[关键词]
[摘要]
目的 在巨噬细胞中确定四逆散中具有I型干扰素(IFN-I)调控作用的中药,并探究其具体作用靶点及主要作用成分。方法 通过实时荧光定量PCR(qRT-PCR)技术检测四逆散组方药材柴胡、枳实、白芍、甘草水提物对IFN-I通路相关基因表达的影响;通过CCK-8法检测甘草水提物对巨噬细胞RAW264.7细胞活力的影响;qRT-PCR法检测甘草水提物对IFNα2诱导的干扰素诱导基因(ISGs)mRNA表达水平的影响;蛋白免疫印迹(Western blotting)法检测甘草水提物对IFNα2诱导的JAK1、TYK2、STAT1、STAT2蛋白磷酸化水平的影响;qRT-PCR法检测甘草水提物中单体成分甘草酸、甘草苷、异甘草素、甘草素、18β甘草次酸对IFNα2诱导的ISGs的mRNA表达水平的影响。结果 四逆散组方药材柴胡、枳实、白芍各水提物对IFNα2诱导的Isg15和Ifit1表达水平无显著影响,而甘草水提物显著增加了IFNα2诱导的Isg15和Ifit1的基因表达水平(P<0.01、0.001);甘草水提物显著增加了IFNα2诱导的JAK1、TYK2、STAT1、STAT2蛋白磷酸化(P<0.05、0.01、0.001);甘草水提物中18β-甘草次酸能够显著增强IFNα2诱导的Isg15和Ifit1的mRNA表达(P<0.001)。结论 甘草水提物能够显著激活JAK-STAT信号通路并诱导IFN-I下游ISGs的表达,具有较强的固有免疫激活作用。18β-甘草次酸可能是甘草水提物发挥激活IFN-I通路的主要有效成分之一。
[Key word]
[Abstract]
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.
[中图分类号]
R285.5
[基金项目]
国家自然科学基金资助项目(82004029);北京市科技新星项目(Z201100006820025,Z211100002121167)