铁通过NF-κB通路调控小鼠破骨样细胞分化机制  

Mechanism of iron regulating differentiation of mouse monocytes to osteoclasts through NF-κB signaling pathway

作　　者：王啸[1] 孙婧悦 华俊[1] 徐又佳[1] WANG Xiao;SUN Jing-yue;HUA Jun;XU You-jia(Department of Orthopedics,the Second Affiliated Hospital of Soochow University,Suzhou 215004,Jiangsu,China;Department of Oncology,the First Affiliated Hospital of Soochow University,Suzhou 215006,Jiangsu,China)

机构地区：[1]苏州大学附属第二医院骨科,骨质疏松症临床中心,苏州大学骨质疏松诊疗技术研究所,江苏苏州215004 [2]苏州大学附属第一医院肿瘤科,江苏苏州215006

出　　处：《中华骨质疏松和骨矿盐疾病杂志》2022年第3期257-263,共7页Chinese Journal Of Osteoporosis And Bone Mineral Research

基　　金：国家自然科学基金(82003473);苏州市科技计划项目(KJXW201815);院科研预研基金项目(SDFEYBS1810)。

摘　　要：目的探讨铁对小鼠单核细胞RAW264.7向破骨样细胞分化过程的影响及核因子κB(nuclear factor kappa-B,NF-κB)信号通路在其中的作用。方法RAW264.7细胞以400μmol/L枸橼酸铁铵(ferric ammonium citrate,FAC)干预,以NF-κB受体活化因子配体(receptor activator of nuclear factor-kappa B ligand,RANKL)诱导向破骨样细胞分化,分化能力采用抗酒石酸酸性磷酸酶(tartrate resistant acid acid phosphatase,TRAP)染色及噬骨陷窝实验检测。应用RNA-Seq对FAC组与对照组细胞进行转录组测序,并在此基础上分析差异表达基因,通过基因本体(gene ontology,GO)分析、京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)通路分析对差异基因进行注释。采用凝胶电泳迁移率实验(electrophoretic mobility shift assay,EMSA)验证NF-κB胞核内结合情况,并通过抑制NF-κB信号通路,研究该通路在其中的作用。结果铁干预后,TRAP阳性染色细胞增多(P<0.01)、噬骨陷窝范围扩大;FAC组与对照组共有994个差异表达基因,其中上调基因520个,下调基因474个。GO分析结果显示,差异基因的功能集中表现在生物学过程蛋白修饰过程的正调控、细胞黏附、酶抑制剂活性等。KEGG通路分析发现差异表达mRNA多富集于NF-κB通路。EMSA结果提示,高铁环境下,细胞NF-κB通路激活。将NF-κB通路抑制后,FAC对破骨细胞的分化作用减弱。结论铁蓄积可能通过激活NF-κB信号通路,刺激破骨细胞分化过程。Objective To investigate the effects of iron accumulation on the differentiation of mouse monocyte RAW264.7 to osteoclast and the roles of nuclear factor kappa-B(NF-κB)signaling pathway.Methods RAW264.7 cells were treated with 400μmol/L ferric ammonium citrate(FAC)and induced to differentiate into osteoclast like cells by receptor activator of nuclear factor-kappa B ligand(RANKL).The differentiation ability of RAW264.7 was detected by tartrate resistant acid phosphatase(TRAP)staining and osteophagocytic lacuna test.RNA-Seq technology was used to complete transcriptome sequencing of the cells in FAC group and control group,and on this basis,the differentially expressed genes were analyzed.The function and signal pathway were explained by gene ontology(GO)analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis.Electrophoretic mobility shift assay(EMSA)was used to verify the nuclear binding of NF-κB.By inhibiting this signaling pathway,the roles of this pathway were studied.Results After iron intervention,TRAP positive staining cells and the number of bone filling pits were obviously increased(P<0.01).There were 994 differentially expressed genes in FAC group and control group,including 520 up-regulated genes and 474 down-regulated genes.GO function showed that the function of differential genes could be divided into three parts:biological process,cellular component,and molecular function,which were mainly manifested in adhesion,positive regulation of protein modification,and enzyme inhibitor activity.KEGG pathway enrichment analysis showed that the differentially expressed mRNA was mainly enriched in NF-κB signaling pathway.The results of EMSA suggested that the NF-κB signaling pathway of osteoclast like cells was significantly activated in high iron environment.After inhibiting the signal pathway,the effects of FAC on osteoclast differentiation were significantly weakened.Conclusion Iron accumulation may stimulate osteoclast differentiation by activating NF-κB signaling pathway.

关 键 词：骨质疏松症 破骨细胞 铁 信号通路 

分 类 号：R681[医药卫生—骨科学]