Real-time monitoring of IL-6 and IL-10 reporter expression for anti-inflammation activity in live RAW 264.7 cells
Real-time monitoring of IL-6 and IL-10 reporter expression for anti-inflammation activity in live RAW 264.7 cells
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DOI：
10.1016/j.bbrc.2018.09.173
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发表时间：
2018-11-02
影响因子：
3.1
通讯作者：
Miyazaki, Koyomi
中科院分区：
文献类型：
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作者：
Saiki, Papawee;Nakajima, Yoshihiro;Miyazaki, Koyomi
在以往的研究中，我们认为IL-6和IL-10可以作为慢性炎症性疾病相关抗炎的良好生物标志物。最近，我们通过对IL-6和IL-10水平的筛选，从天然产物中发现了新的抗炎化合物。虽然，我们可以用多种方法来检测IL-6和IL-10水平。然而，所有方法都不能测量IL-6和IL-10水平的动态变化。大多数方法有多个步骤，耗时长。因此，目前还没有一种合适的筛选方法。为此，我们建立了IL-6和IL-10启动子的检测方法，可以在活的单细胞中以3-磷酸甘油醛脱氢酶(GAPDH)启动子为参照基因进行监测。可在两步内实时连续测定IL-6、IL-10水平。我们用广为人知的抗炎化合物，如栎素、黄原酮、β-D-葡聚糖和地塞米松，评估了脂多糖诱导的RAW 264.7细胞中IL-6和IL-10的表达。结果表明，内毒素可强烈诱导IL-6和IL-10的表达。所有抗炎化合物均能抑制内毒素诱导的RAW 264.7细胞IL-6的表达。在脂多糖诱导的RAW 264.7细胞中，槲皮素、黄原酮和地塞米松可抑制IL-10报告基因的表达。而β-D-葡聚糖可诱导IL-10报告基因表达。这些结果表明，该方法可用于检测内毒素诱导的RAW 264.7细胞中IL-6和IL-10的表达，从而达到抗炎的目的。此外，结果还表明，天然化合物对IL-6和IL-10表达的时程有影响。因此，实时监测对于天然化合物的筛选具有优势。提示该方法可作为抗炎活性的化合物筛选方法。(C)2018 Elsevier Inc.保留所有权利。
In previous study, we suggested that the interleukin (IL)-6 and IL-10 could serve as a good biomarker for anti-inflammation that related to chronic inflammatory disease. Recently, we are finding new anti inflammation compounds from natural products by screening of IL-6 and IL-10 levels. Although, we could measure IL-6 and IL-10 levels by several methods. However, all methods could not measure continuous kinetic of IL-6 and IL-10 levels. Most methods have multiple steps and take a long time. Therefore, there is no a suitable method for screening. To this end, we established IL-6 and IL-10 promoter assay which can monitor with reference gene as Glyceraldehyde 3-phosphate dehydrogenase (gapdh) promoter in living single cell. It could determine IL-6 and IL-10 levels continuously in real-time within two steps. We evaluated IL-6 and IL-10 reporter expression in LPS-induced RAW 264.7 cells with well-known anti-inflammatory compounds such as quercetin, xanthones, beta-D-glucan and dexamethasone. As the results, the expression of IL-6 and IL-10 reporters were strongly induced by LPS. The expression of IL-6 reporter was inhibited by all anti-inflammation compounds in LPS-induced RAW 264.7 cells. The expression of IL-10 reporter was inhibited by quercetin, xanthones and dexamethasone in LPS-induced RAW 264.7 cells. While, expression of IL-10 reporter was induced by beta-D-glucan. These results indicated that this assay could use for determination of IL-6 and IL-10 reporter expression in LPS-induced RAW 264.7 cells for anti-inflammation activity. Moreover, the results showed that natural compounds have an effect on the time course of IL-6 and IL-10 expressions. Therefore, real-time monitoring has a merit for natural compounds screening. We suggested that this assay could serve as a compound screening assay for anti-inflammation activity. (C) 2018 Elsevier Inc. All rights reserved.