GLPK通过H3K23琥珀酰化依赖的线粒体稳态介导RAW264.7巨噬细胞炎症反应  

GLPK mediates inflammatory response in RAW264.7 macrophages through H3K23 succinylation-dependent mitochondrial homeostasis

作　　者：刘宇翔 梅健 朱祥瑞 欧琅琳 庞祥明 孟子童 唐瑜璟 沈傲 文诗晴 马翠 LIU Yuxiang;MEI Jian;ZHU Xiangrui;OU Langlin;PANG Xiangming;MENG Zitong;TANG Yujing;SHEN Ao;WEN Shiqing;MA Cui(Department of Immunology,College of Medical Laboratory Science and Technology,Harbin Medical University(Daqing),Daqing 163319,China)

机构地区：[1]哈尔滨医科大学大庆校区医学检验与技术学院免疫教研室,163319

出　　处：《免疫学杂志》2024年第9期687-693,共7页Immunological Journal

基　　金：国家自然科学基金(82170059);黑龙江省自然科学基金(ZD2023H003)。

摘　　要：目的 探讨甘油激酶(GLPK)对脂多糖(LPS)诱导的小鼠Raw264.7巨噬细胞炎症反应的调控作用。方法 体外培养Raw264.7巨噬细胞,构建LPS诱导的细胞炎症模型。利用RT-qPCR技术检测细胞炎症因子NF-κB、TNF-α、IL-6和IL-1β的转录水平;Western blot、免疫荧光检测GLPK表达与定位;Western blot检测细胞泛琥珀酰修饰与H3K23su的表达水平;染色质免疫沉淀定量PCR(ChIP-qPCR)检测H3K23su表达在抑炎因子IL-10启动子的富集水平;使用DCFH-DA探针检测细胞总活性氧(ROS)生成情况、Mito-SOX探针检测线粒体ROS水平、通过JC-1线粒体膜电位荧光探针检测线粒膜电位变化反映线粒体功能异常情况;采用GLPK过表达质粒转染细胞以检测GLPK对炎症反应、线粒体功能及琥珀酰化修饰的影响。结果 LPS诱导的Raw264.7细胞发生线粒体功能异常、炎症反应加剧、琥珀酰化修饰减少与GLPK蛋白表达下降。LPS处理组给予过表达GLPK后可以改善线粒体功能,减少炎症因子的转录水平。ChIP-qPCR结果显示,过表达GLPK能够逆转LPS抑制的IL-10启动子H3K23su修饰,抑制细胞的炎症反应。结论 LPS通过GLPK依赖的H3K23琥珀酰化修饰介导小鼠Raw264.7巨噬细胞的炎症反应。Objective To elucidate the regulatory effects of Glycerol Kinase(GLPK)on the inflammatory response induced by lipopolysaccharide(LPS)in mouse Raw264.7 macrophages.Methods Raw264.7 macrophages were cultured in vitro,and an inflammatory model was established through LPS induction.The transcriptional levels of inflammatory cytokines NF-κB,TNF-α,IL-6,and IL-1βwere quantified using RT-qPCR.The expression and localization of GLPK were examined via Western blot and immunofluorescence.Additionally,Western blot analysis was employed to detect the levels of cellular pan-succinylation and H3K23su expression.ChIP-qPCR was utilized to assess the enrichment of H3K23su modification at the IL-10 promoter.The total reactive oxygen species production was measured using DCFH-DA probes,while mitochondrial ROS levels were determined with Mito-SOX probes.Mitochondrial membrane potential changes,indicative of mitochondrial dysfunction,were evaluated using JC-1 fluorescent probes.Furthermore,GLPK overexpression plasmids were transfected into cells to investigate the effects of GLPK on inflammatory responses,mitochondrial function,and epigenetic modifications.Results LPS treatment led to mitochondrial dysfunction,inflammatory responses exacerbation,succinylation modifications reduction,and GLPK protein expression decrease in Raw264.7 cells.Overexpression of GLPK in LPS-treated cells improved mitochondrial function and reduced the transcription of pro-inflammatory cytokines.ChIP-qPCR analysis revealed that GLPK overexpression could reverse the LPSinduced suppression of H3K23su modification at the IL-10 promoter,thereby attenuating the inflammatory response.Conclusion LPS mediates inflammatory responses in Raw264.7 macrophages through a GLPK-dependent H3K23 succinylation modification mechanism.

关 键 词：甘油激酶 线粒体稳态 琥珀酰化修饰 RAW264.7巨噬细胞 炎症反应 

分 类 号：R392.1[医药卫生—免疫学]