RANKL通过ROS介导HIF-1α下调促进自噬和破骨细胞分化  

RANKL drives autophagy and osteoclast differentiation via ROS-mediated HIF-1αdown-regulation

作　　者：李松涛 孙靖[2] 初同伟 LI Songtao;SUN Jing;CHU Tongwei(Department of Orthopaedics,Second Affiliated Hospital,Army Medical University(Third Military Medical University),Chongqing;Department of Spinal Surgery,Ninth Medical Center,Chinese PLA General Hospital,Beijing,China)

机构地区：[1]陆军军医大学(第三军医大学)第二附属医院骨科,重庆 [2]中国人民解放军总医院第九医疗中心脊柱外科,北京

出　　处：《陆军军医大学学报》2025年第8期816-825,共10页Journal of Army Medical University

基　　金：重庆英才计划(CQYC2020020347)。

摘　　要：目的 探究核因子κB受体活化因子配体(receptor activator of nuclear factor-κB ligand,RANKL)通过ROS-HIF-1α信号轴调控自噬和破骨细胞分化的分子机制。方法 以RAW264.7小鼠单核/巨噬细胞系为模型,采用随机数字表法将细胞分为Control组、RANKL组及联合干预组[RANKL+CQ组(自噬抑制剂干预)、RANKL+FG-4592组(HIF-1α稳定剂干预)、RANKL+NAC组(ROS清除剂干预)]。通过抗酒石酸酸性磷酸酶(TRAP)染色定量检测成熟多核破骨细胞数量;Western blot检测HIF-1α、LC3蛋白表达水平;qRT-PCR分析破骨细胞分化相关基因(TRAP、Cath K和MMP-9)mRNA表达水平;基于腺病毒mRFP-GFP-LC3标记系统结合激光共聚焦显微镜观察自噬通量活性;DCFH-DA探针检测细胞内活性氧(ROS)水平。结果 与Control组相比,RANKL组中TRAP阳性多核破骨细胞数量显著增加(P<0.05),破骨细胞分化相关基因mRNA表达水平明显上调;同时激活自噬通路,表现为LC3-Ⅱ/LC3-Ⅰ比值升高(P<0.05)及自噬溶酶体数量增加(P<0.05);细胞内ROS水平显著升高(P<0.05),且HIF-1α蛋白表达下调(P<0.05)。与RANKL组比较,RANKL+NAC组显著抑制细胞内ROS蓄积(P<0.05),并逆转HIF-1α蛋白的低表达(P<0.05);同时降低LC3-Ⅱ/LC3-Ⅰ比值(P<0.05)及自噬通量活性(P<0.05);伴随破骨细胞分化相关基因表达水平下降(P<0.05)及成熟破骨细胞数量减少(P<0.05)。与RANKL组比较,RANKL+FG-4592组明显提升HIF-1α蛋白表达水平(P<0.05);同时抑制自噬激活[LC3-Ⅱ/LC3-Ⅰ比值降低(P<0.05);自噬通量活性减少(P<0.05)],进而减少破骨细胞分化相关基因表达(P<0.05)及成熟破骨细胞数量(P<0.05)。与RANKL组比较,RANKL+CQ组显著抑制自噬通量活性(P<0.05);同时明显减少破骨细胞分化相关基因表达水平(P<0.05)及成熟破骨细胞数量(P<0.05)。结论 RANKL通过ROS介导的HIF-1α下调激活自噬,促进破骨细胞分化。Objective To investigate the molecular mechanism by which receptor activator of nuclear factor-κB ligand(RANKL)regulates autophagy and osteoclast differentiation through the ROS-HIF-1αsignaling axis.Methods Mouse monocytic/macrophage RAW264.7 cells were randomly divided into control group,RANKL group,and 3 combined intervention groups,that is,RANKL+CQ group(intervention with autophagy inhibitor,CQ),RANKL+FG-4592 group(intervention with HIF-1αstabilizer,FG-4592),and RANKL+NAC group(intervention with ROS scavenger,NAC).The number of mature multinucleated osteoclasts was quantitatively detected with tartrate-resistant acid phosphatase(TRAP)staining;the protein levels of HIF-1αand LC3 were detected by Western blotting and the mRNA levels of osteoclast differentiation-related genes(TRAP,Cath K and MMP-9)were analyzed by qRT-PCR;autophagy flux and activity were observed by adenovirus mRFP-GFP-LC3 labeling system combined with confocal laser microscopy;and the production of intracellular reactive oxygen species(ROS)was measured by DCFH-DA probe.Results Compared with the control group,the RANKL group obtained significantly increased number of TRAP-positive multinucleated osteoclasts(P<0.05),up-regulated mRNA levels of osteoclast differentiation-related genes,activated autophagy pathway,as evidenced by the increased LC3-Ⅱ/LC3-Ⅰratio(P<0.05)and number of autophagic lysosomes(P<0.05),elevated intracellular ROS generation(P<0.05),and down-regulated HIF-1αprotein level(P<0.05).NAC intervention significantly inhibited intracellular ROS accumulation(P<0.05),reversed the lowered expression of HIF-1αprotein(P<0.05),decreased the LC3-Ⅱ/LC3-Ⅰratio(P<0.05)and autophagy flux and activity(P<0.05),and reduced expression levels of osteoclast differentiation-related genes and the number of mature osteoclasts(P<0.05).In the RANKL+FG-4592 group,HIF-1αprotein level was significantly increased(P<0.05),autophagy activation was inhibited[with decreased LC3-Ⅱ/LC3-Ⅰratio(P<0.05)and autophagy flux and activity(P<0.05)],and the

关 键 词：HIF-1Α 自噬 破骨细胞分化 活性氧 

分 类 号：R322.71[医药卫生—人体解剖和组织胚胎学] R329.28[医药卫生—基础医学] R394.2