基于斑马鱼模型研究天麻多糖的免疫调节作用

Assessment of immunomodulatory effect of Gastrodiae rhizomapolysaccharides in zebrafish embryo

天麻多糖(Gastrodiae rhizoma polysaccharides, GRP)具有一定的免疫调节和抗炎作用,然而目前对其机理的研究相对较少,也缺乏系统性的评价模型。本研究首次使用斑马鱼模型评估了GRP的安全性和免疫调节作用,为未来GRP成分的研究和开发提供了重要的数据支持。首先从GRP给药后斑马鱼的存活率、心率和卵黄囊水肿情况等方面进行了安全性评估。采用吖啶橙、DAF-FM DA和DCFH-DA荧光探针等方法分别检测了凋亡细胞、一氧化氮(NO)和活性氧(ROS)水平;通过实时定量PCR(RT-qPCR)研究GRP对脂多糖(LPS)诱导的免疫相关蛋白的基因表达的影响;使用中性红和苏丹黑B染色评估GRP对斑马鱼巨噬细胞及中性粒细胞数量的影响。结果表明,GRP抑制了LPS诱导的心率加快、卵黄囊水肿和形态畸形等病理反应,表明GRP对LPS诱导的斑马鱼胚胎毒性具有保护作用。此外,GRP表现出免疫调节作用,呈剂量依赖性地抑制了LPS诱导的NO和ROS的产生以及细胞凋亡。qPCR结果表明,GRP的抗炎作用与C3a和TNF-α的下调以及IL-10翻译的上调有关。此外,GRP还减少了中性粒细胞和巨噬细胞在炎症部位的积聚,体现出了其对LPS诱导的斑马鱼的保护作用。

Gastrodiae rhizoma polysaccharides(GRP)have garnered attention primarily for their hepatoprotective and anti-cancer properties,with limited exploration in immunomodulation and anti-inflammation.This study,for the first time,evaluates the safety and immune regulatory effects of GRP by using a zebrafish model,elucidating its mechanism of action and furnishing important data for future GRP components research and development.Safety evaluations encompassing survival rate,heart rate,and yolk sac edema of zebrafish are conducted.Additionally,acridine orange,DAF-FM DA,and DCFH-DA fluorescent probes are employed to detect apoptotic cells,nitric oxide(NO)and reactive oxygen species(ROS)levels respectively.The impact of GRP on the expression of immune genes induced by lipopolysaccharide(LPS)is studied through real-time quantitative PCR(RT-qPCR).The effects of GRP on the quantity of macrophages and neutrophils in zebrafish are assessed by using neutral red and Sudan Black B staining.Our resultsshow co-incubation of GRP inhibits the pathological reactions such as increased heart rate,yolk sac edema,and morphological malformations induced by LPS,demonstrating GRP has a protective effect against LPS-induced toxicity in zebrafish embryos.Furthermore,GRP exhibits dese-dependent immunomodulatory effects,suppressing the production of NO and ROS and cell apoptosis induced by LPS.The results of qPCR indicate the anti-inflammatory action of GRP is associated with the downregulation of C3a and TNF-αand the upregulation of IL-10 translation.Moreover,GRP also reduces the accumulation of neutrophils and macrophages at sites of inflammation,highlighting its protective role against LPS-induced effects in zebrafish.