BACKGROUND Cinobufagin is the major bufadienolide of Bufonis venenum (Chansu), which has been traditionally used for the treatment of chronic pain especially cancer pain. The current study aimed to evaluate its antinociceptive effects in bone cancer pain and explore the underlying mechanisms. METHODS Rat bone cancer model was used in this study. The withdrawal threshold evoked by stimulation of the hindpaw was determined using a 2290 CE electrical von Frey hair. The β-endorphin and IL-10 levels were measured in the spinal cord and cultured primary microglia, astrocytes, and neurons. RESULTS Cinobufagin, given intrathecally, dose-dependently attenuated mechanical allodynia in bone cancer pain rats, with the projected Emax of 90% MPE and ED50 of 6.4 μg. Intrathecal cinobufagin also stimulated the gene and protein expression of IL-10 and β-endorphin (but not dynorphin A) in the spinal cords of bone cancer pain rats. In addition, treatment with cinobufagin in cultured primary spinal microglia but not astrocytes or neurons stimulated the mRNA and protein expression of IL-10 and β-endorphin, which was prevented by the pretreatment with the IL-10 antibody but not β-endorphin antiserum. Furthermore, spinal cinobufagin-induced mechanical antiallodynia was inhibited by the pretreatment with intrathecal injection of the microglial inhibitor minocycline, IL-10 antibody, β-endorphin antiserum and specific μ-opioid receptor antagonist CTAP. Lastly, cinobufagin- and the specific α-7 nicotinic acetylcholine receptor (α7-nAChR) agonist PHA-543613-induced microglial gene expression of IL-10/β-endorphin and mechanical antiallodynia in bone cancer pain were blocked by the pretreatment with the specific α7-nAChR antagonist methyllycaconitine. CONCLUSIONS Our results illustrate that cinobufagin produces mechanical antiallodynia in bone cancer pain through spinal microglial expression of IL-10 and subsequent β-endorphin following activation of α7-nAChRs. Our results also highlight the broad significance of the recently uncovered spinal microglial IL-10/β-endorphin pathway in antinociception.

中文翻译：

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脊柱小胶质细胞IL-10 /β-内啡肽途径是由cbubufagin诱导的α7烟碱乙酰胆碱受体激活后的骨癌疼痛引起的机械性异常性疼痛的机制。

背景技术西诺蟾蜍精是Bufonis venenum（Chansu）的主要丁二烯内酯，传统上已被用于治疗慢性疼痛，特别是癌症疼痛。当前的研究旨在评估其在骨癌疼痛中的镇痛作用并探索其潜在机制。方法采用大鼠骨癌模型。使用2290 CE电子冯·弗雷（von Frey）头发确定后脚刺激引起的退缩阈值。测量脊髓和培养的原发性小胶质细胞，星形胶质细胞和神经元中的β-内啡肽和IL-10水平。结果鞘内给予烟蟾毒素可剂量依赖性地减轻骨癌疼痛大鼠的机械性异常性疼痛，预计Emax为90％MPE，ED50为6.4μg。鞘内注射烟蟾蜍精还刺激骨癌疼痛大鼠脊髓中IL-10和β-内啡肽（而非强啡肽A）的基因和蛋白质表达。此外，在培养的原发性脊柱小胶质细胞中用奇诺蟾蜍而不是星形胶质细胞或神经元处理刺激了IL-10和β-内啡肽的mRNA和蛋白质表达，但用IL-10抗体而不是β-内啡肽抗血清预处理可以防止这种情况。此外，通过鞘内注射小胶质细胞抑制剂米诺环素，IL-10抗体，β-内啡肽抗血清和特异性μ阿片受体拮抗剂CTAP进行预处理，可以抑制脊髓西蟾蜍精诱导的机械性异常性疼痛。最后，骨癌疼痛中的奇诺法汀和特异性α-7烟碱乙酰胆碱受体（α7-nAChR）激动剂PHA-543613诱导的小胶质细胞IL-10 /β-内啡肽基因表达和机械抗异常性疼痛的阻断作用nAChR拮抗剂甲基甘可尼丁。结论我们的结果表明，西诺蟾蜍精通过激活α7-nAChRs后IL-10和随后的β-内啡肽的脊髓小胶质细胞表达在骨癌疼痛中产生机械性抗异常性疼痛。我们的结果还突出了最近发现的脊髓小胶质细胞IL-10 /β-内啡肽途径在抗伤害感受中的广泛意义。结论我们的结果表明，西诺蟾蜍精通过激活α7-nAChRs后IL-10和随后的β-内啡肽的脊髓小胶质细胞表达在骨癌疼痛中产生机械性抗异常性疼痛。我们的结果还突出了最近发现的脊髓小胶质细胞IL-10 /β-内啡肽途径在抗伤害感受中的广泛意义。结论我们的结果表明，西诺蟾蜍精通过激活α7-nAChRs后IL-10和随后的β-内啡肽的脊髓小胶质细胞表达在骨癌疼痛中产生机械性抗异常性疼痛。我们的结果还突出了最近发现的脊髓小胶质细胞IL-10 /β-内啡肽途径在抗伤害感受中的广泛意义。