肿瘤坏死因子受体相关因子6（TRAF6）抑制可调节胶原诱导的类风湿性关节炎大鼠的骨质流失和基质金属蛋白酶表达水平。

Tumor necrosis factor receptor-associated factor 6 (TRAF6) inhibition modulates bone loss and matrix metalloproteinase expression levels in collagen-induced rheumatoid arthritis rat.

作者信息

Guo Jiangtao, Cao Xuqing, Ma Xiaoli, Hao Chunfang, Wu Lili, Zhang Mingzhu, Yang Yashan, Zhao Jingtian, Chen Kunting, Yin Zhe

机构信息

Department of Rheumatology and Immunology, People's Hospital of Ningxia Hui Autonomous Region (the Affiliated People's Hospital of Ningxia Medical University and the First Affiliated Hospital of Northwest Minzu University), Ningxia, China.

2 Department of Neurology, People's Hospital of Ningxia Hui Autonomous Region (the Affiliated People's Hospital of Ningxia Medical University and the First Affiliated Hospital of Northwest Minzu University), Ningxia, China.

出版信息

Ann Palliat Med. 2020 Nov;9(6):4017-4028. doi: 10.21037/apm-20-1894. Epub 2020 Nov 10.

DOI:10.21037/apm-20-1894

PMID:33183053

Abstract

BACKGROUND

Rheumatoid arthritis (RA) is a main characterized by persistent synovitis, systemic inflammation, and autoantibodies. Tumor necrosis factor receptor-associated factor 6 (TRAF6) is an E3 ubiquitin ligase and is a crucial cytoplasm signal adaptor that can regulate critical biological processes. This research aims to explore the function of TRAF6 on bone loss and matrix metalloproteinase (MMP) expression in collagen-induced RA rats.

METHODS

The RA model in rats (Sprague Dawley rat, 5-6 weeks old, weight 246.88±8.31 g) was set up via using collagen-induced RA. The shRNA-TRAF6 knockdown efficiency was tested using real-time reverse transcription-polymerase chain reaction (qRT-PCR) and western blot, respectively. The rats were divided into four groups: the control group, RA group, RA + shRNA-NC group, and RA + TRAF6-shRNA group. The tartrate-resistant acidic phosphatase (TRAP), hematoxylin and eosin (H&E), and Saffron O staining were employed to test the bone injury. The mRNA and protein expressive of Osteoclast-associated receptor (OSCAR), TRAP, Osterix (Osx), Collagen type I alpha 1 (COL1A1), Distal-less homeobox2 (Dlx2), tissue inhibitor of metalloproteinase (TIMP), matrix metalloproteinase-1(MMP-1), Cyclooxygenase 2 (COX2) and qRT-PCR performed MMP-13 and western blot, respectively.

RESULTS

The mRNA and protein expression levels of TRAF6 were down-regulated in the RA + TRAF6- shRNA group. After the levels of TRAF6 were inhibited, the levels of bone volume/total volume (BV/TV), trabecular bone thickness (Tb.Th), and trabecular bone number (Tb.N) were increased, while the levels of trabecular bone space (Tb.Sp), Osteocalcin and ALP were deceased. The mRNA and protein expression levels of OSCAR, TRAP, MMP-1, COX2, and MMP-13 were reduced obviously in the RA + TRAF6- shRNA group compared with the RA + shRNA-NC group, while the levels of TIMP-1, OSX, CoL1A1, and DLx2 were enhanced obviously.

CONCLUSIONS

Inhibition of TRAF6 reduces bone loss and MMP expression levels in collagen-induced RA rat, and supplies an alternative treatment method in RA.

摘要

背景

类风湿关节炎（RA）主要特征为持续性滑膜炎、全身性炎症和自身抗体。肿瘤坏死因子受体相关因子6（TRAF6）是一种E3泛素连接酶，是一种关键的细胞质信号衔接蛋白，可调节关键生物学过程。本研究旨在探讨TRAF6在胶原诱导的RA大鼠骨质流失和基质金属蛋白酶（MMP）表达中的作用。

方法

通过胶原诱导的RA建立大鼠（Sprague Dawley大鼠，5 - 6周龄，体重246.88±8.31 g）RA模型。分别使用实时逆转录 - 聚合酶链反应（qRT - PCR）和蛋白质免疫印迹法检测shRNA - TRAF6的敲低效率。将大鼠分为四组：对照组、RA组、RA + shRNA - NC组和RA + TRAF6 - shRNA组。采用抗酒石酸酸性磷酸酶（TRAP）、苏木精和伊红（H&E）以及番红花O染色检测骨损伤情况。分别通过qRT - PCR检测破骨细胞相关受体（OSCAR）、TRAP、osterix（Osx）、I型胶原α1（COL1A1）、远端缺失同源盒2（Dlx2）、基质金属蛋白酶组织抑制剂（TIMP）、基质金属蛋白酶 - 1（MMP - 1）、环氧合酶2（COX2）以及MMP - 13的mRNA表达，并用蛋白质免疫印迹法检测其蛋白表达。

结果

RA + TRAF6 - shRNA组中TRAF6的mRNA和蛋白表达水平下调。TRAF6水平受到抑制后，骨体积/总体积（BV/TV）、骨小梁厚度（Tb.Th）和骨小梁数量（Tb.N）水平升高，而骨小梁间隙（Tb.Sp）、骨钙素和碱性磷酸酶水平降低。与RA + shRNA - NC组相比，RA + TRAF6 - shRNA组中OSCAR、TRAP.MMP - 1、COX2和MMP - 13的mRNA和蛋白表达水平明显降低，而TIMP - 1、OSX、COL1A1和DLx2水平明显升高。