1α,25-(OH)2D3通过促进RANKL的自噬性降解对抗肾性骨营养不良
1α,25-(OH)2D3 mitigate renal osteodystrophy by promoting autophagic degradation of RANKL
黄杜军 1王建坤 1王嘉嘉 1肖展豪1
作者信息
- 1. 福州市第二总医院骨科,福建 350007
- 折叠
摘要
目的 研究1α,25-二羟维生素D3(1α,25-dihydroxyvitamin D3,1α,25-(OH)2D3)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-KB ligand,RANKL).方法 用1α,25-(OH)2D3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)2D3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)2D3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 real-time PCR,qRT-PCR)检查发现,1α,25-(OH)2D3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)2D3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)2D3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)2D3可促进成骨细胞中RANKL的自噬性降解.
Abstract
Objective To research whether 1α,25-dihydroxyvitamin D3(1α,25-(OH)2D3)can degrade receptor activator of nuclear factor-κB ligand(RANKL)in osteoblasts through autophagy activation.Methods After intervention with 1α,25-(OH)2D3 in osteoblasts,the supernatant for treating osteoclast precursors was collected,and then the differentiation levels of os-teoclasts,the production levels of RANKL in the supernatant of osteoblasts,the mRNA and protein levels of RANKL in osteo-blasts and the mRNA and protein levels of Atg5,Atg7,and BECN1 were detected respectively.Finally,the pharmacological in-hibitor 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)2D3.Results Compared to the direct supernatant of osteoblasts,the super-natant treated with 1α,25-(OH)2D3 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 os-teoblasts and promoted the expression of autophagy genes(Atg5,Atg7 and BECN1)and LC3 conversion(denoted as LC3Ⅱ/Ⅰ).However,1α,25-(OH)2D3 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)2D3 could be reversed with the applica-tion of autophagy inhibitor chloroquine.Conclusion 1α,25-(OH)2D3 promote the autophagic degradation of RANKL in osteo-blasts.
关键词
1α,25-(OH)2D3/成骨细胞/破骨细胞/自噬/RANKL/肾性骨营养不良Key words
1α,25-(OH)2D3/Osteoblast/Osteoclast/Autophagy/RANKL/Renal osteodystrophy引用本文复制引用
基金项目
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)
出版年
2024
NETL
NSTL
万方数据