刺山柑果实正丁醇提取物对树突状细胞成熟的影响及机制研究

Investigation of the Effect of Capparis spinosa Fruit N-Butanol Extract on Dendritic Cell Maturation and Its Mechanism

为探讨刺山柑果实正丁醇相(Capparis spinosa L.fruit n-Butanol Extract,CSBE)对小鼠树突状细胞(Dendritic Cell,DC)成熟的影响及其作用机理,用1、2、3 mg·mL^(-1)的CSBE处理DC,采用流式细胞术和ELISA检测DC表面标志物、抗原吞噬能力和细胞因子分泌验证DC成熟度.采用液相色谱-质谱联用(LC-MS)鉴定CSBE抗炎活性成分,基于网络药理学分析其在类风湿性关节炎(Rheumatoid Arthritis,RA)的相关靶点.利用String数据库和Cytoscape 3.7.2软件构建关键靶点蛋白质-蛋白质互作(Protein-Protein Interaction,PPI)网络,利用Metascape平台进行GO和KEGG富集分析并用western blot检测相关蛋白的表达.实验结果显示:CSBE单独处理时对DC表面共刺激分子和细胞因子没有显著影响,但显著抑制了细菌脂多糖(Lipopolysaccharides,LPS)诱导的DC表面分子CD40和CD86的表达、细胞因子TNF-α和IL-6的分泌以及基质金属蛋白酶MMP9和MMP13的表达,增加了抗原吞噬能力.LC-MS正离子模式下有49种抗炎化合物,负离子模式下有42种抗炎化合物.抗炎化合物在RA中共有核心靶点包括AKT1、TNF、SRC及MMP9等,其中有机酸类化合物主要关联辅助性T细胞的分化、Th17辅助性细胞的分化、Th17型免疫反应等Th17相关的生物学过程,涉及PI3K-AKT信号通路、Th17细胞分化、HIF-1信号通路和IL-17信号通路等类风湿性关节炎相关的信号通路.CSBE能够抑制DC的成熟及促炎细胞因子的表达,通过降低MMP表达抑制DC迁移,从而发挥免疫抑制及治疗RA的功能.

This study investigated the effect and mechanism of Capparis spinosa L.fruit n-butanol extract(CSBE)on the maturation of dendritic cell(DC).DC was treated with 1,2,3 mg·mL^(-1) CSBE.DC surface molecule expression,antigen phagocytosis and cytokine secretion were determined by fow cytometry and ELISA to verify DC maturity.Liquid chromatography-mass spectrometry(LC-MS) was used to identify anti-infammatory components of CSBE and network pharmacology was utilized to analyze their targets related to rheumatoid arthritis(RA).String database and Cytoscape 3.7.2 software were utilized to construct protein-protein interaction(PPI) network maps of the key targets,and Metascape platform was used for GO and KEGG enrichment analysis and western blot was used to identify the expression of related proteins.The experiment results showed that CSBE alone had no significant effect on the expression of DC co-stimulatory molecules and cytokines,but significantly inhibited the expression of CD40 and CD86,matrix metalloproteinases MMP9 and MMP13,and the secretion of TNF-αand IL-6 induced by bacterial lipopolysaccharides(LPS).CSBE also increased antigen phagocytosis of DC.There were 49 anti-infammatory compounds in the positive ion mode of LC-MS,and 42 anti-infammatory compounds in the negative ion mode.Common core targets include AKT1,TNF,SRC and MMP9.Among them,organic acids are mainly associated with Th17-related biological processes such as helper T cell differentiation,Th17 helper cell differentiation,Th17 type immune response,et al..It involves PI3K-AKT signaling pathway,Th17 cell differentiation,HIF-1 signaling pathway,and IL-17 signaling pathway related to RA.CSBE can inhibit the maturation of DC and the expression of pro-infammatory cytokines,and inhibit DC migration by reducing the expression of MMP,thus exerting the function of immunosuppression and treatment of RA.