香菇多糖和黄芪多糖对鲤免疫细胞的活性和IL-1β体外诱生表达的影响

Effects of the activation of immunological cells and the expression of interleukin-1β in the Cyprinus carpio after stimulation by Lentinan and Astragalus polysaccharides

为了探讨香菇和黄芪多糖对鲤免疫细胞的免疫活性作用,采用Percoll密度离心等技术,对鲤的头肾巨噬细胞和外周血白细胞进行分离纯化,并离体培养,从细胞和分子水平上研究了香菇和黄芪多糖的免疫调节作用。巨噬细胞和外周血白细胞分别体外暴露不同浓度的黄芪多糖和香菇多糖后,采用MTT法测定它们对鲤外周血白细胞增殖的影响;NBT还原法和Griess试剂显色法测定对头肾巨噬细胞的呼吸爆发的影响;实时定量PCR法测定头肾巨噬细胞细胞因子IL-1β诱导表达的影响。结果显示,黄芪和香菇多糖作用头肾巨噬细胞24h后,香菇多糖浓度为1,10,100μg·mL-1时能显著诱导巨噬细胞的氧爆发活性,黄芪多糖则没有显著的诱导作用;黄芪和香菇多糖作用头肾巨噬细胞96h后,香菇多糖低浓度时对细胞的氮呼吸爆发活性无显著影响,黄芪多糖浓度10μg·mL-1能显著诱导氮呼吸爆发活性,而在浓度为1000μg·mL-1两者均表现为高浓度抑制作用;两者作用外周血白细胞24h后,香菇多糖浓度为100,1000μg·mL-1和黄芪多糖浓度为10,100μg·mL-1时都能显著促进鲤外周血淋巴细胞的增殖;两种多糖作用头肾巨噬细胞24h后能显著增强IL-1β的体外诱导表达。结果表明黄芪多糖和香菇多糖对离体培养鲤免疫细胞有明显活性作用,对鲤非特异性免疫和特异性免疫具有促进作用。

The polysaccharides in Chinese herbs have been showing to have immunomodulating effects in terrestrial animals, however, the mechanism of the immunomodulating effects of Chinese herbal polysaccharides in fish has not been studied and clarified, especially in the cellular and molecular levels. To investigate the in vitro immunomodulating effects of Lentinan and Astragalus polysaccharides in fish, the macrophages and the peripheral blood leukocytes were isolated from the head kidney and blood of Cyprinus carpio through Percoll gradient density centrifugation. The cells were cultured and stimulated with different concentrations of Astragalus polysaccharides and Lentinan, the proliferation of the peripheral blood granulocytes, respiratory burst of macrophage and IL-1β mRNA expression of macrophages were analyzed by MTT, NBT reduction, Griess reaction and Real time PCR respectively. Results showed that proliferation of peripheral blood leukocytes after 24 h incubation with Astragalus polysaccharides at 10,100μg·mL^-1 and Lentinan at 100,1 000 g. mL 1 was markedly stimulated. Lentinan at 1, 100, 1 000 g·mL^-1 could induce superoxide anion in macrophages markedly, while Astragalus polysaccharides had no effect. Reduction of nitric oxide of head kidney macrophages （HK macrophages） after 96 h incubation with polysaccharides showed that Astragalus polysaccharides at 10 g·mL^-1 had conspicuous effect on nitrogen burst activity of macrophages but Astragalus polysaccharides had no effect, moreover both Astragalus polysaccharides and Lentinan at 1 000μg·mL^-1 inhibited thenitrogen burst activity at higher doses. The expression of interleukin-1β in the HK macrophages after 24 h stimulation by the two polysaccharides showed that Astragalus polysaccharides and Lentinan could stimulate IL-1β expression in carp head kidney. The conclusion is that Astragalus polysaccharides and Lentinan could stimulate the proliferation and respiratory burst activities of the immune cells in carp and upregulate the IL-1β expression in carp head kidney macrophages, hence modulate the non-specific immune responses in carp. The effect of herbal polysaccharides on the cytokine gene expression of macrophages in fish is first reported in this paper. However, more cytokines need to be studied in the future.