Toll样受体4信号通路在脑损伤后小胶质细胞与星形胶质细胞的炎性反应作用机制

Exploration of the mechanism of Toll like receptor 4 signaling pathway in the inflammatory reaction of microglia and astrocyte after brain injury

目的观察Toll样受体4（TLR4）信号通路在脑损伤后小胶质细胞与星形胶质细胞的炎性反应中的作用。方法建立创伤性脑损伤（TBI）C57BL／10ScNJ小鼠动物模型和脂多糖（LPS）刺激的原代小胶质细胞和星形胶质细胞离体研究模型，通过免疫荧光、蛋白印记技术、实时定量聚合酶链反应（Real-time PCR）等技术研究TLR4及其下游信号通路活化。结果小鼠脑损伤后皮质区小胶质细胞数量增多，TLR4／骨髓分化初反应蛋白88（MyD88）／核转录因子-κB（NF-κB）蛋白表达显著上调；在LPS作用后，小胶质细胞和星形胶质细胞TLR4／MyD88／NF-κB蛋白表达均上调，炎性因子白细胞介素-1β（IL-1β）mRNA分别为对照组的577．03倍和888．70倍，而诱导型一氧化氮合成酶（iNOS）mRNA分别为对照组的752．32倍和320．14倍；在TLR4特异性抑制剂TAK242和NF-κB特异性抑制剂BAY117082的分别作用下，小胶质细胞IL-1β和iNOS mRNA表达均下调，在星形胶质细胞中，IL-1β和iNOS的表达也表现出同样的趋势。此外，在利用LPS作用后的小胶质细胞和星形胶质细胞的条件培养基分别刺激星形胶质细胞和小胶质细胞时，后两者的IL-1β和iNOS表达较LPS单独刺激明显上调，且此效应可以被TAK242和BAY117086分别抑制。结论小鼠遭受TBI后，脑皮质损伤区TLR4／NF-κB信号通路明显激活，小胶质细胞和星形胶质细胞通过TLR4／NF-κB信号通路产生促炎性反应，该通路活化后的炎性因子可以进一步促进彼此的炎性反应。

Objective To investigate the role of Toll like receptor 4 （TLR4）/nuclear factor-κB （NF-κB） signaling pathway in the inflammatory response of mieroglia and astroeyte after brain injury. Methods C57BL/10ScNJ mice were used to create a traumatic brain injury （TBI） model in vivo. Primary cultured microglia and primary astrocytes were also used to create an in vitro TBI models by lipopolysaccha-ride （LPS） stimulation. Downstream signaling pathways of TLR4 were detected by immunofluoreseenee, Western blotting technique and real-time quantitative polymerase chain reaction （Real -time PCR） to show the expression of inflammatory cytokines. Results In vivo, the numbers of mieroglia and apoptotie neurons were increased in cortical lesions after TBI, accompanied by TLR4/MyD88/NF-κB signaling pathway related protein up-expression. In vitro, the TLR4/MyD88/NF-κB signaling pathway related protein were up - regulated both in microglia and astroeyte, besides, the mRNA levels of interleukins-1 β （IL-1β） and inducible nitric oxide synthase （iNOS） in mieroglia were 577.03 and 888.70 times compared with control group respectively, which was much same as the astrocyte. What＇ s more, robust micro-glia proliferation also emerged. After administration of TAK242 and BAYllT086, expression of TLR4, MyD88 and phosphorylated NF-κB decreased as well as IL-1β and iNOS were upregulated both in the microglia and astroeyte. Additionally, when using conditioned medium from mieroglia and astrocyte stimulated by LPS to activate astrocytes and microglia, expression of both IL -1β and iNOS was found to significantly increase in comparison with LPS stimulation alone. Such effect can be inhibited by TAK242 and BAY117086 respectively. Conclusion In the TBI model, TLR4/NF-κB signaling pathway were activated significantly near the injured brain area. LPS can mimic TBI in vitro and activate microglia and astroeytes to produce proinflammatory responses via the TLR4/NF-κB pathway, besides, mieroglia and astro-cytes can enhance the pro-inflammation effects in astrocytes and microglia respectively through TLR4/ NF-κB pathway, the pro-inflammatory factors of which can further promote inflammatory reaction.