甲基莲心碱调节HMGB1-RAGE信号通路对神经病理性疼痛大鼠的镇痛作用

Study on the analgesic effect of neferine on neuropathic pain rats by regulating the HMGB1-RAGE signaling pathway

目的探讨甲基莲心碱对神经病理性疼痛大鼠的镇痛作用及HMGB1-RAGE信号通路在其中发挥的作用。方法将SD大鼠分为假手术组、模型组、甲基莲心碱低剂量组、甲基莲心碱高剂量组和甲基莲心碱高剂量+rHMGB1(重组蛋白HMGB1)组,每组10只。脊背神经结扎建立神经病理性疼痛大鼠模型。机械性刺激和热敏实验检测大鼠疼痛;酶联免疫吸附法(ELISA)检测前列腺素E2(PGE2)、P物质(SP)、5-羟色胺(5-HT)、肿瘤坏死因子-α(TNF-α)、白介素-1β(IL-1β)、IL-10水平;六胺银染色观察大鼠脊髓背角形态学变化;TUNEL染色观察脊髓背角神经元凋亡;Western blot检测脊髓背角组织HMGB1、RAGE、NF-κB、p-NF-κB、Bcl-2、Bax蛋白水平。结果与对照组比较,模型组大鼠神经原纤维结构严重损伤,大鼠缩爪阈值和热敏潜伏期、IL-10水平、Bcl-2蛋白表达水平显著降低,PGE2、SP、5-HT、TNF-α、IL-1β水平、脊髓背角神经元凋亡率、HMGB1、RAGE、p-NF-κB/NF-κB、Bax蛋白表达水平显著升高(P<0.05)。与模型组比较,甲基莲心碱低、高剂量组大鼠神经原纤维结构损伤显著减轻,大鼠缩爪阈值和热敏潜伏期、IL-10水平、Bcl-2蛋白表达水平显著升高,PGE2、SP、5-HT、TNF-α、IL-1β水平、脊髓背角神经元凋亡率、HMGB1、RAGE、p-NF-κB/NF-κB、Bax蛋白表达水平显著降低(P<0.05)。重组蛋白rHMGB1可部分逆转甲基莲心碱对神经病理性疼痛大鼠的治疗作用(P<0.05)。结论甲基莲心碱通过抑制HMGB1-RAGE信号通路可减轻神经病理性疼痛大鼠炎症因子和疼痛因子表达、降低脊髓背角神经元凋亡,进而减轻疼痛。

Objective To investigate the analgesic effect of neferine on neuropathic pain rats and the involvement of the high mobility group box-1(HMGB1)-receptor for advanced glycation end-products(RAGE)signaling pathway.Methods Sprague-Dawley rats were randomly divided into sham operation group,model group,low-dose neferine group,high-dose neferine group,and high-dose neferine+rHMGB1(recombinant protein HMGB1)group.A neuropathic pain rat model was established by ligating the spinal nerve.Mechanical stimulation and thermal sensitivity experiments were applied to detect pain behaviors in rats.Enzyme-linked immunosorbent assay was applied to detect levels of prostaglandin E2(PGE2),substance P(SP),serotonin(5-HT),tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),and IL-10.Grocott methenamine silver staining was applied to observe the morphological changes in rat spinal dorsal horn.The terminal deoxynucleotidyl transferase(TdT)-mediated dUTP nick-end labelling staining was applied to observe apoptosis of spinal dorsal horn neurons.Western blot was applied to detect the protein levels of HMGB1,RAGE,nuclear factor-kappaB(NF-κB),p-NF-κB,B-cell lymphoma 2(Bcl-2),and Bcl-2-associated X protein(Bax)in spinal dorsal horn tissue.Results Compared with those of the control group,rats in the model group presented severe damages to the neural fiber structure,significantly lower paw retraction threshold,thermal sensitivity latency,IL-10 level and protein level of Bcl-2,but significantly higher levels of PGE2,SP,5-HT,TNF-αand IL-1β,apoptotic rate of spinal dorsal horn neurons,and protein expressions of HMGB1,RAGE,p-NF-κB/NF-κB and Bax(P<0.05).Compared with those of model group,rats in the low-dose and high-dose neferine groups presented significantly alleviated damages to the neural fiber structure,significantly higher paw retraction threshold,thermal sensitivity latency,IL-10 level and protein level of Bcl-2,but significantly lower levels of PGE2,SP,5-HT,TNF-αand IL-1βlevels,apoptotic rate of spinal dorsal horn neurons,and protein expressions of HMGB1,RAGE,p-NF-κB/NF-κB and Bax(P<0.05).The recombinant protein rHMGB1 was able to partially reverse the therapeutic effect of neferine on neuropathic pain in rats(P<0.05).Conclusion Neferine can reduce inflammatory and pain factor levels and apoptosis of spinal dorsal horn neurons in neuropathic pain rats by inhibiting the HMGB1-RAGE signaling pathway,and thereby alleviates pain.