目的 在巨噬細(xì)胞中確定四逆散中具有I型干擾素（IFN-I）調(diào)控作用的中藥，并探究其具體作用靶點(diǎn)及主要作用成分。方法 通過(guò)實(shí)時(shí)熒光定量PCR（qRT-PCR）技術(shù)檢測(cè)四逆散組方藥材柴胡、枳實(shí)、白芍、甘草水提物對(duì)IFN-I通路相關(guān)基因表達(dá)的影響；通過(guò)CCK-8法檢測(cè)甘草水提物對(duì)巨噬細(xì)胞RAW264.7細(xì)胞活力的影響；qRT-PCR法檢測(cè)甘草水提物對(duì)IFNα2誘導(dǎo)的干擾素誘導(dǎo)基因（ISGs）mRNA表達(dá)水平的影響；蛋白免疫印跡（Western blotting）法檢測(cè)甘草水提物對(duì)IFNα2誘導(dǎo)的JAK1、TYK2、STAT1、STAT2蛋白磷酸化水平的影響；qRT-PCR法檢測(cè)甘草水提物中單體成分甘草酸、甘草苷、異甘草素、甘草素、18β甘草次酸對(duì)IFNα2誘導(dǎo)的ISGs的mRNA表達(dá)水平的影響。結(jié)果 四逆散組方藥材柴胡、枳實(shí)、白芍各水提物對(duì)IFNα2誘導(dǎo)的Isg15和Ifit1表達(dá)水平無(wú)顯著影響，而甘草水提物顯著增加了IFNα2誘導(dǎo)的Isg15和Ifit1的基因表達(dá)水平（P＜0.01、0.001）；甘草水提物顯著增加了IFNα2誘導(dǎo)的JAK1、TYK2、STAT1、STAT2蛋白磷酸化（P＜0.05、0.01、0.001）；甘草水提物中18β-甘草次酸能夠顯著增強(qiáng)IFNα2誘導(dǎo)的Isg15和Ifit1的mRNA表達(dá)（P＜0.001）。結(jié)論 甘草水提物能夠顯著激活JAK-STAT信號(hào)通路并誘導(dǎo)IFN-I下游ISGs的表達(dá)，具有較強(qiáng)的固有免疫激活作用。18β-甘草次酸可能是甘草水提物發(fā)揮激活I(lǐng)FN-I通路的主要有效成分之一。

Objective To identify the Chinese medicine with type I interferon (IFN-I) activities in Si-Ni-San in macrophages and elucidate its specific targets and active components. Methods qRT-PCR analysis was used to detect expression of IFN-I pathway related genes in RAW264.7 cells treated with water extract of Bupleuri Radix, Aurantii Fructus Immaturus, Paeoniae Radix Alba, Glycyrrhizae Radix et Rhizoma in Si-Ni-San. CCK-8 assay was performed to detect cell viability of RAW264.7 cells treated with water extract of Glycyrrhizae Radix et Rhizoma. qRT-PCR analysis was used to detect effect of water extract of Glycyrrhizae Radix et Rhizoma on IFNα2 induced mRNA expression of interferon inducible genes (ISGs). Western blotting was used to detect phosphorylation of JAK1, TYK2, STAT1, and STAT2 in IFNα2-induced RAW264.7 cells treated with water extract of Glycyrrhizae Radix et Rhizoma. qRT-PCR was used to detect effects of glycyrrhizic acid, liquiritin, isoglycyrrhizin, glycyrrhizin, and 18β-glycyrrhetinic acid on IFNα2-induced mRNA expression level of ISGs. Results The water extracts of Bupleuri Radix, Aurantii Fructus Immaturus, and Paeoniae Radix Alba had no significant effects on IFNα2-induced expression levels of Isg15 and Ifit1, while water extract of Glycyrrhizae Radix et Rhizoma significantly increased IFNα2-induced gene expression levels of Isg15 and Ifit1 (P < 0.01, 0.001). Water extract of Glycyrrhizae Radix et Rhizoma significantly increased IFNα2-induced phosphorylation of JAK1, TYK2, STAT1 and STAT2 (P < 0.05, 0.01, and 0.001).18β-glycyrrhetinic acid in water extract of Glycyrrhizae Radix et Rhizoma could significantly enhance IFNα2-induced mRNA expression of Isg15 and Ifit1 (P < 0.001). Conclusion Water extract of Glycyrrhizae Radix et Rhizoma can significantly activate JAK-STAT signaling pathway and induce the expression of ISGs downstream of IFN-I, which has a strong intrinsic immune activation effect. 18β-Glycyrrhetinic acid may be one of the main effective components of Glycyrrhizae Radix et Rhizoma water extract to activate IFN-I pathway.

R285.5

國(guó)家自然科學(xué)基金資助項(xiàng)目（82004029）；北京市科技新星項(xiàng)目（Z201100006820025，Z211100002121167）