HMGB1/TLR4通路在白蛋白结合型紫杉醇致神经病理性疼痛大鼠脊髓的表达

Expression of HMGB1/TLR4 pathway in spinal cord in neuropathic pain induced by nab-paclitaxel in rats

目的探究高迁移率族蛋白B1(HMGB1)/Toll样受体4(TLR4)通路相关蛋白及下游炎性因子在白蛋白结合型紫杉醇所致的神经病理性疼痛大鼠脊髓组织中的表达及其在神经病理性疼痛中的作用。方法36只雄性SD大鼠随机分为紫杉醇组和对照组,每组18只,紫杉醇组实验第1,3,5,7天腹腔注射白蛋白结合型紫杉醇(2.47 mg/kg)建立神经病理性疼痛模型,对照组腹腔注射等剂量生理盐水。实验第8天处死大鼠取L_(4-6)脊髓。以机械刺激缩足反射阈值(MWT)和热刺激缩足反射潜伏期(TWL)作为痛阈观察指标;HE染色观察大鼠L_(4-6)脊髓的病理学改变,免疫组化染色检测脊髓组织中HMGB1、TLR4、星形胶质细胞活化标志物胶质纤维酸性蛋白(GFAP)及小胶质细胞活化标志物离子钙接头蛋白抗体(Iba-1)的表达变化,Western blot检测大鼠L_(4-6)脊髓TLR4通路相关蛋白MyD88、TRIF、IRF3的表达变化,ELISA检测TNF-α、IL-1β和IL-6水平。结果与对照组相比,紫杉醇组大鼠MWT、TWL均降低(P<0.05),提示神经病理性疼痛模型构建成功。HE染色结果显示,对照组大鼠L_(4-6)脊髓组织中,神经元结构完整,核膜结构清晰,核仁明显;而紫杉醇组神经细胞层次排列絮乱,神经元固缩、扭曲,细胞体变形。与对照组相比,紫杉醇组大鼠L_(4-6)脊髓组织中HMGB1、TLR4、GFAP、Iba-1、MyD88、TRIF、IRF3、TNF-α、IL-1β和IL-6的表达水平均升高(P<0.05)。结论白蛋白结合型紫杉醇诱导的神经病理性疼痛大鼠脊髓中HMGB1/TLR4通路相关蛋白及下游炎性因子的表达增加,星形胶质细胞及小胶质细胞活化增强,该变化可能参与了白蛋白结合型紫杉醇所致神经病理性疼痛的发病机制。

Objective To investigate the expression of high-mobility group protein B1(HMGB1)/Toll-like receptor 4(TLR4)pathway-related proteins and downstream inflammatory factors in rat spinal cord tissue after treatment with nab-paclitaxel,and explore its role in the neuropathic pain induced by nab-paclitaxel.Methods Thirty-six SD male rats were randomly divided into two groups:control group and paclitaxel group,with 18 in each group.A neuropathic pain model was established by intraperitoneal injection of nab-paclitaxel(2.47 mg/kg)at days 1,3,5,7.Rats in control group were given an equal dose of normal saline.At day 8,the L_(4-6) spinal cord of rats was collected.The mechanical withdrawal threshold(MWT)and the thermal withdrawal latency(TWL)of rats in each group were detected to assess the pain threshold.HE staining was used to observe the pathological changes of L_(4-6) spinal cord of rats.Immunohistochemical staining was used to detect the expression of HMGB1,TLR4,glial fibrillary acidic protein(GFAP)and ionized calcium binding adaptor molecule-1(Iba-1).Western blot was used to detect the expression of TLR4 pathway-related proteins including MyD88,TRIF and IRF in the L_(4-6) spinal cord tissue of rats.ELISA was used to detect the levels of inflammatory factors including tumor necrosis factorα(TNF-α),interleukin-1β(IL-1β)and interleukin-6(IL-6).Results Compared with control group,MWT and TWL were decreased in paclitaxel group(P<0.05),suggesting that the neuropathic pain model was successfully constructed.HE staining showed that the neuron exhibited structural integrity in L_(4-6) spinal cord tissue of rats in control group,the membrane structure of nuclear was clear,and the nucleolus was obvious.In paclitaxel group,the structure of neural cells was disorderly and unsystematic,and the neurons were pyknotic,distorted and deformed.Compared with control group,the expression of HMGB1,TLR4,GFAP,Iba-1,MyD88,TRIF,IRF3,TNF-α,IL-1βand IL-6 in L_(4-6) spinal cord tissue in paclitaxel group were significantly increased(all P<0.05).Conclusion The expression of HMGB1/TLR4 pathway-related proteins and downstream inflammatory factors are increased in spinal cord of rats with neuropathic pain induced by nab-paclitaxel,and the activation of astrocytes and microglia is enhanced,which may be involved in the pathogenesis of neuropathic pain induced by nab-paclitaxel.