Toll样受体4在同型半胱氨酸诱导巨噬细胞铁死亡中的作用  

Role of Toll⁃like receptor 4 in regulation of homocysteine⁃induced ferroptosis in macrophages

作　　者：翟俊杰 温少莹 李心如 孙睿 祁宁 张启凡 杨力[2] 黄晖[2] 马凌桔 郝银菊 姜怡邓 李桂忠 马胜超[1,4] ZHAI Junjie;WEN Shaoying;LI Xinru;SUN Rui;QI Ning;ZHANG Qifan;YANG Li;HUANG Hui;MA Lingju;HAO Yinju;JIANG Yideng;LI Guizhong;MA Shengchao(Key Laboratory of Metabolic Cardiovascular Disease Research of National Health Commission,Yinchuan 750004,Ningxia,China;不详)

机构地区：[1]国家卫生健康委员会代谢性心血管疾病研究重点实验室,宁夏银川750004 [2]宁夏医科大学总医院老年与特需医学科,宁夏银川750004 [3]宁夏医科大学检验学院,宁夏银川750004 [4]宁夏医科大学基础医学院,宁夏银川750004

出　　处：《实用医学杂志》2025年第3期313-321,共9页The Journal of Practical Medicine

基　　金：国家自然科学基金青年项目(编号:81900273);国家自然科学基金面上项目(编号:82060139,82270492);宁夏回族自治区重点研发计划项目(编号:2019BEG03006,2022BEG02054,2023BEG02074,2022BFH02013);宁夏自然科学基金优秀青年项目(编号:2023AAC05035);宁夏医科大学揭榜挂帅项目(编号:XJKF240301,XJKF240305)。

摘　　要：目的探讨Toll样受体4在调控同型半胱氨酸(homocysteine,Hcy)诱导巨噬细胞铁死亡中的作用。方法培养小鼠巨噬细胞RAW264.7,设置对照组(Control)、同型半胱氨酸干预组(Hcy)和同型半胱氨酸+铁死亡抑制剂组(Hcy+Fer-1);巨噬细胞转染干扰片段后予同型半胱氨酸干预,设置对照组(Control)、同型半胱氨酸干预组(Hcy)、TLR4干扰阴性对照+同型半胱氨酸干预组(si-NC+Hcy)、TLR4干扰+同型半胱氨酸干预组(si-TLR4+Hcy);巨噬细胞转染过表达慢病毒后予同型半胱氨酸干预,对照组(Control)、同型半胱氨酸干预组(Hcy)、TLR4过表达阴性对照+同型半胱氨酸干预组(OE-NC+Hcy)、TLR4过表达+同型半胱氨酸干预组(OE-TLR4+Hcy);干预细胞48 h后,实时荧光定量PCR和Western blot检测Hcy干预的巨噬细胞中TLR4表达水平;Western blot检测巨噬细胞中铁死亡相关蛋白ACSL4、GPX4及FTH1表达水平;采用铁离子试剂盒检测巨噬细胞内Fe^(2+)浓度;采用ROS试剂盒及激光共聚焦显微镜检测细胞内活性氧的含量。结果与Control组相比,Hcy组促铁死亡相关蛋白ACSL4的表达水平升高(P<0.05),抗铁死亡相关蛋白GPX4与FTH1的表达水平降低(P<0.05),Fe^(2+)浓度升高(P<0.05),ROS含量增多,同时Hcy组中TLR4蛋白及mRNA表达水平均升高(P<0.05);巨噬细胞转染TLR4干扰片段后,与si-NC+Hcy组相比,si-TLR4+Hcy组中GPX4与FTH1表达水平增高(P<0.05),ACSL4表达水平降低(P<0.05),Fe^(2+)浓度降低(P<0.05),ROS含量减少;巨噬细胞转染TLR4过表达慢病毒后,与OE-NC+Hcy组相比,OE-TLR4+Hcy组中GPX4与FTH1表达水平降低(P<0.05),ACSL4表达水平升高(P<0.05)。结论Hcy诱导巨噬细胞铁死亡的发生,而Toll样受体4对Hcy诱导的巨噬细胞铁死亡有正向调控作用。Objective To investigate the role of Toll-like receptor 4(TLR4)in the regulation of homocysteine(Hcy)-induced ferroptosis in macrophages.Methods Mouse macrophage cells RAW264.7 were cultured and divided into control group,Hcy intervention group(Hcy group),and Hcy plus ferroptosis inhibitor group(Hcy+Fer-1 group).After transfection with interference fragments,macrophages were treated with Hcy,and then divided into control group,Hcy intervention group(Hcy group),TLR4 interference negative control plus Hcy intervention group(si-NC+Hcy group),and TLR4 interference plus Hcy intervention group(si-TLR4+Hcy group).Macrophages were transfected with overexpression lentivirus and treated with Hcy,then were divided into control group,Hcy intervention group(Hcy group),a TLR4 overexpression negative control plus Hcy intervention group(OE-NC+Hcy group),and a TLR4 overexpression plus Hcy intervention group(OE-TLR4+Hcy group).After 48 hours of intervention,real-time fluorescent quantitative PCR and western blot were used to detect the expression levels of TLR4 in macrophages treated with Hcy;western blot was used to detect the expression levels of ferroptosis-related proteins ACSL4,GPX4,and FTH1 in macrophages,and ferrous ion assay kit to detect the concentration of Fe^(2+)in macrophages;reactive oxygen species(ROS)assay kit and laser confocal microscopy were used to detect the content of intracellular reactive oxygen species.Results Compared with those in the control group,the expression level of the pro-ferroptosis protein ACSL4 was increased in the Hcy group(P<0.05),while the expression levels of anti-ferroptosis proteins GPX4 and FTH1 were decreased(P<0.05);the concentration of Fe^(2+)was increased(P<0.05),and the content of ROS was increased.Meanwhile,the protein and mRNA expression levels of TLR4 were both increased in the Hcy group(P<0.05).After macrophages were transfected with TLR4 interference fragments,compared with those in the si-NC+Hcy group,the expression levels of GPX4 and FTH1 were increased(P<0.05);the expressio

关 键 词：TLR4 巨噬细胞 同型半胱氨酸 铁死亡 

分 类 号：R34[医药卫生—基础医学]