TLR4介导的神经炎性在甲基苯丙胺引起神经损伤中的作用

Role of TLR4-mediated neuroinflammatory response in Methamphetamine-induced neural damage

目的探讨Toll样受体4(Toll like receptor 4,TLR4)介导的神经炎性在甲基苯丙胺(Methamphetamine,Meth)引起神经损伤中的作用。方法51只C57BL/6J雄性小鼠随机分为3个剂量组,分别是对照(Saline)组、低剂量Meth组(10 mg/kg·bw)和高剂量Meth组(30 mg/kg·bw),腹腔给药,4次/d,每次间隔2 h。进一步采用TLR4缺陷型(TLR4^(-/-))小鼠,探讨TLR4介导神经炎性的毒性作用,分组如下:野生型和TLR4^(-/-)小鼠随机分为Saline组和Meth染毒组,染毒后旷场实验观察小鼠运动情况,Y迷宫实验观察小鼠学习记忆能力。Western blot法检测小鼠海马组织TLR4、Peli1、MyD88、TRIF、炎性通路MAPK和NF-κB相关蛋白表达。免疫组化法观察TNF-α表达水平并检测突触蛋白表达。结果与Saline组比较,TLR4、MyD88、TRIF和Peli1在低剂量及高剂量Meth组蛋白表达均明显上调,MAPK和NF-κB通路激活,表现为p-ERK、p-JNK和p-p38及NF-κB磷酸化水平升高。野生型小鼠与TLR4^(-/-)型小鼠分别给予Meth,发现Meth可导致小鼠体重明显下降,而TLR4^(-/-)小鼠染毒后其体重下降程度明显减轻。旷场实验发现,Meth染毒野生型小鼠运动距离变长,Y迷宫中进入新异臂的次数减少,突触后蛋白PSD95表达明显下调,而棘突标志物Drebrin亦有降低趋势,TLR4下游Peil1、TRIF表达上调,MAPK和NF-κB通路激活,而在TLR4^(-/-)小鼠海马组织中,上述现象被逆转。免疫组化结果显示,Meth暴露后野生型小鼠TNF-α表达上调而TLR4^(-/-)小鼠则无明显改变。结论Meth可能通过TLR4下游的TRIF-Peli1炎症相关蛋白介导MAPK和NF-κB通路的激活,促进炎症因子释放,引起神经元炎性损伤。因此,TLR4可作为Meth神经炎性反应干预的潜在靶点,具有一定的治疗意义。

Objective To investigate the role of Toll-like receptor 4(TLR4)-mediated neuroinflammatory response in methamphetamine(Meth)-induced neural damage.Methods 51 C57BL/6J male mice were randomly divided into three groups:the Saline group,the low-dose Meth group(10 mg/kg·bw)and the high-dose Meth group(30 mg/kg·bw).The mice were administrated intraperitoneally 4 times a day with an interval of 2 hours.Furthermore,TLR4-defective(TLR4^(-/-))mice were used to explore the toxic effect of TLR4-mediated neuroinflammation.The groups were as follows:wild-type mice and TLR4-deficient mice were both randomly divided into the saline group and the Meth treated group.After Meth exposure,open field test and Y maze experiment were conducted to observe the movement as well as the learning and memory ability respectively.Western blot was used to detect Peli1,MyD88,TRIF,MAPK and NF-κB in the downstream pathway of TLR4 in mouse hippocampal tissues.Moreover,immunohistochemical assay was applied to investigate the expression level of inflammatory factor TNF-αin hippocampus.Results Compared with the Saline group,the protein expressions of TLR4,MyD88,TRIF and Peli1 were significantly up-regulated at both of low and high doses of Meth,meanwhile,the MAPK and NF-κB pathways were activated,manifesting as increased levels of p-ERK,p-JNK,p-p38 and p-NF-κB.Meth exposure contributed to dramatical decrease of body weight of wild-type mice,an effect was significantly reversed in TLR4-deficient mice.Additionally,the open field test showed that the exercise distance of wild-type mice after Meth exposure increased,and the Y-maze test showed that the number of entering new foreign arms in Meth exposure wild type mice was decreased.Further result revealed that the expression of postsynaptic protein PSD95 in the hippocampus of mice treated with Meth was significantly down-regulated,and the marker of spines,Drebrin,also decreased.In the Meth exposure hippocampal tissues of wild-type mice,the expression of Peil1 and TRIF,which lie downstream of TLR4,was up-regulated and the MAPK and NF-κB pathways were activated in wild-type mice,however,these effects were markedly reversed in TLR4 deficient mice.The immunohistochemistry result showed that the expression of inflammatory factor TNF-αin the hippocampus of wild type mice was up-regulated after Meth exposure,but no significant changes in TLR4 deficient mice were observed.Conclusion Meth may mediate the activation of MAPK and NF-κB pathway through TLR4 downstream TRIF-peli1 axis,promoting the release of inflammatory cytokines,and mediated neuronal inflammatory damage.Therefore,TLR4 can be recognized as a potential target for the intervention of Meth-mediated neuroinflammatory response,which displays certain therapeutic significance.