基于斑马鱼模型的防风多糖调节免疫作用机制研究

Immune regulation mechanism of Saposhnikoviae Radix polysaccharide based on zebrafish model

利用斑马鱼模型评价防风多糖的免疫调节作用,并利用转录组测序和实时荧光定量PCR技术探讨其作用机制。以免疫细胞荧光标记的转基因斑马鱼Tg(lyz:DsRed)为实验动物,选用长春瑞滨诱导免疫低下模型,评价防风多糖对斑马鱼体内巨噬细胞密度和分布的影响。以野生型AB系斑马鱼为实验动物,采用中性红和苏丹黑B染色法,检测防风多糖对斑马鱼体内巨噬细胞和中性粒细胞数目的影响;采用DAF-FM DA荧光探针法,检测各组斑马鱼体内NO含量的变化;ELISA法检测斑马鱼体内IL-1β和IL-6的含量变化。利用转录组测序分析空白对照组、模型组和防风多糖处理组斑马鱼的差异表达基因,通过Gene Ontology(GO)和Kyoto Encyclopedia of Genes and Genomes(KEGG)富集分析其免疫调节作用机制,并利用RT-qPCR法验证关键基因的表达水平。结果显示,防风多糖能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目,并降低免疫低下斑马鱼体内NO、IL-1β和IL-6含量。转录组测序分析结果显示防风多糖通过影响Toll样受体通路和单纯疱疹感染通路上免疫相关基因的表达水平,进而影响下游的细胞因子以及干扰素的释放,完成T细胞的活化过程,发挥调节机体免疫活性的作用。

The immunomodulatory effect of Saposhnikoviae Radix polysaccharide(SRP)was evaluated based on the zebrafish model,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-1βand 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.