AIM To elucidate mechanisms by which mineral trioxide aggregate (MTA) suppresses pro-inflammatory cytokine mRNA expression in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. METHODOLOGY Mineral trioxide aggregate extracts were prepared by immersing set ProRoot MTA in culture medium. RAW264.7 cells were cultured in the presence of LPS and MTA extracts. mRNA expression levels of interleukin (IL)-1α, IL-6, early growth response 2 (Egr2), suppressor of cytokine signalling 3 (Socs3) and IL-10 were quantified with reverse transcription-quantitative polymerase chain reaction. Phosphorylation of nuclear factor-kappa B (NF-κB) p65 in RAW264.7 cells was analysed by Western blotting. Intracellular calcium imaging was performed with Fluo-4 AM. The activity of nuclear factor of activated T cells (NFAT) was determined by luciferase assays. Enforced expression and silencing of Egr2 in RAW264.7 cells were carried out using an expression vector and specific RNAi, respectively. In vivo kinetics of Egr2+ cells in MTA-treated rat molar pulp tissues were examined using immunohistochemistry. Data were analysed by one-way analysis of variance, followed by the Tukey-Kramer test (P < 0.05). RESULTS Exposure to MTA extracts resulted in reduced mRNA expression levels of IL-1α and IL-6, as well as reduced expression of phosphorylated NF-κB, in LPS-stimulated RAW264.7 cells. Exposure to MTA extracts induced Ca2+ influx, which was blocked by NPS2143, an antagonist of calcium-sensing receptor (CaSR); Ca2+ influx then triggered activation of calcineurin/NFAT signalling and enhanced mRNA expression of Egr2. Enforced expression of Egr2 in RAW264.7 cells promoted the expression of both IL-10 and Socs3. In vivo application of MTA onto rat molar pulp tissue resulted in the appearance of Egr2-expressing cells that coexpressed CD163, a typical M2 macrophage marker. CONCLUSIONS Mineral trioxide aggregate extracts induced downregulation of IL-1α and IL-6 in LPS-stimulated RAW264.7 cells via CaSR-induced activation of calcineurin/NFAT/Egr2 signalling and subsequent upregulation of IL-10 and Socs3.

中文翻译：

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三氧化二矿物质聚集体通过钙调神经磷酸酶/活化的T细胞核因子/脂多糖刺激的巨噬细胞的早期生长反应2途径抑制促炎性细胞因子的表达。

目的阐明三氧化二矿聚集体（MTA）抑制脂多糖（LPS）刺激的RAW264.7巨噬细胞中促炎性细胞因子mRNA表达的机制。方法学通过将固定的ProRoot MTA浸入培养基中来制备三氧化二矿骨料提取物。在LPS和MTA提取物的存在下培养RAW264.7细胞。白细胞介素（IL）-1α，IL-6，早期生长反应2（Egr2），细胞因子信号传导抑制剂3（Socs3）和IL-10的mRNA表达水平通过逆转录定量聚合酶链反应进行定量。通过蛋白质印迹分析RAW264.7细胞中核因子κB（NF-κB）p65的磷酸化。细胞内钙成像使用Fluo-4 AM进行。通过荧光素酶测定法测定了活化的T细胞（NFAT）的核因子的活性。分别使用表达载体和特异性RNAi在RAW264.7细胞中进行Egr2的强制表达和沉默。使用免疫组织化学检查了经MTA处理的大鼠磨牙牙髓组织中Egr2 +细胞的体内动力学。数据通过方差的单向分析进行分析，然后进行Tukey-Kramer检验（P <0.05）。结果暴露于MTA提取物可降低LPS刺激的RAW264.7细胞中IL-1α和IL-6的mRNA表达水平，并降低磷酸化的NF-κB的表达。暴露于MTA提取物中会引起Ca2 +内流，这被钙敏感受体（CaSR）的拮抗剂NPS2143阻止。Ca2 +大量涌入随后触发了钙调神经磷酸酶/ NFAT信号的激活，并增强了Egr2的mRNA表达。Egr2在RAW264中的强制表达。7个细胞促进了IL-10和Socs3的表达。在大鼠磨牙牙髓组织上体内应用MTA导致共同表达CD163（典型的M2巨噬细胞标志物）的Egr2表达细胞的出现。结论三氧化二矿聚集体提取物通过CaSR诱导的钙调神经磷酸酶/ NFAT / Egr2信号转导激活以及随后的IL-10和Socs3上调，诱导LPS刺激的RAW264.7细胞中IL-1α和IL-6的下调。