扎冲十三味丸抗炎质量标志物筛选及作用机制研究

Identification of anti-inflammatory substances in Zhachong shisanwei pills and investigation of the underlying mechanisms

该实验拟通过扎冲十三味丸(Zhachong shisanwei pills,ZC-13)抗炎活性物质的筛选,明确其抗炎质量标志物并进一步探究其可能的作用机制。首先采用超高效液相色谱与串联四级杆飞行时间质谱仪联用技术(ultra performance liquid chromatography/quadrupole-time-of-flight-mass spectrum,UPLC/Q-TOF-MS)结合核因子-κB(nuclear factor kappa-B,NF-κB)双荧光报告基因系统及一氧化氮(NO)含量检测筛选扎冲十三味丸中的抗炎活性成分,明确其抗炎质量标志物,并通过网络药理学及生物信息学方法预测其质量标志物主要作用靶点和通路,验证木香烃内酯主要抗炎通路。结果从扎冲十三味丸中筛选到NF-κB抑制相关质量标志物4种:没食子酸、鞣花酸、芹糖甘草苷、甘草酸;NO释放抑制相关质量标志物4种:没食子酸、甘草素、木香烃内酯、去氢木香内酯。上述成分主要通过调控磷酸肌醇依赖性蛋白激酶1(3-phosphoinositide-dependent protein kinase 1,PDPK1)、丝裂原活化蛋白激酶14(mitogen-activated protein kinase 14,MAPK14)、糖原合成激酶3β(glycogen synthase kinase-3β,GSK3β)等抗炎相关靶点,进而调控胞内磷脂酰肌醇激酶-蛋白激酶B(phosphoinositide 3-kinase-protein kinase B,PI3K-AKT)、MAPK、哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)等相关通路发挥抗炎作用,其中,木香烃内酯可通过抑制AKT磷酸化及NF-κB核转移发挥抗炎作用。以上结果初步确定了扎冲十三味丸抗炎质量标志物及可能的作用机制,为规范扎冲十三味丸的临床应用及完善其质量评价标准提供了理论依据。

The aim of this study was to identify the anti-inflammatory markers of Zhachong shisanwei pills(ZC-13)and characterize their mechanisms.UPLC/Q-TOF-MS combined with an NF-κB dual fluorescence reporter gene system and NO content detection were utilized to identify the anti-inflammatory bioactive substances in ZC-13.Network pharmacology and bioinformatics methods were used to predict the main targets and pathways of these anti-inflammatory markers,and to verify the main anti-inflammatory pathways of costunolide.Results showed that in ZC-13,four kinds of markers related to NF-κB inhibition were identified:gallic acid,ellagic acid,liquiritin apioside,glycyrrhizic acid,and four kinds of markers related to NO release inhibition were found:gallic acid,liquiritigenin,costunolide,and dehydrocostus lactone.The above components exert anti-inflammatory activities mainly through the regulation of PDK1(3-phosphoinositide-dependent protein kinase 1),MAPK14(mitogenactivated protein kinase),GSK3β(glycogen synthase kinase-3β)and other anti-inflammatory-related targets,and further adjust the PI3 K-AKT(phosphoinositide 3-kinase-protein kinase B),MAPK,mTOR(mammalian target of rapamycin)pathways.Among them,costunolide can inhibit AKT phosphorylation and NF-κB nuclear transfer.The above results identified the anti-inflammatory markers and possible mechanisms of ZC-13,and provide a theoretical basis for standardizing the clinical application and quality of ZC-13.