抑制IRE1α通路影响破骨细胞增殖分化的体外研究  

In Vitro Study on Effect of Inhibiting IRE1αPathway on Osteoclast Proliferation and Differentiation

作　　者：于成博 张志翔 玉伊琳 曹颖光[1,2,3] 宋珂 YU Chengbo;ZHANG Zhixiang;YU Yilin;CAO Yingguang;SONG Ke(Department of Stomatology,Tongji Hospital Affiliated to Tongji Medical College,Huazhong University of Science and Technology,Wuhan 430030,China;School of Stomatology,Tongji Medical College,Huazhong University of Science and Technology,Wuhan 430030,China;Key Laboratory of Oral and Maxillofacial Development and Regeneration,Hubei Province,Wuhan 430022,China)

机构地区：[1]华中科技大学同济医学院附属同济医院口腔科,湖北武汉430030 [2]华中科技大学同济医学院口腔医学院,湖北武汉430030 [3]湖北省口腔颌面发育与再生重点实验室,湖北武汉430022

出　　处：《口腔医学研究》2025年第2期122-127,共6页Journal of Oral Science Research

基　　金：国家自然科学基金面上项目(编号:82170933、82470958);湖北省杰出青年项目(编号:2023AFA106)。

摘　　要：目的:探究肌醇需要酶1α(insitol-requiring enzyme 1α,IRE1α)通路在破骨细胞分化中的作用。方法:通过使用巨噬细胞集落刺激因子和核因子κB受体活化因子配体处理小鼠单核巨噬细胞系RAW264.7,诱导其向破骨细胞分化。使用Western blot和实时荧光定量聚合酶链反应(real-time fluorescence quantitative polymerase chain reaction,qRT-PCR)检测破骨细胞分化相关基因和蛋白表达水平,同时检测IRE1α通路的表达情况。使用IRE1α的核糖核酸内切酶抑制剂4μ8c处理RAW264.7,CCK-8法检测其对细胞增殖的影响。在诱导RAW264.7细胞破骨分化的同时使用4μ8c处理细胞,抗酒石酸酸性磷酸酶染色评估4μ8c对破骨细胞形成的影响,Western blot检测破骨细胞分化相关蛋白和IRE1α通路蛋白的表达水平。使用4μ8c处理RAW264.7,同时使用细菌脂多糖刺激细胞模拟炎症环境,qRT-PCR检测破骨分化相关炎性因子表达。结果:IRE1α通路的表达在破骨细胞分化过程中增加,4μ8c不仅显著抑制了破骨前体细胞的增殖,同时抑制了破骨细胞的形成,并下调了破骨细胞分化相关因子:活化T细胞核因子1、基质金属蛋白酶-9和组织蛋白酶K的蛋白表达水平。结论:破骨细胞分化过程中IRE1α通路被激活。抑制IRE1α通路不仅能抑制破骨细胞的形成和增殖,还能下调破骨细胞标志蛋白和破骨分化相关促炎因子的表达。Objective:To investigate the role of inositol demanding enzyme 1α(IRE1α)pathway in osteoclast differentiation.Methods:Mouse monocyte macrophage line RAW264.7 was induced to differentiate into osteoclasts by treating it with macrophage colony stimulating factor(M-CSF)and receptor activator of nuclear factor kappa-B ligand(RANKL).Western blot and real-time fluorescence quantitative polymerase chain reaction(qRT-PCR)were used to detect the expression levels of osteoclast differentiation related genes and proteins,as well as the expression of the IRE1αpathway.RAW264.7 was treated with IRE1αRNase inhibitor 4μ8c,and its effect on cell proliferation was detected by CCK-8 assay.During the induction of osteoclast differentiation,4μ8c was used to treat the cells.The effect of 4μ8c on osteoclast formation was evaluated by tartrate resistant acid phosphatase(TRAP)staining,and the expression levels of osteoclast differentiation related proteins and IRE1αpathway proteins were detected by Western blot.RAW264.7 was treated with 4μ8c and stimulated with bacterial lipopolysaccharides(LPS)to simulate an inflammatory environment.qRT-PCR was used to detect the expression of inflammatory factors related to osteoclast differentiation.Results:The expression of IRE1αpathway increased during osteoclast differentiation,and 4μ8c not only significantly inhibited the proliferation of osteoclast precursor cells,but also inhibited osteoclast formation,and downregulated the protein levels of osteoclast differentiation related factors:nuclear factor of activated T-cells(NFATc1),matrix metalloproteinase-9(MMP-9),and cathepsin K(CTSK).Conclusion:The IRE1αpathway is activated during osteoclast differentiation.Inhibiting the IRE1αpathway not only inhibits the formation and proliferation of osteoclasts,but also downregulates the expression of osteoclast marker proteins and pro-inflammatory factors related to osteoclast differentiation.

关 键 词：肌醇需要酶1α 破骨细胞 牙周炎 

分 类 号：R78[医药卫生—口腔医学]