沙利度胺对大鼠紫杉醇诱发神经痛及其脊髓背角核因子-κB通路表达的影响

Effect of thalidomide on nuclear factor-κB pathway in the spinal cord dorsal horn in rats with paclitaxel-induced neuropathic pain

目的:观察沙利度胺对大鼠紫杉醇诱发神经痛及其脊髓背角核因子-κB(NF-κB)通路表达的影响。方法:清洁级健康雄性SD大鼠(河北医科大学实验动物中心提供),体重180~200 g,4~6周龄,采用随机数字表法分成4组,每组12只。紫杉醇模型组(P组):腹腔注射紫杉醇2 mg/kg,隔日1次注射,共注射4次,注射持续7 d;沙利度胺(Thalidomide)组(T组):每天口服Thalidomide 100 mg/kg,隔日1次,连续4次;紫杉醇+Thalidomide(P+T)组:腹腔注射紫杉醇2 mg/kg+每天口服Thalidomide 100 mg/kg,隔日1次,连续4次。空白对照组(C组):腹腔注射等量生理盐水并口服等量橄榄油。4组大鼠于给药前1 d (T1),给药后第7天(T2),第14天(T3)测机械缩足阈值(MWT)。于腹腔给药第14天MWT测完后腹腔注射戊巴比妥钠麻醉,快速取出脊髓背角,采用蛋白质印迹法(Western blot)法检测NF-κB p65、裂解的半胱氨酰天冬氨酸特异性蛋白酶-3(cleaved Caspase-3)蛋白表达水平;将脊髓背角组织匀浆,采用酶联免疫吸附试验(ELISA)法检测其中白细胞介素(IL)-1β和肿瘤坏死因子-α(TNF-α)蛋白表达水平。重复测量设计的计量资料比较采用重复测量的方差分析,随机区组设计的计量资料比较采用单因素方差分析。结果:与C组(14.8±1.6)比较,P组、P+T组大鼠MWT在T2(P组:5.2±0.8;P+T组:9.8±0.8, t=3.345、6.753, P<0.05)及T3 (P组:2.7±0.6;P+T组:9.7±0.7)时点均显著降低( t=5.642、7.865, P<0.05),与P组比较,T2,T3时点T组(T2:14.9±1.1;T3:15.2±1.2, t=6.483、8.964, P<0.05),P+T组大鼠MWT升高( t=3.483、5.236, P<0.05);与C组比较,P组脊髓背角NF-κBp65 (0.77±0.08)、cleaved Caspase-3 (0.88±0.11)、TNF-α (152.97±4.78) ng/L和IL-1β (83.64±3.49) ng/L均表达上调( t=15.694、6.875、10.877、7.964, P<0.05),P+T组的NF-κBp65 (0.44±0.07)、TNF-α(81.79±3.89) ng/L和IL-1β(39.76±3.31) ng/L表达也显著上调( t=6.483、12.567、5.674, P<0.05);与P组比较,T组及P+T组脊髓背角NF-κBp65 (T组:0.33±0.06;P+T组:0.44±0.07, t=9.587、10.566, P<0.05)、cleaved Caspase-3(T组:0.49±0.07;P+T组:0.51±0.09, t=9.443、7.568, P<0.05)、TNF-α(T组:(66.49±1.78) ng/L,P+T组;(81.79±3.89) ng/L, t=14.573、9.861, P<0.05)和IL-1β(T组:(24.83±2.38) ng/L;P+T组:(39.76±3.31) ng/L, t=19.313、15.465, P<0.05)表达却显著下调。 结论:沙利度胺可通过下调NF-κB及下游炎性因子IL-1β和TNF-α表达,减少cleaved Caspase-3生成,预防紫杉醇诱发神经痛发生。

Objective To investigate the effect of thalidomide on nuclear factor-κB(NK-κB)pathway in the spinal cord dorsal horn in rats with paclitaxel-induced neuropathic pain.Methods Pathogen-free male Sprague-Dawley rats,aged 4-6 weeks,weighing 180-200 g,were used in the study.All the 48 rats were randomly divided into four groups by random digital table method:normal control group(C group,n=12)intraperitoneally injected with the same volume of normal saline and orally given the same volume of olive oil,paclitaxol-treated group(P group,n=12)intraperitoneally injected with paclitaxol(2 mg/kg,once every two days for 4 consecutive times at 1,3,5 and 7 days),thalidomide-treated group(T group,n=12)intraperitoneally injected with thalidomide(100 mg/kg,once every two days for 4 consecutive times at 1,3,5 and 7 days),and paclitaxol+thalidomide group(P+T group,n=12)given intraperitoneal injection of paclitaxol(2 mg/kg)+oral administration of thalidomide(100 mg/kg),once every two days for 4 consecutive times at 1,3,5 and 7 days.Mechanical paw withdrawal threshold(MWT)was measured at time point T1(1 day before intraperitoneal administration),T2(7 days after intraperitoneal administration)and T3(14 days after intraperitoneal administration)respectively.Then the spinal cord dorsal horns were removed immediately for determination of the expression of NF-κB p65,cleaved cysteinyl aspartate-specific protease(Caspase)-3,interleukin(IL)-1βand tumor necrosis factor-α(TNF-α)by Western blotting or enzyme linked immunosorbent assay(ELISA).Results As compared with C group(14.8±1.6),Compared with group C,MWT of P,P+T group were reduced at point T2(P:5.2±0.8;P+T:9.8±0.8,t=3.345,6.753,P<0.05)and T3(P:2.7±0.6;P+T:9.7±0.7,t=5.642,7.865,P<0.05);compared with group P,MWT of rats in group T(T2:14.9±1.1;T3:15.2±1.2,t=6.483,8.964,P<0.05)and group P+T t=3.483,5.236,P<0.05)were increased at point T2 and T3.The expression of NF-κBp65(0.77±0.08),cleaved Caspase-3(0.88±0.11),TNF-α(152.97±4.78)and IL-1β(83.64±3.49)in group P in the spinal cord dorsal horn were up-regulated(t=15.694,6.875,10.877,7.964,P<0.05),the expression of NF-κBp65(0.44±0.07),TNF-α(81.79±3.89)and IL-1β(39.76±3.31)were increased in group P+T(t=6.483,12.567,5.674,P<0.05),compared with group C.Compared with group P,the expression of NF-κBp65(T:0.33±0.06;P+T:0.44±0.07,t=9.587,10.566,P<0.05),cleaved Caspase-3(T:0.49±0.07;P+T:0.51±0.09,t=9.443,7.568,P<0.05),TNF-α(T:66.49±1.78;P+T:81.79±3.89,t=14.573,9.861,P<0.05)and IL-1β(T:24.83±2.38;P+T:39.76±3.31;t=19.313,15.465,P<0.05)were decreased in the spinal cord dorsal horn of rats in group T and P+T.Conclusion Up-regulation of NF-κB and boost of inflammatory response led to activated cleaved Caspase-3 in the spinal cord dorsal horn,which may play an important role in paclitaxel-induced neuropathic pain.Thalidomide orally can inhibit nuclear factor-κB,IL-1β,TNF-αand cleaved Caspase-3 and then prevent the formation of neuralgia.