Yoshida Lucia Satiko, Kakegawa Tomohito, Yuda Yasukatsu, Takano-Ohmuro Hiromi
Research Institute of Pharmaceutical Sciences, Musashino University, 1-1-20 Shinmachi, Nishitokyo, 202-8585, Japan.
Faculty of Pharmaceutical Sciences, Josai International University, 1 Gumyo, Togane, 283-8555, Japan.
J Nat Med. 2017 Oct;71(4):723-734. doi: 10.1007/s11418-017-1106-5. Epub 2017 Jul 11.
We aimed to find candidate molecules possibly involved in the anti-inflammatory activity of shikonin (active compound of "Shikon") by analyzing its effects on gene expression of lipopolysaccharide (LPS)-treated THP-1 macrophages. Polysome-associated mRNAs (those expected to be under translation: translatome) from cells treated with LPS alone (LPS: 5 µg/mL), shikonin alone (S: 100 nM), or LPS plus shikonin (LPS&S) for 3 h were analyzed by DNA microarray followed by detection of enriched pathways/gene ontologies using the tools of the STRING database. Candidate genes in enriched pathways in the comparison of LPS&S cells vs. LPS cells were analyzed by reverse-transcription quantitative real-time PCR (RT-qPCR; 1, 2, and 3 h). DNA microarray showed shikonin significantly influences gene expression. Gene expression changes between LPS&S cells and LPS cells were compared to detect relevant proteins and/or mRNAs underlying its anti-inflammatory effects: shikonin downregulated pathways which were upregulated in LPS cells, for example, 'innate immune response'. Within changed pathways, three genes were selected for RT-qPCR analyses as key candidates influencing inflammatory responses: CYBA (component of the superoxide-generating Nox2 enzyme), GSK3B (controller of cell responses after toll-like receptor stimulation), and EIF4E (a key factor of the eukaryotic translation initiation factor 4F complex that regulates abundance of other proteins involved in immune functions). All three mRNAs were decreased at 2 h, and CYBA continued low at 3 h relative to LPS cells. Given that shikonin decreased the expression of CYBA gene of Nox2, in addition to the direct inhibition of the Nox2 activity that we have previously shown, it is suggested that one of its anti-inflammatory mechanisms could be attenuation of oxidative stress.
我们旨在通过分析紫草素（“紫草根”的活性成分）对脂多糖（LPS）处理的THP-1巨噬细胞基因表达的影响，寻找可能参与其抗炎活性的候选分子。用单独的LPS（LPS：5μg/mL）、单独的紫草素（S：100 nM）或LPS加紫草素（LPS&S）处理细胞3小时后，通过DNA微阵列分析多聚体相关mRNA（预计正在翻译的mRNA：翻译组），然后使用STRING数据库工具检测富集的通路/基因本体。通过逆转录定量实时PCR（RT-qPCR；1、2和3小时）分析LPS&S细胞与LPS细胞比较中富集通路中的候选基因。DNA微阵列显示紫草素显著影响基因表达。比较LPS&S细胞和LPS细胞之间的基因表达变化，以检测其抗炎作用背后的相关蛋白质和/或mRNA：紫草素下调了在LPS细胞中上调的通路，例如“先天免疫反应”。在变化的通路中，选择三个基因进行RT-qPCR分析，作为影响炎症反应的关键候选基因：CYBA（产生超氧化物的Nox2酶的成分）、GSK3B（Toll样受体刺激后细胞反应的控制器）和EIF4E（真核翻译起始因子4F复合物的关键因子，调节参与免疫功能的其他蛋白质的丰度）。相对于LPS细胞，所有三种mRNA在2小时时均下降，CYBA在3小时时持续较低。鉴于紫草素除了我们之前所示的直接抑制Nox2活性外，还降低了Nox2的CYBA基因表达，提示其抗炎机制之一可能是减轻氧化应激。
