Zhu D W, Xue D, Lai W, Xu W N, Jiang S Y
Department of Oral and Maxillofacial Surgery, Institute of Stomatology, School and Hospital of Stomatology, Tianjin Medical University, Tianjin 300070, China.
Department of Periodontics, Institute of Stomatology, School and Hospital of Stomatology, Tianjin Medical University, Tianjin 300070, China(Present address: Department of Dentistry, Xuanwu Hospital, Capital Medical University, Beijing 100053, China).
Zhonghua Kou Qiang Yi Xue Za Zhi. 2018 Nov 9;53(11):753-759. doi: 10.3760/cma.j.issn.1002-0098.2018.11.007.
To investigate the effects and mechanisms of microRNA-146a (miR-146a) on osteogenic differentiation of human periodontal ligament cells (hPDLC) stimulated by lipopolysaccharide (LPS) of (Pg). hPDLC were cultured and induced to the phase of osteogenic differentiation. These cells were divided into five groups: non-osteogenic differentiation cells, osteogenic differentiation cells, osteogenic differentiation cells treated with Pg LPS, osteogenic differentiation cells treated with Pg LPS and miR-146a mimic, osteogenic differentiation cells treated with Pg LPS and miR-146a negative control. Osteogenic markers and mineralization were detected via quantitative real-time PCR (qPCR) and alizarin red staining, respectively. Meanwhile, non-radioactive transcription factor assay was applied to explore the nuclear activity of nuclear factor kappa B (NF-κB) p65 in nuclear extracts of hPDLC. Compared with cells of osteogenic differentiation in non-LPS-stimulated groups, Pg LPS could decrease the markers of osteogenic differentiation of hPDLC such as collagen Ⅰ (Col-Ⅰ), alkaline phosphatase (ALP), Runt-related transcription factor-2 (RUNX2) and osteocalcin (OCN) (0.05), inhibit mineralization, and stimulate NF-κB p65 nuclear activity expression (non-LPS stimulated group: 1.023±0.217, LPS stimulated group: 6.252±0.613, 0.008). However, compared with cells in Pg LPS/miR-146a negative control group, miR-146a increased Col-Ⅰ (0.007) and OCN (0.049) mRNA expression, rather than ALP (0.167) and RUNX2 (0.580) at day 3; miR-146a also upregulated mRNA levels of Col-Ⅰ, ALP, RUNX2 and OCN (0.05) at day 7 and day 14, and enhance mineralization. Meanwhile, miR-146a mimic could decrease the nuclear activity of NF-κB p65 induced by Pg LPS in hPDLC (miR-146a: 2.427±0.354, negative control: 5.863±0.482, 0.019). miR-146a could reverse the inhibitory effects of Pg LPS on osteogenic differentiation of hPDLC through enhancing the expression of osteogenic markers and decreasing inflammatory pathway in hPDLC.
为研究微小RNA-146a（miR-146a）对牙龈卟啉单胞菌（Pg）脂多糖（LPS）刺激的人牙周膜细胞（hPDLC）成骨分化的影响及机制。培养hPDLC并诱导其进入成骨分化阶段。将这些细胞分为五组：非成骨分化细胞组、成骨分化细胞组、经Pg LPS处理的成骨分化细胞组、经Pg LPS和miR-146a模拟物处理的成骨分化细胞组、经Pg LPS和miR-146a阴性对照处理的成骨分化细胞组。分别通过定量实时聚合酶链反应（qPCR）和茜素红染色检测成骨标志物和矿化情况。同时，应用非放射性转录因子检测法探究hPDLC核提取物中核因子κB（NF-κB）p65的核活性。与非LPS刺激组的成骨分化细胞相比，Pg LPS可降低hPDLC的成骨分化标志物如Ⅰ型胶原（Col-Ⅰ）、碱性磷酸酶（ALP）、 runt相关转录因子2（RUNX2）和骨钙素（OCN）的表达（P<0.05），抑制矿化，并刺激NF-κB p65核活性表达（非LPS刺激组：1.023±0.217，LPS刺激组：6.252±0.613，P<0.008）。然而，与Pg LPS/miR-146a阴性对照组的细胞相比，miR-146a在第3天时增加了Col-Ⅰ（P<0.007）和OCN（P<0.049）的mRNA表达，但未增加ALP（P=0.167）和RUNX2（P=0.580）的表达；在第7天和第14天时，miR-146a还上调了Col-Ⅰ、ALP、RUNX2和OCN的mRNA水平（P<0.05），并增强了矿化。同时，miR-146a模拟物可降低Pg LPS诱导的hPDLC中NF-κB p65的核活性（miR-146a组：2.427±0.354，阴性对照组：5.863±0.482，P<0.019）。miR-146a可通过增强hPDLC中成骨标志物的表达并减少炎症信号通路来逆转Pg LPS对hPDLC成骨分化的抑制作用。
