1α,25-(OH)_(2)D_(3)通过促进RANKL的自噬性降解对抗肾性骨营养不良  

1α,25-(OH)_(2)D_(3) mitigate renal osteodystrophy by promoting autophagic degradation of RANKL

作　　者：黄杜军 王建坤 王嘉嘉 肖展豪 HUANG Dujun;WANG Jiankun;WANG Jiajia;XIAO Zhanhao(Department of Orthopedics,Fuzhou Second General Hospital,Fuzhou,Fujian 350007,China)

机构地区：[1]福州市第二总医院骨科,福建350007

出　　处：《中国骨与关节损伤杂志》2024年第9期943-948,共6页Chinese Journal of Bone and Joint Injury

基　　金：福建省自然科学基金项目(2020J011198);福建省创伤骨科急救与康复临床医学研究中心(2020Y2014)。

摘　　要：目的研究1α,25-二羟维生素D3(1α,25-dihydroxyvitamin D3,1α,25-(OH)_(2)D_(3))是否可通过自噬的激活降解成骨细胞当中的核因子κB受体活化因子配体(Receptor activator of nuclear factor-κB ligand,RANKL)。方法用1α,25-(OH)_(2)D_(3)干预成骨细胞后,收集上清液用于干预破骨前体细胞,再分别检测破骨细胞分化水平、成骨细胞上清液中RANKL的产生水平、成骨细胞中RANKL mRNA和蛋白表达水平以及Atg5、Atg7、BECN1的mRNA和蛋白水平。最后利用自噬的药理学抑制剂氯喹和拯救(Rescue)实验来验证自噬机制对于1α,25-(OH)_(2)D_(3)调控的成骨细胞RANKL产生状态的影响。结果研究结果表明,相比直接的成骨细胞上清液,1α,25-(OH)_(2)D_(3)作用后的上清液明显抑制破骨细胞的分化。酶联免疫吸附实验(Enzyme-linked immunosorbnent assay,ELISA)和实时荧光定量PCR(Quantitative realtime PCR,qRT-PCR)检查发现,1α,25-(OH)_(2)D_(3)的干预抑制成骨细胞中RANKL分泌,并促进自噬基因(Atg5、Atg7、BECN1)的表达和LC3转换(表示为LC3II/I)。但是,1α,25-(OH)_(2)D_(3)未影响成骨细胞中RANKL基因的表达,却抑制RANKL的蛋白水平。此外,1α,25-(OH)_(2)D_(3)抑制的成骨细胞RANKL的产生和蛋白水平可随自噬抑制剂氯喹的应用而逆转。结论1α,25-(OH)_(2)D_(3)可促进成骨细胞中RANKL的自噬性降解。Objective To research whether 1α,25-dihydroxyvitamin D3(1α,25-(OH)_(2)D_(3))can degrade receptor activator of nuclear factor-κB ligand(RANKL)in osteoblasts through autophagy activation.Methods After intervention with 1α,25-(OH)_(2)D_(3)in osteoblasts,the supernatant for treating osteoclast precursors was collected,and then the differentiation levels of osteoclasts,the production levels of RANKL in the supernatant of osteoblasts,the mRNA and protein levels of RANKL in osteoblasts and the mRNA and protein levels of Atg5,Atg7,and BECN1 were detected respectively.Finally,the pharmacological inhibitor of autophagy,chloroquine and the rescue experiment were used to verify the effect of autophagy on the production status of RANKL in osteoblasts regulated by 1α,25-(OH)_(2)D_(3).Results Compared to the direct supernatant of osteoblasts,the supernatant treated with 1α,25-(OH)_(2)D_(3)significantly inhibited osteoclast differentiation.Enzyme linked immunosorbent assay(ELISA)and quantitative real-time PCR(qRT-PCR)revealed that intervention with osteoclast inhibited RANKL secretion in osteoblasts and promoted the expression of autophagy genes(Atg5,Atg7 and BECN1)and LC3 conversion(denoted as LC3II/I).However,1α,25-(OH)_(2)D_(3)did not affect the expression of RANKL gene in osteoblasts,but inhibited RANKL protein levels.In addition,RANKL production and protein levels in osteoblasts inhibited by 1α,25-(OH)_(2)D_(3)could be reversed with the application of autophagy inhibitor chloroquine.Conclusion 1α,25-(OH)_(2)D_(3)promote the autophagic degradation of RANKL in osteoblasts.

关 键 词：1α 25-(OH)_(2)D_(3) 成骨细胞 破骨细胞 自噬 RANKL 肾性骨营养不良 

分 类 号：R681.3[医药卫生—骨科学]