舒芬太尼通过AMPK/mTOR调控自噬对小鼠神经病理性疼痛的镇痛效应机制研究

田聪贵; 徐妹; 潘海波; 王小敏

舒芬太尼通过AMPK/mTOR调控自噬对小鼠神经病理性疼痛的镇痛效应机制研究

Study on the analgesic effects of sufentanil on neuropathic pain in mice by regulating autophagy through AMPK/mTOR

摘要

摘要:
目的： 探讨舒芬太尼对小鼠神经病理性疼痛的镇痛效应具体机制。
方法： 建立小鼠神经病理性疼痛模型，将其随机分为Sham组、脊神经结扎术（SNL）组、SNL + 苏芬太尼组、SNL + 氯喹组。SNL + 舒芬太尼组小鼠灌胃给予1 μg/kg舒芬太尼，SNL + 氯喹组小鼠腹腔注射0.02 g/kg 氯喹，其余给予等体积0.9%氯化钠注射液,每天1次。用HE染色、尼氏染色观察舒芬太尼对NP小鼠脊髓背角的组织结构的影响；蛋白免疫印迹法（Western blotting）、免疫荧光检测各组小鼠神经组织中AMPK、mTOR、ULK1 蛋白表达水平，RT-qPCR检测各组小鼠神经组织中AMPK、mTOR、ULK1基因表达水平。
结果： 手术建模后，SNL组小鼠机械刺激缩足阈值和热刺激缩足阈值较Sham组均明显降低（P ＜ 0.01）；与SNL小组相比，SNL + 舒芬太尼组和SNL + 氯喹组机械刺激缩足阈值和热刺激缩足阈值升高（P ＜ 0.01）。与Sham组相比，SNL组小鼠脊髓背角细胞结构紊乱细胞肿胀，细胞严重固缩，核仁明显消失，神经元之间空隙增大。与SNL组相比，SNL + 舒芬太尼组和SNL + 氯喹组的小鼠神经组织上述病理损伤显著缓解。Western blotting、免疫荧光和q-PCR结果显示，相比于Sham组小鼠，SNL组mTOR的表达水平下降（P ＜ 0.05），AMPK、ULK1表达水平升高（P ＜ 0.05）。与SNL组相比，SNL + 舒芬太尼组小鼠的AMPK、ULK1表达水平降低（P ＜ 0.05），mTOR的表达水平升高（P ＜ 0.05）。
结论： 舒芬太尼能够通过AMPK/ mTOR 信号通路介导的自噬,从而提高小鼠的疼痛阈值达到镇痛效果。

Abstract:
Objective To investigate the specific mechanism of analgesic effects of sufentanil on neuropathic pain in mice.
Methods The model of neuropathic pain in mice was established, and randomly divided into the sham operation group (Sham group), spinal nerve ligation (SNL) group, SNL + sufentanil group and SNL + chloroquine group. The SNL + sufentanil group were given 1 μg/kg sufentanil by intragastric administration, the SNL + chloroquine group were intraperitoneally injected with 0.02g/kg chloroquine, and the rest were given the same volume of normal saline once a day. HE staining and Nissl staining were used to observe the pathological changes of spinal dorsal horn and degenerative neurons in mice with neuropathic pain, Western blotting and immunofluorescence were used to detect the protein expression levels of AMPK, mTOR and ULK1, and RT-qPCR was used to detect the expression levels of AMPK, mTOR and ULK1 genes in nerve tissues of mice in each group.
Results After surgical modeling, the mechanical stimulation foot-retraction threshold and thermal stimulation foot-retraction threshold in the SNL group were significantly lower than those in Sham group (P ＜ 0.01). Compared with the SNL group, the mechanical stimulation foot-retraction threshold and thermal stimulation foot-retraction threshold in the SNL + Sufentanil group and SNL + Chloroquine group increased (P ＜ 0.01). Compared with the Sham group, the disordered spinal dorsal horn cell structure, cell swelling, severe cell shrinkage, obvious disappearance of nucleoli and increased space between neurons were observed in the SNL group. Compared with the SNL group, the above pathological damage of nerve tissue in SNL + sufentanil group and SNL + chloroquine group significantly relieved. The results of Western blotting, immunofluorescence and q-PCR showed that compared with the Sham group, the expression levels of mTOR in the SNL group decreased (P ＜ 0.05), and the expression levels of AMPK and ULK1 increased (P ＜ 0.05). Compared with the SNL group, the expression levels of AMPK and ULK1 in SNL + Sufentanil group decreased (P ＜ 0.05), and the expression level of mTOR increased (P ＜ 0.05).
Conclusions Sufentanil can increase the pain threshold of mice through autophagy mediated by AMPK/ mTOR signaling pathway to achieve analgesic effect.

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