Abstract
OBJECTIVE To investigate the structural characteristics of Gynostemma longipes oligo-saccharide(GLO)and explore their immunomodulatory effects against vinorelbine(VNR)-induced immunotoxicity in a zebrafish model.METHODS ① Oligosaccharide fractions were isolated from the roots of Gynostemma longipes and characterized using ultra-high performance liquid chromatography-charged aerosol detection(UHPLC-CAD)and ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF/MS).② At 4 hours post-fertilization(hpf),normally hatched AB wild-type zebrafish embryos were exposed to GLO at the concentrations of 50,100,200,400 and 800 mg·L-1,while the control group was treated with fresh E3 medium.Mortality was statistically analyzed using GraphPad Prism 10,and morphological changes were observed under a microscope over a 7-day period.③ At 48 hpf,normally hatched zebrafish embryos with fluorescently labeled neutro-phils and macrophages and 120 hpf healthy embryos with fluorescently labeled T cells were divided into control(0.1%DMSO),model(200 mg·L-1 VNR),model+80 mg·L-1 levamisole hydrochloride(LH),and model+GLO(50,100,200 mg·L-1)groups.After 24 h of treatment,fluorescence intensities of neutrophils,macrophages,and T cells were measured using confocal microscopy.④ At 48 hpf,fluores-cently labeled embryos(neutrophils and macrophages)were grouped and treated as described in step③.Following treatment,the medium was replaced with fresh E3,and a tail transection was performed to induce local acute inflammation.At 6 h post-injury,the fluorescence intensities of neutrophils and macrophages at the injury site were quantified under a fluorescence microscope to assess their recruit-ment function.⑤ At 48 hpf,normally hatched AB wild-type zebrafish embryos were grouped and treated as described in step ③.After treatment,transcriptome sequencing was conducted for Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analyses to explore the immunomodulatory mechanisms.The expression levels of Toll-like receptor 4(TLR4),myeloid differen-tiation factor 88(Myd88),interleukin-1β(IL-1β),IL-6,IL-10,IL-8,tumor necrosis factor-α(TNF-α)and nuclear factor κB p65 subunit(NF-κB p65)were measured by RT-qPCR.TLR4 protein levels were detected by Western blotting and IL-1β and IL-6 protein levels by ELISA.RESULTS ① The oligosac-charide fraction was isolated from G.longipes and identified as a mixture of oligosaccharides with degrees of polymerization ranging from 2 to 6.② GLO showed no toxicity to zebrafish embryos at concen-trations of 50-200 mg·L-1,but induced embryonic malformations at 400-800 mg·L-1.Based on embryo survival,an effect-response curve was fitted,and the median lethal concentration(LC50)of GLO was determined to be 471.3 mg·L-1.③ Compared with the control group,fluorescence intensities of neutro-phils,macrophages,and T cells were significantly reduced in the model group,which were markedly restored in model+LH and model+GLO groups.④ Compared with the control group,the fluorescence intensities of neutrophils and macrophages at the injury site of tails were significantly decreased in the model group,suggesting a marked reduction in the recruitment capacity of these immune cells to the injured site.Compared with the model group,the VNR-induced impairment of immune cell recruitment was effectively restored at varying doses in both the LH+model group and the GLO+model groups.⑤ Transcriptomic analysis revealed significant enrichment of the Toll-like receptor signaling pathway in both GO and KEGG annotations.RT-qPCR results showed that,compared with control group,the gene expressions of TLR4,Myd88,and downstream inflammatory factors(IL-1β,IL-6,TNF-α,NF-κB p65,IL-10 and IL-8)were significantly upregulated in the model group.However,these increases were significantly inhibited in model+LH and model+GLO groups.Protein levels of TLR4,IL-1β,and IL-6 were also elevated in the model group compared with the control group,which was significantly attenu-ated in model+LH and model+GLO groups.CONCLUSION This study has confirmed that GLO consist of oligosaccharides with polymerization degrees ranging from 2 to 6,with relatively high contents of disac-charides and pentasaccharides.In a zebrafish model,GLO exert a dual immunomodulatory effect by regulating the Toll-like receptor signaling pathway,promoting the recovery of immune cell numbers and recruitment function while inhibiting excessive inflammatory responses.关键词
长梗绞股蓝/寡糖/免疫调节/斑马鱼/Toll样受体信号通路/免疫细胞Key words
Gynostemma longipes/oligosaccharide/immunomodulation/zebrafish/Toll-like receptor signaling pathway/immune cells分类
医药卫生
