蕨麻多糖组分抑制过氧化氢诱导小鼠脾淋巴细胞凋亡的作用

Inhibitory action of Potentilla anserine polysaccharide fraction on H_2O_2-induced apoptosis of murine splenic lymphocytes

分离提取蕨麻多糖组分(Potentilla anserine polysaccharide fraction,PAPF),进行理化性质检测、红外光谱和气相色谱分析。用过氧化氢(H2O2)建立小鼠体外淋巴细胞凋亡模型,通过形态学、琼脂糖凝胶电泳、流式细胞术等方法研究不同浓度的PAPF对H2O2介导的小鼠脾淋巴细胞凋亡的影响。结果表明PAPF得率为6.22%,不含淀粉、氨基酸和蛋白质,该多糖组分由鼠李糖、阿拉伯糖、葡萄糖和半乳糖组成,红外光谱扫描呈现多糖类物质的特征吸收峰。形态学、琼脂糖凝胶电泳、流式细胞仪分析结果表明,200μmol·L–1H2O2可诱导脾淋巴细胞出现典型凋亡。PAPF(50、100、200和400μg·mL–1)能拮抗过氧化氢所诱导的细胞凋亡,抑制作用随多糖组分浓度的增加而增强。提示PAPF对过氧化氢介导的淋巴细胞凋亡具有显著的抑制作用,且呈浓度依赖关系。

A water-soluble polysaccharide fraction from root of Potentilla anserine was obtained. Gas chromatogram, FT-IR, physical and chemical characteristics of the Potentilla anserine polysaccharide fraction （PAPF） were analyzed. The protective effects of PAPF against the H2O2 induced process of apoptosis of murine splenic lymphocytes were investigated in vitro. Morphological assessment of apoptosis was performed with light microscope and laser scanning confocal microscope. DNA fragmentation was visualized by agarose gel electrophoresis. The amount of apoptotic cells was measured by flow cytometry. The results showed that PAPF is composed of rhamnose, arabinose glucose and galactose. H2O2 （200 μmol·L^-1） induced apoptosis of murine splenic lymphocytes with the cell volume reduced, cytoplasm and nuclear shrunk and DNA stained non-uniformly. Condensed chromatin and formation of apoptotic body were observed in the apoptotic cells. Apoptotic bodies in the cells treated with PAPF and H2O2 were less than those in H2O2 treatment alone. DNA fragmentation assay showed that PAPF （50, 100, 200, and 400μg·mL^-1） obviously reduced H2O2-induced ladder bands. Flow cytometry analysis showed that H2O2 increased the populations of apoptotic sub-G1 cells from 5.60% （control） to 45.40%, and PAPF decreased H2O2-induced apoptosis to 37.80%, 22.70%, 17.70%, and 8.50%, respectively. In conclusion, PAPF reduced H2O2-induced oxidative damage in a dose dependent manner.