胞外信号调节激酶抑制剂对慢性压迫损伤大鼠痛觉过敏的作用

The effect of the inhibitor of extracellular signal-regulated kinase on pain hypersensitivity in rats with chronic constriction injury

目的探讨脊髓背角胞外信号调节激酶(ERK)在慢性压迫性损伤(CCI)大鼠中的作用。方法大鼠48只随机分为6组(n=8):假手术+5%二甲基亚砜(DMSO)(S组)、CCI+5%DMSO(C组)、CCI+U0126(ERK上游激酶抑制剂)0.2μg(U0.2组)、CCI+U01260.5μg(U0.5组)、CCI+U01261μg(U1组)、CCI+U01265μg(U5组),分别鞘内注射5%DMSO或U0126。记录注射后0.5,2,6,12,14h大鼠机械缩爪阈值(MWT)和热缩爪潜伏期(TWL)。另取50只CCI5d大鼠随机分为5组:G1、G2、G3、G4、G5组,鞘内注射U01265μg后0.5,2,6,12,24h取材,免疫组化法(n=6)和免疫印迹法(n=4)检测脊髓背角磷酸化胞外信号调节激酶(pERK)含量。结果U0.5组给药后0.5,2,6hMWT为(12.3±2.8)g、(11.9±3.3)g、(9.5±2.7)g;U1组给药后0.5,2,6,12hMWT为(12.6±2.6)g、(13.3±3.2)g、(11.4±3.3)g、(10.5±3.7)g;U5组给药后各时点MWT分别为(14.6±1.1)g、(13.4±1.6)g、(12.3±2.8)g、(11.7±2.7)g、(10.8±2.5)g,与C组相比明显升高有统计学差异(P<0.05,P<0.01)。U0.5组0.5、2hTWL为(15.8±2.6)s、(15.8±2.4)s;U1组0.5,2,12hTWL为(17.7±3.8)s、(16.9±4.2)s、(15.6±5.3)s;U5组各时点TWL与C组相比明显升高差异有显著性(P<0.05,P<0.01)。G1、G2、G3组大鼠脊髓结扎侧背角浅层pERK阳性神经元细胞与CCI组(23.8±4.2)比明显减少,差异有显著性(P<0.05)。G1组胞浆与胞核pERK1分别为(0.6±0.2)、(0.88±0.3),与CCI组(1.97±0.3)、(2.03±0.6)相比明显降低,差异有显著性(P<0.05)。G1组胞浆与胞核pERK2分别为(0.83±0.5)、(0.92±0.3),与CCI组(2.92±1.1)、(2.63±0.8)相比均显著减少,差异有显著性(P<0.05)。结论脊髓背角ERK级联系统参与神经病理性疼痛的信号转导过程。

Objective To determine the role of spinal cord＇ s extracellular signal-regulated kinase（ERK） in neuropathic pain. Methods In this study, model were established chronic constriction injury （CCI） in rats. 48 rats were divided into 6 group randomly （ n = 8） ： sham ＋ DMSO（ group S） , CCI ＋ DMSO（ group C） and CCI ＋ different doses of U0126 （0.2,0.5,1,5 μg/10 μl, group U0.2, U0.5, U1, U5 ）. The changes of mean withdral threshold （MWT） and thermal withdral latency（TWL） were observed at 0.5,2,6,12,24h timepoints after injection. Another 50 CCI5d rats were randomly divided into 5 groups： G1, G2, C3, C4, G5, which were received a bolus of 5.0μg U0126 and were killed at 0. 5 ,2,6 ,12 ,24h after injection respectively. The expression of spinal cord phospho-ERK （pERK） were assessed by Immunohistochemistry（ n = 6） and western blot（ n = 4）. Results The MWT of 0.5, 2, 6h of group U0.5 ; 0.5,2,6,12h of group U1 ; 0.5,2,6,12,24h of group U5 was 12.3 ± 2.8g, 11.9±3.3g, 9.5±2.7g, 14.6±1.1g, 13.4±1.6g, 12.3 ±2.8g, 11.7 ±12.7g, 10.8 ±2.5g, 12.6 ±2.6g, 13.3 ± 3.2g, 11.4 ± 3.3g, 10.5 ± 3.7g respeetively. Compared with group C were significantly promoted （ P 〈 0.05 or P〈0.01）. The TWL of 0.5, 2, 6h of group U0.5; 0.5,2,6,12h of group U1; 0.5,2,6,12,24h of group U5 was 15.8 ±2.6s, 15.8 ±2.4s, 17.7 ±3.8s, 16.9 ±4.2s, 15.6 ±5.3s, 20.7 ±4.4s, 17.1 ±5.8s, 16.6 ± 4.6s, 17.7 ± 5.5s, 15.4 ± 3.8s respectively. Compared with group C were signifieantly promoted （ P 〈 0.05 or P 〈 0.01 ）. The numbers of positive neurons of G1, G2, G3 group were 2.0 ± 1.4,9.7 ± 4.4,15.5 ± 4.2. Compared with CCI group（23.8 ±4.2） were markedly decreased （P〈0.05）. Similarly resμlt can be obtained in western blot. The cytosolic and nuclear pERK1 of G1 group were 0.64 ±0.2,0.88 ± 0.3. The cytosolic and nuclear pERK2 of G1 group were 0.83 ± 0.5,0.92 ± 0.3. Compared with CCI group were significantly decreased （ P 〈 0.05 ）. Conclusion These data suggested that ERK caspase may involve in the signal transductor of dorsal horn in neuropathic pain.