白细胞介素1α诱导破骨细胞活化和骨流失  

Interleukin-1 alpha induces osteoclast activation and bone loss

作　　者：杨锐娟 李阳阳 蔡瑞艳[1] 刘慧兵[1] 郭春[1,2] Yang Ruijuan;Li Yangyang;Cai Ruiyan;Liu Huibin;Guo Chun(Henan Key Laboratory of Neural Regeneration,First Affiliated Hospital of Xinxiang Medical University,Weihui 453100,Henan Province,China;Department of Orthopedics,First Affiliated Hospital of Xinxiang Medical University,Weihui 453100,Henan Province,China)

机构地区：[1]新乡医学院第一附属医院河南省神经修复重点实验室,河南卫辉453100 [2]新乡医学院第一附属医院骨科,河南卫辉453100

出　　处：《中国组织工程研究》2022年第23期3691-3699,共9页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金资助项目(81672190),项目负责人:郭春;河南省卫生健康委员会项目(SB201903013),项目负责人:郭春;河南省科学技术厅项目(182102310260),项目负责人:郭春;新乡医学院第一附属医院青年基金项目(QN-2020-B11),项目负责人:杨锐娟。

摘　　要：背景:白细胞介素1是一种重要的促炎细胞因子,已被证实在调节骨炎症和骨重建中发挥重要作用。有研究表明,白细胞介素1α可诱导MC3T3-E1细胞凋亡,同时抑制成骨细胞分化。目的:探讨白细胞介素1α在小鼠破骨细胞活化和骨流失中的作用及机制。方法:①细胞实验:分别以白细胞介素1α单独或与核因子κB受体活化因子配体(receptor activator of nuclear factor-κB ligand,RANKL)联合作用于RAW264.7细胞1 d和4 d。CCK8检测细胞活力,抗酒石酸酸性磷酸酶染色检测多核破骨细胞,实时荧光定量PCR、免疫荧光染色及Western blot检测破骨形成相关的特异基因及核转录因子κB和Wnt/β-catenin信号通路相关基因的mRNA和蛋白表达情况;分别以白细胞介素1α单独或与RANKL和巨噬细胞集落刺激因子联合作用于骨髓源性巨噬细胞7 d,抗酒石酸酸性磷酸酶染色检测多核破骨细胞,Western blot检测破骨形成相关的特异基因的蛋白水平的表达情况。②动物实验:将小鼠随机分为2组:对照组腹腔注射PBS,实验组腹腔注射白细胞介素1α溶液,每周2次,5周后取材,采用μCT、苏木精-伊红染色、抗酒石酸酸性磷酸酶染色和免疫荧光分析小鼠股骨骨组织变化及相关基因的表达情况。结果与结论:①细胞实验结果显示,白细胞介素1α单独干预可显著促进RAW264.7细胞增殖,而白细胞介素1α与RANKL联合作用可刺激RAW264.7细胞向破骨细胞分化(P<0.05);在RANKL或RANKL+巨噬细胞集落刺激因子存在的情况下,白细胞介素1α明显上调RAW264.7细胞和骨髓源性巨噬细胞中破骨细胞相关标志物的表达(P<0.05),并增加抗酒石酸酸性磷酸酶阳性多核破骨细胞的数量(P<0.05);白细胞介素1α显著激活RAW264.7细胞的核转录因子κB和Wnt/β-catenin信号通路(P<0.05);阻断核转录因子κB或Wnt3信号通路不仅逆转了白细胞介素1α引起的RAW264.7细胞的核转录因子κB和Wnt3信号通路的激�BACKGROUND:Interleukin-1 is an important pro-inflammatory cytokine that has been documented in the regulation of bone inflammation and bone remodeling.A previous study has demonstrated that interleukin-1αcan induce apoptosis while inhibiting osteoblast differentiation in MC3T3-E1 cells.OBJECTIVE:To investigate the role and mechanism of interleukin-1αon osteoclast activation and bone loss in mice.METHODS:(1)Cell test:RAW264.7 cells were either treated with interleukin-1αalone or with receptor activator of nuclear factor-κB ligand(RANKL)for 1 and 4 days.Cell viability was tested by cell counting kit-8 assay.The number of multinuclear osteoclasts was detected by tartrate resistant acid phosphatase assay.The mRNA and protein levels of osteoclast-specific genes and genes related to nuclear factor-κB pathway and Wnt/β-catenin pathway were tested by real-time fluorescence quantitative PCR,immunofluorescence staining or western blot.Bone marrow-derived macrophages were either treated with interleukin-1αalone or with RANKL and macrophage colony-stimulating factor for 7 days.The number of multinuclear osteoclasts was detected by tartrate resistant acid phosphatase assay.The protein levels of osteoclast-specific genes were tested by western blot.(2)Animal test:Twenty-four male C57BL/6J mice(6-8 weeks old)were assigned into two groups at random:control group and test group.Mice were subsequently treated with interleukin-1αsolution or PBS by intraperitoneal injection twice a week for 5 weeks.Bone tissues from the femurs were performed with micro-computed tomography analysis and hematoxylin-eosin staining,tartrate resistant acid phosphatase,and immunofluorescence analysis.RESULTS AND CONCLUSION:Cell test:Interleukin-1αalone significantly increased RAW264.7 cell proliferation,but stimulated cell differentiation into osteoclasts in combination with RANKL(P<0.05).Interleukin-1αsignificantly increased the expression of osteoclast-related markers and the number of tartrate resistant acid phosphatasepositive multinuclear c

关 键 词：白细胞介素1Α 破骨细胞 RAW264.7细胞 骨髓来源巨噬细胞 抗酒石酸酸性磷酸酶 核因子ΚB受体活化因子配体 骨丢失 μCT 核转录因子κB信号通路 Wnt/β-catenin信号通路 

分 类 号：R363[医药卫生—病理学]