S100A9激活NF-кB促进小胶质细胞TLR7表达和炎症因子释放  

S100A9 activates NF-κB to promote TLR7 expression andinflammatory cytokine release in microglia

作　　者：白巧 周鑫 张小印 赵珊珊 陈立[2] 刘永刚 BAI Qiao;ZHOU Xin;ZHANG Xiaoyin;ZHAO Shanshan;CHEN Li;LIU Yonggang(Laboratory of Stem Cell and Tissue Engineering,College of Basic Medicine,Chongqing Medical University,Chongqing 400016,China;Children’s Hospital,Chongqing Medical University,Chongqing 400016,China)

机构地区：[1]重庆医科大学基础医学院干细胞与组织工程研究室,重庆400016 [2]重庆医科大学附属儿童医院,重庆400016

出　　处：《神经解剖学杂志》2023年第6期624-632,共9页Chinese Journal of Neuroanatomy

基　　金：重庆医科大学未来医学青年创新团队(W0037)。

摘　　要：目的:S100钙结合蛋白A9(S100A9)激活核因子κB(NF-κB)促进小胶质细胞toll样受体7(TLR7)的表达和炎症因子释放的作用及其机制研究。方法:CCK-8实验检测BV2小胶质细胞的增殖率;转录组测序并结合GO分析、KEGG富集分析和STRING数据库对差异基因(DEGs)进行比对并从差异表达基因中筛选出目标基因;Real time RT-PCR验证TLR7的表达;免疫荧光染色检测CD68、CD206的表达;Western Blot检测CD68、CD206、TLR7、p65、p-p65的表达;ELISA检测白细胞介素-6(IL-6)和肿瘤坏死因子-α(TNF-α)的表达。结果:中等浓度的S100A9对小胶质细胞无增殖抑制效应;实验组CD68蛋白的表达水平较对照组明显增加,而CD206蛋白的表达水平明显下降,提示S100A9促进BV2小胶质细胞向促炎型激活;Toll样受体4(TLR4)的抑制剂TAK-242明显抑制S100A9刺激BV2小胶质细胞后TNF-α和IL-6的表达水平;TLR4/NF-κB通路激活促进TLR7蛋白表达。结论:中等浓度的S100A9可以促进小胶质细胞向促炎型极化,通过激活TLR4/NF-κB通路促进TLR7表达和包括TNF-α和IL-6在内的多种炎症因子的释放,S100A9具有明显的促炎作用。Objective:To investigate the effect of S100 calcium-binding protein A9(S100A9)activation of nuclear factor kappa-B(NF-κB)on the upregulation of toll-like receptor 7(TLR7)expression and the release of inflammatory factors in microglia,as well as its underlying mechanism.Methods:The viability of BV2 microglia was assessed using CCK-8 kit.Transcriptome sequencing was employed to compare differential genes(DEGs)and identify target genes from the pool of differentially expressed genes.This analysis was complemented by GO analysis,KEGG enrichment analysis and the STRING database.The expression of TLR7 mRNA was verified by real time RT-PCR.The expressions of CD68 and CD206 were detected using immunofluorescence.The expressions of CD68,CD206,TLR7,p65,and p-p65 were detected using Western Blot.The level of interleukin 6(IL-6)and tumor necrosis factor alpha(TNF-α)were verified by ELISA.Results:Moderate concentrations of S100A9 had no inhibitory effect on microglial viability.Compared to the control group,the experimental group showed a significant increase in the expression level of CD68 protein,while the CD206 protein was decreased.This suggests that S100A9 promotes the activation of BV2 microglia into pro-inflammatory types.TAK-242,an inhibitor of toll-like receptor 4(TLR4),significantly inhibited the expression levels of TNF-αand IL-6 after S100A9 stimulated BV2 cells.Activation of the TLR4/NF-κB pathway promoted the expression of TLR7 protein.Conclusion:The moderate concentration of S100A9 can promote the polarization of microglia towards a proinflammatory direction.It also promotes the expression of TLR7 and the release of various inflammatory factors,including TNF-αand IL-6,through the activation of the TLR4/NF-κB pathway.This activation has an obvious proinflammatory effect.

关 键 词：S100钙结合蛋白A9(S100A9) 小胶质细胞 toll样受体7(TLR7) 肿瘤坏死因子-α(TNF-α) 白细胞介素-6(IL-6) 

分 类 号：R741[医药卫生—神经病学与精神病学]