBP大鼠模型的机械异常性疼痛试验显示，给予α-眼镜蛇毒素以剂量依赖性方式产生显着的镇痛作用，并且通过鞘内注射M4 mAChR-siRNA或腹腔注射托吡卡胺（M4毒蕈碱胆碱能受体拮抗剂）预处理来阻断镇痛作用。全细胞膜片钳记录表明，α-眼镜蛇毒素可以降低 CIBP 大鼠 SDH 神经元的自发放电和自发兴奋性突触后电流。在原代腰部 SDH 神经元中，细胞内钙测量显示 α-眼镜蛇毒素降低了细胞内钙浓度，免疫荧光显示 M4 毒蕈碱胆碱能受体和 CaMKII/CREB 共表达。在CIBP模型和原代SDH神经元中，Western blot显示α-眼镜蛇毒素增加了p-CaMKII和p-CREB的水平，并且α-眼镜蛇毒素的作用被M4 mAChR-siRNA拮抗。定量聚合酶链反应（qPCR）结果显示，α-眼镜蛇毒素通过SDH中的M4毒蕈碱胆碱能受体下调促炎细胞因子的表达。这些结果表明，α-眼镜蛇毒素可能激活M4毒蕈碱胆碱能受体，通过CaMKII信号通路触发SDH神经元兴奋性的抑制，从而在CIBP大鼠模型中产生拮抗作用。
Cancer-induced bone pain (CIBP) is a common pain in clinics, which can reduce the quality of life and increase the mortality of patients, but the treatment of CIBP is limited. This study was designed to investigate the analgesic effect of alpha-cobratoxin on CIBP and further to explore the molecular target and potential signal pathway. As shown by the mechanical allodynia test in a CIBP rat model, administration of alpha-cobratoxin produced significant analgesia in a dose-dependent manner, and the analgesic effects were blocked by pretreatment with an intrathecal injection of M4 mAChR-siRNA or intraperitoneal injection of tropicamide, an antagonist of M4 muscarinic cholinergic receptor. Whole-cell patch-clamp recording showed that alpha-cobratoxin can decrease the spontaneous firing and spontaneous excitatory postsynaptic currents of SDH neurons in CIBP rats. In primary lumber SDH neurons, intracellular calcium measurement revealed that alpha-cobratoxin decreased intracellular calcium concentration, and immunofluorescence demonstrated that M4 muscarinic cholinergic receptor and CaMKII/CREB were co-expressed. In the CIBP model and primary SDH neurons, Western blot showed that the levels of p-CaMKII and p-CREB were increased by alpha-cobratoxin and the effect of alpha-cobratoxin was antagonized by M4 mAChR-siRNA_ The quantitative polymerase chain reaction (qPCR) results showed that alpha-cobratoxin downregulated the expression of proinflammatory cytokines through M4 muscarinic cholinergic receptor in SDH. These results suggest that alpha-cobratoxin may activate M4 muscarinic cholinergic receptor, triggering the inhibition of SDH neuronal excitability via CaMKII signaling pathway, thereby resulting in antagonistic effects in the CIBP rat model.