School/Hospital of Stomatology, Lanzhou University, Lanzhou, P.R.China.
Department of Clinical Dentistry, Faculty of Medicine, University of Bergen, Bergen, Norway.
J Periodontal Res. 2022 Oct;57(5):1034-1042. doi: 10.1111/jre.13044. Epub 2022 Aug 9.
Periodontal ligament cells (PDLCs) are critical for wound healing and regenerative capacity of periodontal diseases. Within an inflammatory periodontal pocket, a hypoxic environment can aggravate periodontal inflammation, where PDLCs response to the inflammation would change. Resolvin D1 (RvD1) is an endogenous lipid mediator, which can impact intracellular inflammatory pathways of periodontal/oral cells and periodontal regeneration. It is not clear how hypoxia and RvD1 impact the inflammatory responses of pro-inflammatory PDLCs phenotype. Therefore, this study aimed to test hypoxia could induce changes in pro-inflammatory phenotype of PDLCs and RvD1 could reverse it.
Human PDLCs were cultured from periodontal tissues from eight healthy individuals and were characterized by immunofluorescence staining of vimentin and cytokeratin. Cell viability was examined by Methyl-thiazolyl-tetrazolium (MTT) assay. To examine the effects of hypoxia and RvD1 on the inflammatory responses of pro-inflammatory PDLCs phenotype, protein levels and gene expressions of inflammatory cytokines and signal transduction molecules were measured by enzyme-linked immunosorbent assay (ELISA), western blotting (WB), and real-time quantitative reverse transcription PCR (real-time qRT-PCR). Alizarin red S staining and real-time qRT-PCR were employed to study the effects of hypoxia and RvD1 on the osteogenic differentiation of pro-inflammatory PDLCs phenotype.
It was found that hypoxia increases the expression of inflammatory factors at the gene level (p < .05). RvD1 reduced the expression of IL-1β (p < .05) in PDLCs under hypoxia both at the protein and RNA levels. There were increases in the expression of p38 mitogen-activated protein kinase (p38 MAPK, p < .01) and protein kinase B (Akt, p < .05) in response to RvD1. Also, a significantly higher density of calcified nodules was observed after treatment with RvD1 for 21 days under hypoxia.
Our results indicate that hypoxia up-regulated the inflammatory level of PDLCs. RvD1 can reduce under-hypoxia-induced pro-inflammatory cytokines in the inflammatory phenotype of PDLCs. Moreover, RvD1 promotes the calcium nodules in PDLCs, possibly by affecting the p38 MAPK signaling pathway through Akt and HIF-1α.
牙周膜细胞（PDLCs）对牙周病的伤口愈合和再生能力至关重要。在炎症性牙周袋内，缺氧环境会加重牙周炎症，PDLCs 对炎症的反应也会发生变化。解析素 D1（RvD1）是一种内源性脂质介质，可影响牙周/口腔细胞的细胞内炎症途径和牙周再生。目前尚不清楚缺氧和 RvD1 如何影响促炎 PDLCs 表型的炎症反应。因此，本研究旨在测试缺氧是否会诱导 PDLCs 的促炎表型发生变化，以及 RvD1 是否可以逆转这种变化。
从 8 名健康个体的牙周组织中培养人牙周膜细胞，并通过波形蛋白和细胞角蛋白的免疫荧光染色进行鉴定。通过甲基噻唑基四唑（MTT）测定法检测细胞活力。为了研究缺氧和 RvD1 对促炎 PDLCs 表型炎症反应的影响，通过酶联免疫吸附测定（ELISA）、蛋白质印迹（WB）和实时定量逆转录聚合酶链反应（real-time qRT-PCR）测量炎症细胞因子和信号转导分子的蛋白水平和基因表达。通过茜素红 S 染色和实时 qRT-PCR 研究缺氧和 RvD1 对促炎 PDLCs 表型成骨分化的影响。
结果发现，缺氧会增加基因水平的炎症因子表达（p<0.05）。RvD1 降低了缺氧条件下 PDLCs 中 IL-1β 的表达（p<0.05），无论是在蛋白质和 RNA 水平。p38 丝裂原活化蛋白激酶（p38 MAPK，p<0.01）和蛋白激酶 B（Akt，p<0.05）的表达增加。此外，在缺氧条件下用 RvD1 处理 21 天后，观察到钙化结节的密度显著增加。
我们的结果表明，缺氧上调了 PDLCs 的炎症水平。RvD1 可以降低缺氧诱导的 PDLCs 炎症表型中的促炎细胞因子。此外，RvD1 通过 Akt 和 HIF-1α 影响 p38 MAPK 信号通路，促进 PDLCs 中的钙结节形成。
