猫爪草多糖的理化性质及其对ANA-1细胞的免疫调节作用

Physicochemical properties of PRT and its immunoregulatory effect on ANA-1 cells

目的探讨猫爪草多糖(PRT)对半贴壁巨噬细胞株ANA-1细胞免疫功能的影响。方法采用水提醇沉法提取和分离PRT;红外光谱法测定PRT分子结构;MTT法检测ANA-1细胞增殖能力;不同浓度的猫爪草多糖作用于脂多糖(LPS)诱导的ANA-1细胞,采用Griess法检测一氧化氮(NO)生成量;一氧化氮合酶(i NOS)测定试剂盒检测活性;RT-PCR法检测i NOS、白介素-6(IL-6)和肿瘤坏死因子α(TNF-α)m RNA水平的表达。结果得到灰白色粉末状猫爪草多糖,多糖含量98.17%;红外光谱显示PRT的糖苷键为α-构型,具有吡喃型糖环;PRT增强ANA-1细胞增殖能力,上调IL-6和TNF-αm RNA的表达,且浓度为200μg/m L时作用最强;PRT呈浓度依赖性抑制LPS诱导的ANA-1细胞分泌NO和i NOS活性,当多糖浓度为100μg/m L时,能够完全抑制i NOS m RNA的表达。结论 PRT能够增强ANA-1细胞增殖能力,上调IL-6和TNF-αm RNA水平的表达,抑制LPS诱导的ANA-1细胞分泌NO和i NOS的表达。

Objective To investigate the effect of polysaccharide of Radix Ranunculi ternati（PRT）on the immune function in semi-adherent macrophage cell line ANA-1 cells.Methods Polysaccharide was extracted from Radix Ranunculi ternati by means of water extracting-alcohol precipitating method.Infrared spectrum was used to analyse molecular structure of PRT.Cell proliferation of ANA-1 cells was measured by MTT colorimetry.Nitric oxide（NO）levels were measured by Griess method.The activity of induced nitric oxide synthase（i NOS）was measured by NOS kit.The expression levels of i NOS,IL-6 and TNF-α m RNA were detected by RT-PCR.Results PRT was a gray power;the purity of the polysaccharides reached98.17%.IR spectra demonstrate polysaccharides molecular structure：the glycoside bond configuration of PRT was α.Its monosaccharide contained pyranose structure.PRT could boost the cell proliferation of ANA-1 cells and the phagocytosis of peritoneal macrophage in mice.At the dose of 25~400 μg/m L,PRT could up-regulate the expression levels of IL-6 and TNF-α m RNA;PRT exhibits optimal bioactivities at the dose of 200 μg/m L.Moreover,PRT exhibits optimal bioactivities at the dose of 200 μg/m L,presenting immuno-reagent manner.PRT could inhibit NO levels and the activity of i NOS in LPSinduced ANA-1 cells in dose dependent manner.At the dose of 100 μg/m L,PRT completely inhibit the expression of i NOS m RNA.Conclusion PRT could boost the proliferation of ANA-1 cells and the expression of IL-6 and TNF-α m RNA,but could inhibit NO production and the activity of i NOS in LPS-induced ANA-1 cells in dose dependent manner.