独一味单体抑制脊髓背角内AKT-mTOR信号通路缓解大鼠神经病理性痛

Neuropathic Pain Relieving in Rats by the Inhibition of Monomer of Lamiophlomis Rotata on AKT-mTOR Signaling Pathway in Spinal Dorsal Horn

目的:观察鞘内给予独一味单体8-O-乙酰山栀苷甲酯(8-O-acetyl-SM,8-OaS)对慢性神经病理性痛大鼠脊髓背角内丝苏氨酸蛋白激酶B(protein kinase B,AKT)-哺乳动物雷帕霉素靶点(the mammalian target of rapamycin,mTOR)信号转导通路表达的影响。方法:采用大鼠腰5脊神经结扎(spinal nerve ligation,SNL)构建神经病理性痛模型,使用Von-Frey细丝连续观察造模后大鼠术侧后足的痛阈改变;应用Westernblot法定量分析大鼠腰膨大节段AKT和mTOR的磷酸化水平;采用免疫荧光组织化学染色观察pAKT和磷酸化mTOR(pmTOR)在脊髓背角内的细胞定位。结果:由行为学数据可见,SNL模型大鼠机械性痛阈明显降低(P<0.01)。术后1~7 d鞘内连续给予8-OaS进行人为干预,与给药前对比差异有统计学意义(P<0.05);免疫荧光双重标记记实验结果显示,脊髓背角内pAKT与星形胶质细胞标记物胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)存在大量共表达,pmTOR在星形胶质细胞和神经元上均观察到大量阳性染色。Western blot实验结果提示,8-OaS能显著下调脊髓背角内pAKT和pmTOR蛋白的表达水平。结论:鞘内给予8-OaS可有效缓解由SNL诱导的机械性痛阈,其机制可能是8-OaS通过下调脊髓背角内AKT-mTOR信号通路的表达水平,进而抑制了星形胶质细胞的活化,从而达到缓解神经病理性痛的效果。

Objective： To observe the effect of intrathecal administration of 8-O-acetyl-SM （8-OaS） on chronic neuropathic pain in rats by inhibiting the expression of protein kinase B （AKT）-mammalian target of rapamycin （mTOR） signaling pathway in spinal dorsal horn after spinal nerve ligation. Methods ： The rat model of neuropathic pain was established by lumbar 5 spinal nerve ligation （ SNL）, and Von Frey filament was used to investigate the mechanical allodynia. Immunofiuorescent histochemistry was adopted to investigate the distribution of pAKT and pmTOR in spinal dorsal horn. The protein levels of pAKT and pmTOR in spinal dorsal horn after drug administration were quantitatively determined by Western blot. Results： Compared with that in the sham group, the paw withdrawal threshold （PWT） significantly decreased （P 〈 0.01 ）, and the intrathecal administration of 8-OaS attenuated mechanical allodynia obviously during the first day and the seventh day after the operation（ P 〈 0.05 ）. Meanwhile, double immunofluorescent staining showed the co-expression of pAKT and astrocytes marker glial fibrillary acidic protein （GFAP） , and positive labeling of pmTOR was expressed in spinal astrocytes and neurons. The results of Western blot revealed that the protein levels of pAKT and pmTOR in spinal dorsal horn were significantly reduced after the treatment of 8-OaS. Conclusion： Intrathecal administration of 8-OaS attenuates the PWT of SNL-induced chronic neuropathic pain. The underlying mechanism of the potential anti-allodynia effect of 8-OaS may be related to the suppression of spinal astrocytes via decreasing the phosphorylation of AKT-mTOR signaling pathway resulting in attenuating the development of neuropathic pain.