大鼠海马神经元TLR4介导的MyD88依赖途径在神经炎症中的作用  

The role of TLR4-mediated MyD88-dependent pathway in neuroinflammation in hippocampal neurons of rats

作　　者：张国霞[1] 周爱玲[1] 张贵萍[1] 胡亚娥[1] 茅家慧[1] 

机构地区：[1]南通大学医学院病理生理学系,江苏南通226001

出　　处：《中国应用生理学杂志》2013年第1期42-46,共5页Chinese Journal of Applied Physiology

基　　金：江苏省普通高校研究生科研创新项目(CXZZ11-0640);南通市应用研究科技计划项目(K2010036);江苏省南通大学研究生科技创新计划项目(YKC11033);江苏省高校优势学科建设工程资助项目

摘　　要：目的:研究大鼠海马神经元是否有Toll样受体4(TLR4)介导的的髓样分化因子88(MyD88)依赖途径及该途径的激活在神经炎症中的作用。方法:采用体外培养7 d的新生大鼠海马神经元,细胞免疫荧光双标法鉴定海马神经元纯度。用TLR4配体脂多糖(LPS)或TLR4抗体预处理海马神经元,以激活或阻断TLR4的作用。实时定量PCR(RT-qPCR)方法检测海马神经元中MyD88、肿瘤坏死因子受体相关因子6(TRAF6)mRNA的表达;Westernblot方法测定海马神经元MyD88和TRAF6蛋白水平;细胞免疫荧光双标法观察海马神经元中核因子κB/P65(NF-κB/P65)的表达定位及TLR4激活或阻断后NF-κB/P65核易位情况;ELISA检测培养上清液中肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)和一氧化氮(NO)的水平。结果:LPS能上调海马神经元MyD88和肿瘤坏死因子受体相关因子(TRAF6)mRNA水平;促使NF-κB/P65转位至核;增加MyD88和TRAF6蛋白的表达;增加海马神经元培养上清中TNF-α、IL-1β和NO含量;TLR4抗体预处理能减弱LPS对海马神经元NF-κB/P65核易位作用及降低培养上清中TNF-α、IL-1β和NO的水平。结论:大鼠海马神经元有TLR4介导的的MyD88依赖途径,该途径的激活能导致TNF-α、IL-lβ和NO含量的增加。海马神经元TLR4介导的MyD88依赖途径参与了神经炎症反应,神经元不是神经炎症反应中的被动者。Objective： To investigate weather there is a toll-like receptor 4（TLR4）-mediated myeloid differentiation factor 88（MyD88）-dependent pathway in hippocampal neurons of rats and the probable role of the pathway in neuroinflammation .Methods： To establish the proper model,primarily cultured hippocampal neurons were treated with lipopolysaccharides（LPS）,or pretreated with TLR4 antibody then co-treated with LPS.The expression of mRNA of MyD88 and TNF-α receptor associated factor 6（TRAF6）were tested by RT-qPCR.The content of MyD88 and TRAF6 were tested by Western blot.The nuclear translocation of nuclear factor-κB/P65（NFκB/p65） was tested by immunofluorescence.The content of tumor necrosis factorα（TNF-α）,interleukin-1β（IL-1β）and nitric oxide（NO）were tested by ELISA.Results： LPS could increase MyD88 and TRAF6 mRNA,upregulate protein level of MyD88 and TRAF6 and increase the level of TNF-α、 IL-1β and NO in cell cultrue supernatant.LPS also could promote NF-κ B/p65 translation to the nucleus.The pretreatment with TLR4 antibody reduced the translocation to nucleus for NF-κB/P65 and the contents of TNF-α,IL-1β and NO in the culture supernatant.Conclusion： There is a TLR4-mediated MyD88-dependent pathway in hippocampal neurons.The activation of this pathway can increase the level of TNF-α,IL-1β and NO in cell culture supernatant.TLR4-mediated MyD88-dependent pathway in hippocampal neurons participate in neuroinflammation,that means neurons are not passive in inflammation.

关 键 词：海马神经元 TLR4 神经炎症 MYD88 

分 类 号：R742[医药卫生—神经病学与精神病学]