Department of Orthopaedic Surgery, Mie University Graduate School of Medicine, 2-174 Edobashi, Tsu City, Mie, 514-8507, Japan.
BMC Musculoskelet Disord. 2019 May 17;20(1):225. doi: 10.1186/s12891-019-2609-x.
The expression of the receptor activator of nuclear factor kappa B (RANK) /RANK ligand (RANKL) /osteoprotegerin (OPG) system and its association with the progression of intervertebral disc (IVD) degeneration has recently been reported in a human IVD. However, the effect of the RANK/RANKL/OPG system on the matrix metabolism of human IVD cells, especially on the expression of catabolic factors relevant to IVD degeneration, remains unknown. The purpose of this study was to examine the expression of the RANK/RANKL/OPG system, and then to evaluate the effect of this system on the expression of catabolic factors by human IVD cells.
Annulus fibrosus (AF) and nucleus pulposus (NP) cells isolated by sequential enzyme digestion from human IVD tissues obtained during spine surgeries were monolayer cultured. The expression of the RANK/RANKL/OPG system was determined using immunohistochemical methods and real-time polymerase chain reaction (PCR). To evaluate the influence of interleukin-1 beta (IL-1β) stimulation on the mRNA expression of RANK, RANKL, and OPG, recombinant human IL-1β (rhIL-1β) was administered in the culture media of IVD cells. To examine the influence of RANKL signaling on the expression of matrix metalloprotease-3 (MMP-3), MMP-13, and IL-1β, the cells were cultured with exogenous recombinant human RANKL (rhRANKL), recombinant human OPG (rhOPG) or anti-human RANKL mouse monoclonal antibody (ahRANKL-mAB) with or without rhIL-1β.
Immunoreactivity to RANK/RANKL/OPG and the mRNA expression of the three genes were obviously identified in both AF and NP cells. rhIL-1β stimulation significantly upregulated the mRNA expression level of RANK/RANKL/OPG. The mRNA expression of catabolic factors was significantly upregulated by stimulation of rhRANKL in the presence of rhIL-1β. On the other hand, the administration of either rhOPG or ahRANKL-mAB significantly suppressed the mRNA expression of catabolic factors that had been upregulated by rhIL-1β stimulation. The suppressive effect of ahRANKL-mAB against rhIL-1β stimulation was also confirmed by the protein expression of MMP-3.
The present study showed that the RANK/RANKL/OPG system may be involved in the progression of IVD degeneration. This study also suggested the potential use of anti-RANKL monoclonal antibody and OPG as therapeutic agents to suppress the progression of IVD degeneration.
核因子 κB 受体激活剂（RANK）/RANK 配体（RANKL）/护骨素（OPG）系统的表达及其与椎间盘（IVD）退变的关系最近在人类 IVD 中有所报道。然而，RANK/RANKL/OPG 系统对人 IVD 细胞基质代谢的影响，特别是对与 IVD 退变相关的分解代谢因子的表达，仍不清楚。本研究旨在检测 RANK/RANKL/OPG 系统的表达，并评估该系统对人 IVD 细胞分解代谢因子表达的影响。
通过连续酶消化法从脊柱手术中获得的人 IVD 组织中分离出纤维环（AF）和髓核（NP）细胞，进行单层培养。采用免疫组织化学方法和实时聚合酶链反应（PCR）检测 RANK/RANKL/OPG 系统的表达。为了评估白细胞介素-1β（IL-1β）刺激对 RANK、RANKL 和 OPG mRNA 表达的影响，在 IVD 细胞的培养基中添加重组人白细胞介素-1β（rhIL-1β）。为了研究 RANKL 信号对基质金属蛋白酶-3（MMP-3）、MMP-13 和 IL-1β表达的影响，用外源性重组人 RANKL（rhRANKL）、重组人 OPG（rhOPG）或抗人 RANKL 单克隆抗体（ahRANKL-mAB）与或不与 rhIL-1β 培养细胞。
在 AF 和 NP 细胞中均明显检测到 RANK/RANKL/OPG 的免疫反应性和三种基因的 mRNA 表达。rhIL-1β 刺激显著上调了 RANK/RANKL/OPG 的 mRNA 表达水平。rhRANKL 刺激在 rhIL-1β 存在下显著上调了分解代谢因子的 mRNA 表达。另一方面，rhOPG 或 ahRANKL-mAB 的给药显著抑制了 rhIL-1β 刺激上调的分解代谢因子的 mRNA 表达。ahRANKL-mAB 对 rhIL-1β 刺激的抑制作用也通过 MMP-3 的蛋白表达得到证实。
本研究表明，RANK/RANKL/OPG 系统可能参与了 IVD 退变的进展。本研究还表明，抗 RANKL 单克隆抗体和 OPG 可能作为治疗药物，抑制 IVD 退变的进展。
