Receptor activator of nuclear factor-κB ligand and tumor necrosis factor-α promotes osteoclast differentiation through the exosomes of inflammatory periodontal ligament stem cells

Abstract

Objectives: Pathological bone resorption is common in chronic periodontitis. However, the effect of exosomes (Exo) secreted by periodontal ligament stem cells (PDLSCs) on bone resorption is unclear. This study explored the Exo of inflammatory PDLSCs, their protein components, and their effects on osteoclast differentiation.

Methods: PDLSCs were isolated from the periodontal ligament tissues of orthodontic patients and those with chronic periodontitis. The surface markers of PDLSCs were detected by flow cytometry. Exo were characterized by Western blot, transmission electron microscope (TEM), bicinchoninic acid assay (BCA), nanosight tracking analysis (NTA). The protein components of Exo were detected by protein profiling. The expression levels of differentially expressed proteins tumor necrosis factor-α (TNF-α), receptor activator of nuclear factor-κB ligand (RANKL), interleukin (IL)-1α, transforming growth factor β (TGF-β), and bone morphogenetic protein 2 (BMP-2) were verified by enzyme linked immunosorbent assay (ELISA). Then, 10, 100, and 1 000 μg·mL^-1 of Exo-CP or Exo-WT were added to RAW264.7 medium, and the expression levels of osteoclast-related indicators were detected by real time quantitative polymerase chain reaction (RT-qPCR), Western blot, and tartrate resistant acid phosphatase (TRAP) staining at 5 days. Experimental data were statistically analyzed using SPSS 24.0 software.

Results: The differentially expressed proteins enriched in Exo-CP were mainly related to the tumor necrosis factor (TNF) signaling, osteoclast differentiation, and nuclear transcription factor κB (NF-κB) signaling pathways. ELISA experiments confirmed Exo-CP had high expression of TNF-α, RANKL, and IL-1α and low expression of TGF-β1 and BMP-2 (P<0.05). Adding Exo-CP to RAW264.7 significantly increased the expression of mRNA and proteins related to osteoclast differentiation of cells. In a concentration-dependent manner, the effect of Exo-CP on osteoclast differentiation at concentrations of 100 and 1 000 μg·mL^-1 was significantly higher than that on the 10 μg·mL^-1 concentration group (P<0.05).

Conclusions: Pathological bone resorption of chronic periodontitis may be caused by the activation of Exo-CP to promote osteoclast differentiation. The main protein in Exo may be RANKL and TNF-α. This research provides a new perspective on pathological bone resorption in chronic periodontitis.

Keywords: chronic periodontitis; exosomes; osteoclast differentiation; periodontal ligament stem cells; receptor activator of nuclear factor-κB ligand; tumor necrosis factor-α.

图 1. PDLSCs细胞及其Exo鉴定结果

Fig 1 Characterization of PDLSCs and Exo A：PDLSCs表面标记物流式检测；B：PDLSCs及Exo特异性标记物Western blot检测；C：Exo的检测结果 TEM × 70 000；D：BCA法检测Exo浓度，NS：P>0.05；E：纳米粒径追踪检测Exo粒径。

图 2. Exo蛋白质谱分析

Fig 2 Analysis of Exo protein profile A：蛋白亚细胞定位；B：2倍差异表达蛋白热图；C：差异表达蛋白的GO富集分析；D：差异表达蛋白的KEGG富集分析。

图 3. RAW264.7细胞的破骨细胞分化检测

Fig 3 Osteoclast differentiation detection of RAW264.7 cells A：RT-qPCR检测TRAP、Cath-K、CTR的mRNA表达水平，*：P<0.05，NS：P>0.05，#：与Control组相比P<0.05；B：Western blot检测TRAF2、IKKβ、NF-κB-p65、Integrin β3蛋白表达水平；C：TRAP染色 × 200；D：细胞长度测量，*：P<0.05，**：P<0.01，NS：P>0.05。