髓核致炎大鼠背根神经节中NF-κB和ASICs上调机制的探讨

Effect of NF-κB and ASIC3 in nerve root pain induced by exposure to nucleus pulposus in rats and related mechanisms

目的通过观察核因子-κB(NF-κB)和酸敏感离子通道(ASIC)信号通路阻断剂PDTC和阿米洛利对髓核致炎大鼠背根神经节(DRG)中NF-κB和ASIC3表达的影响,探索NF-κB和ASIC3在髓核组织诱导神经根性疼痛的可能机制。方法将80只大鼠采用随机数字表法分为5组,对照组、模型组、阿米洛利组、PDTC组和阿米洛利组+PDTC组,每组16只。对照组在暴露L5DRG后直接缝合。模型组取出大鼠尾部的髓核组织置于暴露的L5DRG上,缝合切口。阿米洛利组和PDTC组在模型组的基础上,分别于术后2周内每天腹腔内注射20μg/kg阿米洛利和20μg/kg PDTC。阿米洛利+PDTC组在模型组基础上,术后2周内每天腹腔内注射20μg/kg阿米洛利和20μg/kg PDTC。对照组和模型组不进行药物干预。造模成功后分别于术后2、4、6、8、10、12d检测大鼠后足机械刺激缩足阈值(PWT)。采用RT-PCR法和Western blot法测定ASIC3、NF-κB p65、TNF-α和IL-6 m RNA和蛋白表达水平。术后1、3、7、14d采用ELISA法检测外周血及DRG上清液中的TNF-α和IL-6水平,并检测一氧化氮(NO)水平。结果与对照组比较,术后12d时模型组中大鼠后足的PWT显著下降(P<0.01),术后14d模型组DRG中NF-κB p65和ASIC3的m RNA和蛋白表达显著上调(均P<0.05),外周血和DRG中NO、TNF-α、IL-6水平均显著上调(均P<0.01)。与模型组比较,阿米洛利组、PDTC组和阿米洛利+PDTC组大鼠后足PWT在8d内下降,之后逐渐增加,术后10、12d大鼠后足PWT明显高于模型组(均P<0.01),术后14d DRG中NF-κB p65和ASIC3的m RNA和蛋白表达明显下降(均P<0.05),外周血和DRG中NO、TNF-α、IL-6水平均显著降低(均P<0.01)。结论 NF-κB和ASIC3可相互影响,通过抑制NO及多种细胞炎性因子(TNF-α和IL-6)的表达,减轻髓核组织对DRG的炎症损伤所致的痛觉过敏,减缓神经根性疼痛。

Objective To determine the effect of NF-κB and ASIC3 in nerve root pain induced by exposure to nucleus pulposus in rats and the related mechanisms. Methods SD rats were randomly divided into control group(sham, n=18),inflammatory group(NP, n=16), inflammatory+amiloride group(Ami, n=16), inflammatory+PDTC group(PDTC, n=16), inflammatory+amiloride+PDTC group(Ami+PDTC, n=16). The nerve root inflammation model was induced by exposure of L5 dorsal root ganglion(L5 DRG) to rat nucleus pulposus(NP). NF-κB signaling pathway blocker PDTC(20μg/kg) or/and ASIC3 signaling pathway blocker amiloride(20μg/kg) were injected intraperitonealy to rats in PDTC group, Ami group and Ami+PDTC group, respectively. The mechanical pain withdrawal threshold(PWT) was detected at 2, 4, 6, 8, 10 and 12 days after modeling, respectively. The expression of ASIC3, NF-κB p65, TNF-α, IL-6 m RNA and protein in DRG were examined by real-time PCR and Western blot, respectively.TNF-α and IL-6 concentrations in peripheral blood and DRG were determined at 1, 3, 7 and14 days after the surgical procedures with ELISA method, and NO levels were examined with biochemistry test. Results Rats exposed to the NP showed decreased PWT for 12 days(P <0.01), and the expression of ASIC3, NF-κB and the levels of NO, TNF-α, IL-6 were up-regulated in DRG induced by NP 14 days after surgery(all P<0.01). After administration of amiloride and PDTC, the mechanical PWT decreased for8 days after surgery and then gradually increased in amiloride groups and/or PDTC groups. Furthermore, a significant increase in PWT in amiloride and/or PDTC groups compared with the NP model group were observed at day 10 and day 12 after surgery(all P<0.01). The expression of ASIC3 and NF-κB in DRG was down-regulated(both P<0.05), and the serum levels of NO, TNF-α,IL-6 were significantly decreased for 14 days(all P<0.01). Conclusion The interaction of NF-κB and ASIC3 may alleviate nerve root pain induced by exposure to NP through inhibition of NO and inflammatory cytokines TNF-α and IL-6 levels.