背景:牙周炎是牙齿脱落的主要原因,并可加剧各种全身性炎症。牙周膜干细胞(PDLSCs)是促进牙周组织再生的突出和有利的候选者。本研究旨在探讨蛋白酶激活受体1(PAR1)是否可以减轻丁酸钠(NaB)诱导的PDLSCs成骨抑制,并阐明其潜在机制。
方法:利用来自基因表达综合(GEO)的公共数据集来分析牙周炎中的差异表达基因(DEG)以及随后的京都基因和基因组百科全书(KEGG)途径富集。将PDLSCs在作为阴性对照的对照培养基(CM)中或在成骨培养基(OM)中正常培养以诱导成骨。使用选择性激动剂或拮抗剂(OM+激动剂和OM+拮抗剂)激活或抑制PAR1。PDLSCs成骨的评价是基于成骨相关标志物的水平,包括runt相关转录因子2(RUNX2),osterix(OSX),骨钙蛋白(OCN),和骨桥蛋白(OPN),碱性磷酸酶(ALP)活性,和钙浓度。此外,通过3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四唑溴化物(MTT)测定和茜素红染色测量细胞增殖和成骨分化。确定PAR1靶向肢体发育膜蛋白1(LMBR1)/骨形态发生蛋白(BMP)通路,LMBR1通过细胞转染上调,并且使用选择性抑制剂Noggin蛋白抑制BMP2。最后,将NaB引入PDLSCs以研究对NaB诱导的PDLSCs成骨抑制的影响。
结果:PAR1,RUNX2,OSX,OCN,OPN,扩散,ALP活性,钙浓度,成骨分化,在OM中培养的PDLSC中,BMP2和BMP4表现出显著增加(p<0.01)。这些参数被PAR1激动剂进一步升高,并且相反被PAR1拮抗剂降低(p<0.01)。相反,在OM中培养的PDLSCs中LMBR1降低(p<0.001),PAR1激动剂诱导的进一步减少和PAR1拮抗剂观察到的反向增加(p<0.001)。OE-LMBR1转染成功升高LMBR1水平,随后抑制BMP2和BMP4(p<0.001)。同时,Noggin蛋白有效抑制BMP2和BMP4(p<0.001)。所有观察到的骨生成相关的变化被增加的LMBR1或BMP途径的抑制所逆转(p<0.001)。此外,NaB抑制OM培养的PDLSCs成骨相关变化(p<0.001),这些效应被PAR1激动剂完全逆转(p<0.001)。相反,增加的LMBR1或抑制的BMP途径破坏了PAR1激动剂诱导的骨生成逆转(p<0.001)。
结论:PAR1的激活,通过抑制LMBR1信号传导和激活BMP通路,证实了增强PDLSCs的骨生成和减轻由NaB引起的对PDLSCs骨生成的抑制作用的能力。
BACKGROUND: Periodontitis is the leading cause of tooth loss and can exacerbate various systemic inflammatory conditions. Periodontal ligament stem cells (PDLSCs) stand out as prominent and favorable candidates for promoting periodontal tissue regeneration. This study aimed to investigate whether the protease-activated receptor type 1 (PAR1) can mitigate the sodium butyrate (NaB)-induced PDLSCs osteogenesis inhibition and unravel the underlying mechanism.
METHODS: Public datasets from the Gene Expression Omnibus (GEO) were utilized to analyze differentially expressed genes (DEGs) in periodontitis and subsequent Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment. PDLSCs were cultured normally in control medium (CM) as the negative control or in osteogenic medium (OM) to induce osteogenesis. PAR1 was either activated or suppressed using a selective agonist or antagonist (OM+agonist and OM+antagonist). The evaluation of PDLSCs osteogenesis was based on the levels of osteogenesis-related markers, including runt-related transcription factor 2 (RUNX2), osterix (OSX), osteocalcin (OCN), and osteopontin (OPN), alkaline phosphatase (ALP) activity, and calcium concentration. Additionally, cell proliferation and osteogenic differentiation were measured through the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and Alizarin Red Staining. To determine the PAR1 targeting the limb development membrane protein 1 (LMBR1)/bone morphogenetic protein (BMP) pathway, LMBR1 was upregulated through cell transfection and BMP2 was inhibited using the selective inhibitor Noggin protein. Finally, NaB was introduced into PDLSCs to investigate the effect on NaB-induced inhibition of PDLSCs osteogenesis.
RESULTS: PAR1, RUNX2, OSX, OCN, OPN, proliferation, ALP activity, calcium concentration, osteogenic differentiation, BMP2, and BMP4 exhibited significant increases in PDLSCs cultured in OM (p < 0.01). These parameters were further elevated by PAR1 agonist and conversely reduced by PAR1 antagonist (p < 0.01). Conversely, LMBR1 was decreased in PDLSCs cultured in OM (p < 0.001), with further reduction induced by PAR1 agonist and a reverse increase observed with PAR1 antagonist (p < 0.001). OE-LMBR1 transfection successfully elevated LMBR1 levels, subsequently inhibiting BMP2 and BMP4 (p < 0.001). Meanwhile, the Noggin protein effectively suppressed BMP2 and BMP4 (p < 0.001). All observed osteogenesis-related changes were reversed by the increased LMBR1 or inhibition of the BMP pathway (p < 0.001). Furthermore, NaB suppressed osteogenesis-related changes in OM-cultured PDLSCs (p < 0.001), and these effects were entirely reversed by PAR1 agonist (p < 0.001). Conversely, the increased LMBR1 or inhibited BMP pathway disrupted the osteogenesis reversion induced by PAR1 agonist (p < 0.001).
CONCLUSIONS: The activation of PAR1, through suppressing LMBR1 signaling and activating BMP pathway, demonstrates the ability to enhance the osteogenesis of PDLSCs and mitigate the inhibitory effects on PDLSCs osteogenesis caused by NaB.