Li J Y, Wang S N, Dong Y M
Department of Cariology and Endodoontology, Peking University School and Hospital of Stomatology & National Clinical Research Center for Oral Diseases & National Engineering Laboratory for Digital and Material Technology of Stomatology & Beijing Key Laboratory of Digital Stomatology, Beijing 100081, China.
Beijing Da Xue Xue Bao Yi Xue Ban. 2020 Feb 18;52(1):24-29. doi: 10.19723/j.issn.1671-167X.2020.01.004.
To study the effects of non-steroidal anti-inflammatory drugs (NSAIDs) on anti-inflammation and repair of human dental pulp cells (hDPCs).
Primary hDPCs from the freshly extracted human third molars were cultured and passaged in vitro, and the following experiments were performed using the 4th-6th generations of hDPCs. HDPCs were cultured in Dulbecco's modified eagle medium (DMEM) containing 1 mg/L lipopolysaccharide (LPS) to obtain LPS irritated hDPCs (LPS-hDPCs), which served as the inflammatory positive group. LPS-hDPCs in the experimental group were cultured in DMEM containing different concentrations (1, 10, and 100 μmol/L) of NSAIDs (aspirin or meloxicam). HDPCs cultured in DMEM were used as the negative control group. The effects of NSAIDs on the proliferation of hDPCs were assessed on the 1st, 3rd, 5th, and 7th day by MTT assay. The effects of NSAIDs on the expression of inflammation related genes interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) of LPS-hDPCs were detected at the 6th hour by real-time PCR. The expression of differentiation related markers dentin matrix protein-1 (DMP-1) and dentin sialophosphoprotein (DSPP) were detected on the 7th day by real-time PCR. The effects of NSAIDs on the mineralization of LPS-hDPCs were assesd on the 14th day by alizarin red staining. Calcium mineralized nodules were semi-quantitatively determined by cetyl pyridine chloride.
MTT assay showed that 1-100 μmol/L aspirin or meloxicam significantly promoted the proliferation of hDPC in a concentration dependent manner (P<0.05). Real-time PCR showed that 1-100 μmol/L meloxicam or 100 μmol/L aspirin down-regulated significantly the mRNA expression of TNF-α and IL-6 of LPS-hDPCs (P<0.05), and 100 μmol/L meloxicam down-regulated IL-6 and TNF-α more significantly than 100 μmol/L aspirin of LPS-hDPCs (P<0.05). Real-time PCR showed that 100 μmol/L meloxicam up-regulated the mRNA expression of DMP-1 and DSPP of LPS-hDPCs significantly (P<0.05). Alizarin red staining showed the meloxicam at the concentration of 100 μmol/L significantly promoted the mineralization of LPS-hDPCs (P<0.05).
In this study, meloxicam promoted the proliferation of hDPCs, inhibited the inflammatory reaction and promoted differentiation and mineralization of hDPCs under LPS irritation. The present results suggest that meloxicam may play a role in anti-inflammation and repair of pulp inflammation.
研究非甾体抗炎药（NSAIDs）对人牙髓细胞（hDPCs）抗炎及修复的影响。
从新鲜拔除的人第三磨牙获取原代hDPCs并进行体外培养及传代，使用第4 - 6代hDPCs进行以下实验。将hDPCs培养于含1 mg/L脂多糖（LPS）的杜氏改良 Eagle培养基（DMEM）中，以获得LPS刺激的hDPCs（LPS - hDPCs），作为炎症阳性组。实验组的LPS - hDPCs培养于含不同浓度（1、10和100 μmol/L）NSAIDs（阿司匹林或美洛昔康）的DMEM中。培养于DMEM中的hDPCs作为阴性对照组。通过MTT法在第1、3、5和7天评估NSAIDs对hDPCs增殖的影响。通过实时PCR在第6小时检测NSAIDs对LPS - hDPCs炎症相关基因白细胞介素 - 6（IL - 6）和肿瘤坏死因子 - α（TNF - α）表达的影响。通过实时PCR在第7天检测分化相关标志物牙本质基质蛋白 - 1（DMP - 1）和牙本质涎磷蛋白（DSPP）的表达。通过茜素红染色在第14天评估NSAIDs对LPS - hDPCs矿化的影响。用十六烷基吡啶氯化物对钙矿化结节进行半定量测定。
MTT法显示1 - 100 μmol/L阿司匹林或美洛昔康以浓度依赖性方式显著促进hDPCs增殖（P<0.05）。实时PCR显示1 - 100 μmol/L美洛昔康或100 μmol/L阿司匹林显著下调LPS - hDPCs中TNF - α和IL - 6的mRNA表达（P<0.05），且100 μmol/L美洛昔康比100 μmol/L阿司匹林更显著地下调LPS - hDPCs中IL - 6和TNF - α（P<0.05）。实时PCR显示100 μmol/L美洛昔康显著上调LPS - hDPCs中DMP - 1和DSPP的mRNA表达（P<0.05）。茜素红染色显示100 μmol/L美洛昔康显著促进LPS - hDPCs矿化（P<0.05）。
本研究中，美洛昔康促进hDPCs增殖，抑制LPS刺激下的炎症反应，并促进hDPCs分化和矿化。目前结果表明美洛昔康可能在牙髓炎症的抗炎及修复中发挥作用。
