HMGN1激活TLR4/MyD88/NF-κB p65/IKK-β信号通路诱导小鼠BV2小胶质细胞活化上调其促炎介质表达  

High mobility group nucleosome binding protein 1(HMGN1)induces activation of mouseeBV22 microglia andupregulates their pro-inflammatoryymediator expression by activating TLR4/MyD88/NF-kB p65/IKK-βsignal pathway

作　　者：毛燕 俞佳丽 袁静[1,2,3] 达静静 余福勋[1,2,3] 查艳 MAO Yan;YU Jiali;YUAN Jing;DA Jingjing;YU Fuxun;ZHA Yan(Department of Nephrology,Guizhou Provincial People's Hospital,Guiyang 550002;School of Medicine,Guizhou University,Guiyang 550025;NHC Key Laboratory of Pulmonary Immunological Disease,Guizhou Provincial People's Hospital,Guiyang 550002,China)

机构地区：[1]贵州省人民医院肾内科,贵州贵阳550002 [2]贵州大学医学院生物医学系,贵州贵阳550025 [3]国家卫生健康委员会肺脏免疫性疾病诊治重点实验室,贵州贵阳550002

出　　处：《细胞与分子免疫学杂志》2024年第2期135-141,共7页Chinese Journal of Cellular and Molecular Immunology

基　　金：中国医学科学院中央级公益性科研院所基本科研业务费专项资金(2019PT320003);贵州省高层次创新人才项目(黔科合平台人才[2018]5636-2);贵州省肾脏病临床医学研究中心(黔科合平台人才[2020]2201号);2021年贵州省卫生健康委员会科学技术基金(gzwkj2021-136);2021年贵州省人民医院青年基金(GZSYQN[2021]12号);2022年贵州省基础研究计划(自然科学项目)(黔科合基础-ZK[2022]一般265);贵州省中医药管理局中医药、民族医药科学技术课题(QZYY-2023-018)。

摘　　要：目的 探究高迁移率族核小体结合蛋白1(HMGN1)对小鼠BV2细胞炎症反应的影响并探究其可能的机制。方法使用(0、 100、 200、 500、 1000、 2000)ng/mL的重组HMGN1孵育BV2细胞6 h。用显微镜观察细胞形态变化,实时定量PCR检测细胞中肿瘤坏死因子α(TNF-α)、白细胞介素6(IL-6)、 IL-1β、单核细胞趋化蛋白1(MCP-1) mRNA水平。随后把小胶质细胞随机分成对照组、模型组、抑制剂组和拮抗剂组。模型组用500 ng/mL的HMGN1处理BV2细胞,拮抗剂组在模型组的基础上加入瑞沙托维(resatorvid)/TAK-242进行干预,使用实时定量PCR和免疫荧光细胞化学染色检测细胞中的M1/M2型标志物的表达情况,Western blot法检测诱导型一氧化氮合酶(iNOS)、 Toll样受体4(TLR4)、髓样分化因子88(MyD88)、核因子κB p65(NF-κB p65)和NF-κB抑制物激酶β(IKK-β)的蛋白表达。结果 HMGN1处理后,BV2细胞形态发生明显变化,呈阿米巴样;与0 ng/mL HMGN1组相比,TNF-α、 IL-6、 IL-1β、 MCP-1的mRNA水平随HMGN1剂量的增加而升高;M1型标志物iNOS的mRNA水平随HMGN1剂量的增加而升高,M2型标志物CD206的水平随HMGN1剂量的升高而降低。与模型组相比,拮抗剂组M1型标志物iNOS的mRNA水平显著降低,M2型标志物CD206的水平显著升高。免疫荧光细胞化学染色结果也显示MI型标志物iNOS在拮抗剂组中的表达降低。Western blot的结果提示,拮抗剂组iNOS、 TLR4、 MyD88、 NF-κB p65和IKK-β的蛋白表达显著降低。结论 HMGN1可能通过激活TLR4/MyD88/NF-κB p65/IKK-β信号通路诱导BV2小胶质细胞活化来上调其促炎介质的水平。Objective eTo explore the effects and mechanism of high-mobility group nucleosome-binding protein 1(HMGN1)on the inflammatory response of mouse BV2 microglia.Methods BV2 cells were incubated with recombinant HMGN1 at different concentrations(0,100,200,500,1000,2000 ng/mL)for 6 hours,and the morphological changes were observed under a microscope.The mRNA levels of tumor necrosis factorα(TNF-α),interleukin-6(IL-6),interleukin-1β(IL-1β)and monocyte chemotactic protein 1(MCP-1)were detected by real time quantitative PCR.Microglial cells were then randomly divided into a control group,model group,inhibitor group and antagonist group.The cells in the model group were treated with 500 ng/mL HMGN1,while the antagonist group was treated with 500 ng/mL TAK-242(resatorvid),a Toll-like receptor 4(TLR4)antagonist,in addition to HMGN1.Real time quantitative PCR and immunofluorescence were used to detect the expression of M1/M2 markers in the four groups,and Western blot analysis was used to measure the protein expression levels of inducible nitric-oxide synthase(iNOS),TLR4,myeloid differentiation factor88(MyD88),nuclear factor kB p65(NF-kB p65)and inhibitor of NF-kB(IkB)kinaseβ(IKK-β).Results After the treatment of HMGN1,the morphology of BV2 cells changed significantly,showing an amoeba-like appearance.The mRNA levels of TNF-α,IL-6,IL-1βand MCP-1 increased with the HMGN1 concentration,with a statistically significant difference compared to the 0 ng/mL HMGN1 group.At the same time,the mRNA level of iNOS,a M1 phenotype marker,increased with the HMGN1 concentration,while the level of CD206,a M2 phenotype marker,decreased with HMGN1 concentration,showing a statistically significant difference compared to the O ng/mL HMGN1 group.Compared with the model group,the mRNA level of M1 phenotypic marker iNOS in the antagonist group was significantly lower,and the level of M2 phenotypic marker CD206 was significantly higher.The results of immunofluorescence cytochemistry also showed that the expression of M1 phenotypic marker iNos

关 键 词：高迁移率族核小体结合蛋白1(HMGN1) BV2小胶质细胞 下丘脑 炎症 Toll样受体4(TLR4) 

分 类 号：R364.5[医药卫生—病理学] R338.27[医药卫生—基础医学] R392-33