HMGB1在去传入神经病理性疼痛模型大鼠中的表达及其与神经炎症的关系研究

Expression of high mobility group box 1 protein in deafferentation pain rat models and its relation with neuroinflammation

目的探讨高迁移率族蛋白1(HMGB1)在去传入神经病理性疼痛模型大鼠中的表达特点及其与神经炎症的关系。方法将60只SD大鼠按随机数字表法分为空白对照组(n=10)和模型组(n=50),后者通过剪断一侧C5~T1脊神经后根制备成去传入神经病理性疼痛模型。于造模后3 d、7 d、10 d、14 d和21 d时评估大鼠的疼痛相关行为学(自发性疼痛评分、机械对抗痛阈值和自噬评分),并应用免疫组化染色检测脊髓组织中HMGB1、离子钙接头蛋白抗原-1(IBA-1)及磷酸化核因子-κB(pNF-κB)的阳性表达,应用Western blotting检测脊髓组织中HMGB1、Toll样受体(TLR)2、pNF-κB蛋白的表达。结果(1)模型组大鼠造模后14 d、21 d的自发性疼痛评分、自噬评分均明显高于造模后3 d、7 d、10 d,造模后21 d的自发性疼痛评分、自噬评分均明显高于造模后14 d,差异均有统计学意义(P<0.05)。(2)免疫组化染色检测显示,造模后3 d开始模型组大鼠脊髓组织中HMGB1、IBA-1和pNF-κB即均有阳性表达,且随时间延长三者在受损侧脊髓背角区域的阳性细胞数呈升高趋势,并较未受损侧明显升高;空白对照组大鼠脊髓背角区域的三者阳性细胞数均较少,且受损侧与未受损侧间无明显差异。(3)Western blotting检测显示,与空白对照组比较,模型组造模后3 d、7 d、10 d、14 d、21 d时脊髓组织中HMGB1、TLR2、pNF-κB蛋白的表达均明显升高,差异均有统计学意义(P<0.05),并且随时间延长呈升高趋势。结论去传入神经病理性疼痛模型大鼠局部脊髓组织中HMGB1表达的逐渐升高使HMGB1/TLR2/NF-κB通路相关分子高表达和小胶质细胞激活,导致脊髓局部神经炎症的发生,最终产生疼痛相关行为学变化。

Objective To investigate the expression characteristics of high mobility group box 1 protein(HMGB1)in rat models of deafferentation pain induced by posterior root injury of spinal nerves,and its relation with neuroinflammation.Methods Sixty SD rats were divided into a blank control group(n=10)and a model group(n=50)according to random number table method.Neuropathic pain rat models in the model group were established by cutting the posterior root of C5-T1 spinal nerve,while rats in the control group were performed the same operation without cutting the posterior root of C5-T1 spinal nerve.Three,7,10,14,and 21 d after modeling,behavioral changes,including spontaneous pain scale scores,mechanical antagonistic pain threshold,and autophagy scale scores,were evaluated in the two groups of rats.Immunohistochemical staining was used to detect the HMGB1,ionized calcium binding adapter molecule 1(IBA-1)and phosphorylated nuclear factorκB(pNF-κB)positive cells in the spinal cord of the two groups.Western blotting was used to detect the protein expressions of HMGB1,toll-like receptor(TLR)2 and pNF-κB in the spinal cord of the two groups.Results(1)The scores of spontaneous pain scale and autophagy scale 14 and 21 d after modeling were significantly higher than those 3,7 and 10 d after modeling(P<0.05),and those 21 d after modeling were significantly higher than those 14 d after modeling(P<0.05).(2)Immunohistochemical staining showed that HMGB1,IBA-1 and pNF-κB all expressed in the spinal cord tissues of rats in the model group 3 d after modeling,and the number of positive cells in the dorsal horn of the spinal cord on the injured side became larger with prolongation of exposure time,and that was obviously larger as compared with that on the opposite side;in the spinal cord tissues of the blank control group,the number of positive cells in the spinal dorsal horn area was small,and there was no significant difference in the number of positive cells in the spinal dorsal horn area on both sides.(3)Western blotting showed that,as compared with those in the blank control group,HMGB1,TLR2 and pNF-κB protein expressions in the spinal cord tissues of the model group were significantly increased 3,7,10,14 and 21 d after modeling(P<0.05),and which showed an increasing trend with prolongation of exposure time.Conclusion The gradual increase in HMGB1 expression in the local spinal cord of rats with deafferentation pain leads to HMGB1/TLR2/NF-κB pathway high expression and activation of microglia cells,which induces the occurrence of local neuroinflammation in the spinal cord and eventually results in pain behavioral changes.