Sun Meng, Wang Wen-di, Li Yan, Liu Ke-Chun, Xia Qing, Jiang Yan-Yan, Liu Bin
School of Chinese Materia Medica, Beijing University of Chinese Medicine Beijing 102488, China.
Biology Institute, Qilu University of Technology (Shandong Academy of Sciences) Ji'nan 250103, China.
Zhongguo Zhong Yao Za Zhi. 2023 Apr;48(7):1916-1926. doi: 10.19540/j.cnki.cjcmm.20221201.702.
The immunomodulatory effect of Saposhnikoviae Radix polysaccharide(SRP) was evaluated based on the zebrafish mo-del, and its mechanism was explored by transcriptome sequencing and real-time fluorescence-based quantitative PCR(RT-qPCR). The immune-compromised model was induced by navelbine in the immunofluorescence-labeled transgenic zebrafish Tg(lyz: DsRed), and the effect of SRP on the density and distribution of macrophages in zebrafish was evaluated. The effect of SRP on the numbers of macrophages and neutrophils in wild-type AB zebrafish was detected by neutral red and Sudan black B staining. The content of NO in zebrafish was detected by DAF-FM DA fluorescence probe. The content of IL-1β and IL-6 in zebrafish was detected by ELISA. The differentially expressed genes(DEGs) of zebrafish in the blank control group, the model group, and the SRP treatment group were analyzed by transcriptome sequencing. The immune regulation mechanism was analyzed by Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment, and the expression levels of key genes were verified by RT-qPCR. The results showed that SRP could significantly increase the density of immune cells in zebrafish, increase the number of macrophages and neutrophils, and reduce the content of NO, IL-1β, and IL-6 in immune-compromised zebrafish. The results of transcriptome sequencing analysis showed that SRP could affect the expression level of immune-related genes on Toll-like receptor pathway and herpes simplex infection pathway to affect the release of downstream cytokines and interferon, thereby completing the activation process of T cells and playing a role in regulating the immune activity of the body.
基于斑马鱼模型评估防风多糖(SRP)的免疫调节作用,并通过转录组测序和实时荧光定量PCR(RT-qPCR)探究其作用机制。在免疫荧光标记的转基因斑马鱼Tg(lyz:DsRed)中用长春瑞滨诱导免疫缺陷模型,评估SRP对斑马鱼巨噬细胞密度和分布的影响。通过中性红和苏丹黑B染色检测SRP对野生型AB斑马鱼巨噬细胞和中性粒细胞数量的影响。用DAF-FM DA荧光探针检测斑马鱼中NO的含量。用ELISA检测斑马鱼中IL-1β和IL-6的含量。通过转录组测序分析空白对照组、模型组和SRP处理组斑马鱼的差异表达基因(DEG)。通过基因本体论(GO)和京都基因与基因组百科全书(KEGG)富集分析免疫调节机制,并用RT-qPCR验证关键基因的表达水平。结果表明,SRP可显著增加斑马鱼免疫细胞密度,增加巨噬细胞和中性粒细胞数量,并降低免疫缺陷斑马鱼中NO、IL-1β和IL-6的含量。转录组测序分析结果表明,SRP可影响Toll样受体途径和单纯疱疹感染途径上免疫相关基因的表达水平,从而影响下游细胞因子和干扰素的释放,进而完成T细胞的激活过程,发挥调节机体免疫活性的作用。