Protease-activated receptor type 2 activation downregulates osteogenesis in periodontal ligament stem cells

Abstract Protease-activated receptor-2 (PAR2) is associated with the pathogenesis of many chronic diseases with inflammatory characteristics, including periodontitis. This study aimed to evaluate how the activation of PAR2 can affect the osteogenic activity of human periodontal ligament stem cells (PDLSCs) in vitro. PDLSCs collected from three subjects were treated in osteogenic medium for 2, 7, 14, and 21 days with trypsin (0.1 U/mL), PAR2 specific agonist peptide (SLIGRL-NH2) (100 nM), and PAR2 antagonist peptide (FSLLRY-NH2) (100 nM). Gene (RT-qPCR) expression and protein expression (ELISA) of osteogenic factors, bone metabolism, and inflammatory cytokines, cell proliferation, alkaline phosphatase (ALP) activity, alizarin red S staining, and supernatant concentration were assessed. Statistical analysis of the results with a significance level of 5% was performed. Activation of PAR2 led to decreases in cell proliferation and calcium deposition (p < 0.05), calcium concentration (p < 0.05), and ALP activity (p < 0.05). Additionally, PAR2 activation increased gene and protein expression of receptor activator of nuclear factor kappa-Β ligand (RANKL) (p < 0.05) and significantly decreased the gene and protein expression of osteoprotegerin (p <0. 05). Considering the findings, the present study demonstrated PAR2 activation was able to decrease cell proliferation, decreased osteogenic activity of PDLSCs, and upregulated conditions for bone resorption. PAR2 may be considered a promising target in periodontal regenerative procedures.

摘要：蛋白酶激活受体2（Protease-activated receptor-2，PAR2）与多种炎症性慢性疾病的发病机制密切相关，其中包括牙周炎。本研究旨在探讨体外环境下，PAR2激活对人牙周膜干细胞（human periodontal ligament stem cells，PDLSCs）成骨活性的影响。研究从3名受试者体内分离获取PDLSCs，将其置于成骨培养基中，分别用胰蛋白酶（0.1 U/mL）、PAR2特异性激动肽（SLIGRL-NH2，100 nM）及PAR2特异性拮抗肽（FSLLRY-NH2，100 nM）处理2、7、14及21天。随后对成骨因子、骨代谢及炎症细胞因子的基因（RT-qPCR）表达与蛋白表达（酶联免疫吸附试验，ELISA）、细胞增殖能力、碱性磷酸酶（alkaline phosphatase，ALP）活性、茜素红S染色结果及细胞上清液浓度进行检测与评估。以5%为显著性水平对实验结果开展统计学分析。结果表明，PAR2激活可显著降低细胞增殖水平与钙沉积量（p<0.05）、钙浓度（p<0.05）及ALP活性（p<0.05）。此外，PAR2激活可上调核因子κB受体活化因子配体（receptor activator of nuclear factor kappa-Β ligand，RANKL）的基因与蛋白表达水平（p<0.05），并显著下调骨保护素（osteoprotegerin）的基因与蛋白表达水平（p<0.05）。综合上述研究结果，本研究证实PAR2激活可抑制细胞增殖、降低PDLSCs的成骨活性，并增强骨吸收相关的病理状态。PAR2有望成为牙周再生治疗中的潜在干预靶点。