自噬在骨保护素对破骨细胞及其前体凋亡过程的调控机制

doi:10.11843/j.issn.0366-6964.2018.08.022

畜牧兽医学报 ›› 2018, Vol. 49 ›› Issue (8): 1761-1769.doi: 10.11843/j.issn.0366-6964.2018.08.022

自噬在骨保护素对破骨细胞及其前体凋亡过程的调控机制

刘庆羊^1,2,3,4, 孙自强^1,2,3,4, 赵鸿雁^1,2,3,4, 宋瑞龙^1,2,3,4, 顾建红^1,2,3,4, 刘宗平^1,2,3,4*

1. 扬州大学兽医学院, 扬州 225009;
2. 扬州大学比较医学研究院, 扬州 225009;
3. 江苏高校动物重要疫病与人兽共患病防控协同创新中心, 扬州 225009;
4. 扬州大学教育部农业与农产品安全国际合作联合实验室, 扬州 225009

收稿日期:2018-02-02 出版日期:2018-08-23 发布日期:2018-08-23
通讯作者: 刘宗平(1964-),男,博士,教授,主要从事动物营养代谢病与中毒病研究,Tel:0514-87991448,E-mail:liuzongping@yzu.edu.cn
作者简介:刘庆羊(1991-),女,江苏淮安人,硕士生,主要从事动物营养代谢病与中毒病研究,E-mail:jz1893652@163.com
基金资助:
国家自然科学基金（31672620；31502128；31702304）；江苏省高校优势学科建设工程资助项目（PAPD）

Effects of Autophagy on the Apoptosis of Osteoclasts and Osteoclast Precursors Mediated by Osteoprotegerin

LIU Qing-yang^1,2,3,4, SUN Zi-qiang^1,2,3,4, ZHAO Hong-yan^1,2,3,4, SONG Rui-long^1,2,3,4, GU Jian-hong^1,2,3,4, LIU Zong-ping^1,2,3,4*

1. College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China;
2. Institute of Comparative Medicine, Yangzhou University, Yangzhou 225009, China;
3. Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou 225009, China;
4. Joint International Research Laboratory of Agriculture and Agri-Product Safety of the Ministry of Education of China, Yangzhou University, Yangzhou 225009, China

Received:2018-02-02 Online:2018-08-23 Published:2018-08-23

摘要/Abstract

摘要：

为了探讨骨保护素（osteoprotegerin，OPG）对破骨细胞及其前体自噬与凋亡的影响，本研究以RAW264.7细胞诱导分化形成的破骨细胞（osteoclasts，OCs）及其前体（osteoclast precursors，OCPs）为研究对象，通过自噬促进剂雷帕霉素（rapamycin，RAP）或自噬抑制剂氯喹（chloroquine，CQ）分别预处理1 h和30 min后，再添加80 ng·mL^-1OPG作用细胞6 h，试验分为对照组（Con）、OPG、CQ、CQ+OPG、RAP和RAP+OPG组，利用免疫印迹法（Western blot）检测细胞自噬相关蛋白；流式细胞术Annexin V-FITC/PI双标记法检测细胞凋亡率。结果显示，与对照组相比，OPG组OCPs的Atg5和Beclin-1水平极显著或显著上调（P<0.01或P<0.05），但LC3-Ⅱ无显著差异（P>0.05）；与OPG组相比，RAP+OPG组OCPs中的LC3-Ⅱ水平极显著上调（P<0.01）；与对照组相比，OPG组OCs中LC3-Ⅱ、Atg5和Beclin-1水平极显著下调（P<0.01）；与OPG组相比，RAP+OPG组OCs的Atg5和Beclin-1水平极显著下调（P<0.01）。与对照组相比，OPG处理组和RAP+OPG组OCPs凋亡率极显著上升（P<0.01），CQ+OPG组凋亡率无显著差异（P>0.05）；RAP+OPG比OPG组OCPs凋亡率极显著增加（P<0.01）；与对照组相比，OPG组OCs凋亡率极显著降低（P<0.01），CQ+OPG组凋亡率极显著下降（P<0.01），RAP+OPG组凋亡率极显著下降（P<0.01）。表明OPG能够促进OCPs发生自噬，而自噬的激活促进了OCPs的凋亡；OPG抑制OCs自噬的发生，并且抑制和激活自噬都能抑制OCs的凋亡。

Abstract:

In order to investigate the effects of osteoprotegerin (OPG) on autophagy and apoptosis in osteoclasts (OCs) and osteoclast precursors (OCPs), RAW264.7 cells were pretreated with autophagy promoter rapamycin (RAP) for 1 h or autophagy inhibitor chloroquine (CQ) for 30 min, followed by treatment with 80 ng·mL^-1 OPG for another 6 h, and cells were divided into the Control group (Con), the OPG group, the CQ group, the CQ +OPG group, the RAP group and the RAP+OPG group, Western blot was applied to detect the proteins related to autophagy; Flow cytometry was used to observe the apoptosis rate. The results of Western blot showed that in OCPs treated with OPG the expression of Atg5 was increased significantly (P<0.01) and Beclin-1 was increased (P<0.05), while the expression of LC3-Ⅱ showed no significant difference (P>0.05) compared with Con group; Compared with OPG treatment group, the expression of LC3-Ⅱ was significantly increased in the RAP+OPG group. In OCs, compared with Con group, OPG treatment markedly reduced (P<0.01) the expression of LC3-Ⅱ, Atg5 and Beclin-1; Compared with the OPG group, the expression of Atg5 and Beclin-1 were significantly reduced by the combination use of RAP+OPG. The results of flow cytometry showed that in OCPs, OPG and RAP, alone or in combination, markedly increased the apoptosis rate (P<0.01),while there was no difference (P>0.05) in the CQ+OPG group; The effect of RAP+OPG was stronger compared to the OPG treatment group (P<0.01). While in OCs, the apoptosis level was greatly reduced by OPG (P<0.01), both CQ+OPG and RAP+OPG presented significantly decreased apoptosis level (P<0.01). The results of our study demonstrate that OPG induces apoptosis of OCPs by activating autophagy. Nevertheless OPG reduces apoptosis of OCs through restraining the autophagy in OCs. Interestingly, either the inhibition or promotion of autophagy results in the apoptosis of OCs.

中图分类号:

S813.1

刘庆羊, 孙自强, 赵鸿雁, 宋瑞龙, 顾建红, 刘宗平. 自噬在骨保护素对破骨细胞及其前体凋亡过程的调控机制[J]. 畜牧兽医学报, 2018, 49(8): 1761-1769.

LIU Qing-yang, SUN Zi-qiang, ZHAO Hong-yan, SONG Rui-long, GU Jian-hong, LIU Zong-ping. Effects of Autophagy on the Apoptosis of Osteoclasts and Osteoclast Precursors Mediated by Osteoprotegerin[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(8): 1761-1769.

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自噬在骨保护素对破骨细胞及其前体凋亡过程的调控机制

Effects of Autophagy on the Apoptosis of Osteoclasts and Osteoclast Precursors Mediated by Osteoprotegerin

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