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

doi:10.7518/hxkq.2022.04.002

Abstract

Abstract:

Objective 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). Conclusion 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.

Key words: chronic periodontitis, periodontal ligament stem cells, exosomes, receptor activator of nuclear factor-κB ligand, tumor necrosis factor-α, osteoclast differentiation

CLC Number:

Q 254

Dai Zhenning, Zheng Weihan, Li Shiyu. Receptor activator of nuclear factor-κB ligand and tumor necrosis factor-α promotes osteoclast differentiation through the exosomes of inflammatory periodontal ligament stem cells[J]. West China Journal of Stomatology, 2022, 40(4): 377-385.

Figures/Tables 4

References 26

1	Baima G, Romandini M, Citterio F, et al. Periodontitis and accelerated biological aging: a geroscience approach[J]. J Dent Res, 2022, 101(2): 125-132.
2	Zhao Y, Li Z, Su L, et al. Characterization and regulation of osteoclast precursors following chronic Porphyromonas gingivalis infection[J]. J Leukoc Biol, 2020, 108(4): 1037-1050.
3	Wang Q, Duan M, Liao J, et al. Are osteoclasts mechanosensitive cells[J]. J Biomed Nanotechnol, 2021, 17(10): 1917-1938.
4	Pathak JL, Fang Y, Chen Y, et al. Downregulation of macrophage-specific act-1 intensifies periodontitis and alveolar bone loss possibly via TNF/NF-κB signaling[J]. Front Cell Dev Biol, 2021, 9: 628139.
5	Seidel A, Seidel CL, Weider M, et al. Influence of natural killer cells and natural killer T cells on periodontal disease: a systematic review of the current literature[J]. Int J Mol Sci, 2020, 21(24): 9766.
6	Li J, Sun Z, Lin Y, et al. Syndecan 4 contributes to osteoclast differentiation induced by RANKL through enhan-cing autophagy[J]. Int Immunopharmacol, 2021,91: 107275.
7	Sun Y, Liu G, Zhang K, et al. Mesenchymal stem cells-derived exosomes for drug delivery[J]. Stem Cell Res Ther, 2021, 12(1): 561.
8	Chen H, Wang L, Zeng X, et al. Exosomes, a new star for targeted delivery[J]. Front Cell Dev Biol, 2021, 9: 751079.
9	Walsh SA, Hoyt BW, Rowe CJ, et al. Alarming cargo: the role of exosomes in trauma-induced inflammation[J]. Biomolecules, 2021, 11(4): 522.
10	Abuarqoub DA, Aslam N, Barham RB, et al. The effect of platelet lysate in culture of PDLSCs: an in vitro comparative study[J]. PeerJ, 2019, 7: e7465.
11	秦子顺, 殷丽华, 王开娟, 等. 淫羊藿苷促进人牙周膜干细胞增殖及骨向分化的作用[J]. 华西口腔医学杂志, 2015, 33(4): 370-376.
	Qin ZS, Yin LH, Wang KJ, et al. Effects of Icariin promotion on proliferation and osteogenic differentiation of human periodontal ligament stem cells[J]. West China J Stomatol, 2015, 33(4): 370-376.
12	He Q, Yang S, Gu X, et al. Long noncoding RNA TUG1 facilitates osteogenic differentiation of periodontal ligament stem cells via interacting with Lin28A[J]. Cell Death Dis, 2018, 9(5): 455.
13	Iwasaki K, Komaki M, Akazawa K, et al. Spontaneous differentiation of periodontal ligament stem cells into myofibroblast during ex vivo expansion[J]. J Cell Physiol, 2019, 234(11): 20377-20391.
14	Luo T, von der Ohe J, Hass R. MSC-derived extracellular vesicles in tumors and therapy[J]. Cancers, 2021, 13(20): 5212.
15	Valenzuela Alvarez M, Gutierrez LM, Correa A, et al. Metastatic niches and the modulatory contribution of mesenchymal stem cells and its exosomes[J]. Int J Mol Sci, 2019, 20(8): 1946.
16	Gebeyehu A, Kommineni N, Meckes DG Jr, et al. Role of exosomes for delivery of chemotherapeutic drugs[J]. Crit Rev Ther Drug Carrier Syst, 2021, 38(5): 53-97.
17	Zhang Z, Shuai Y, Zhou F, et al. PDLSCs regulate angiogenesis of periodontal ligaments via VEGF transferred by exosomes in periodontitis[J]. Int J Med Sci, 2020, 17(5): 558-567.
18	Liu T, Hu W, Zou X, et al. Human periodontal ligament stem cell-derived exosomes promote bone regeneration by altering microRNA profiles[J]. Stem Cells Int, 2020, 2020: 8852307.
19	Fei D, Xia Y, Zhai Q, et al. Exosomes regulate interclonal communication on osteogenic differentiation a-mong heterogeneous osteogenic single-cell clones th-rough PINK1/parkin-mediated mitophagy[J]. Front Cell Dev Biol, 2021, 9: 687258.
20	Xie L, Chen J, Ren X, et al. Alteration of circRNA and lncRNA expression profile in exosomes derived from periodontal ligament stem cells undergoing osteogenic differentiation[J]. Arch Oral Biol, 2021, 121: 104984.
21	Zheng Y, Dong C, Yang J, et al. Exosomal microRNA-155-5p from PDLSCs regulated Th17/Treg balance by targeting sirtuin-1 in chronic periodontitis[J]. J Cell Phy-siol, 2019, 234(11): 20662-20674.
22	Lorenzo J. Sexual dimorphism in osteoclasts[J]. Cells, 2020, 9(9): 2086.
23	Chellaiah MA. L-plastin phosphorylation: possible regulation by a TNFR1 signaling cascade in osteoclasts[J]. Cells, 2021, 10(9): 2432.
24	Elango J, Bao B, Wu W. The hidden secrets of soluble RANKL in bone biology[J]. Cytokine, 2021, 144: 155559.
25	Kalluri R, Lebleu VS. The biology, function, and biomedical applications of exosomes[J]. Science, 2020, 367(6478): eaau6977.
26	杨倩茹, 汤润泽, 周菁. 炎症反应中巨噬细胞的力学生物学[J]. 生理科学进展, 2021, 52(5): 335-340.
	Yang QR, Tang RZ, Zhou Q. Mechanobiology of macrophages: how pro-inflammatory stimuli elicit changes in cell mechanics and cellular behavior[J]. Progress Physiol Sci, 2021, 52(5): 335-340.

ELISA指标	Exo-WT	Exo-CP
TNF-α	1.33±0.2	14.21±0.82**
RANKL	0.75±0.06	6.38±0.13*
IL-1α	1.41±0.68	8.41±0.35**
TGF-β1	12.44±2.08	0.58±0.16***
BMP-2	5.36±1.38	1.61±0.22*

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