不同分子量枸杞多糖对RAW264.7巨噬细胞的免疫调节作用

Immunomodulatory effect of lycium barbarum polysaccharide of different molecular weights on macrophage RAW264.7

目的:研究不同分子量枸杞多糖(lycium barbarum polysaccharides,LBP)对小鼠巨噬细胞RAW264.7的免疫调节作用。方法:本研究通过水提醇沉及柱分离法以获得不同分子量的LBP成分。运用CCK8法检测不同给药浓度的LBP对RAW 264.7细胞增殖的影响;采用中性红吞噬实验测定LBP对细胞吞噬能力的影响;采用实时荧光定量PCR法检测肿瘤坏死因子α(TNF-α)、白细胞介素(IL)-6、IL-1β和诱导型一氧化氮合酶(iNOS)mRNA的表达情况,采用Western Blot法检测丝裂原活化蛋白激酶(MAPK)信号通路相应蛋白质的磷酸化水平。结果:通过分离纯化获得2个高纯度均一组分,分别为LBP1[重均分子量(Mw)=1207 kDa]和LBP2(Mw=125 kDa)。与对照组相比,不同浓度的LBP1和LBP2组分对巨噬细胞的增殖和吞噬能力均有明显的促进作用(P<0.05);LBP1和LBP2在50~200μg·mL-1浓度范围内,可促进巨噬细胞中TNF-α,IL-1β,IL-6和iNOS mRNA的表达,且呈现剂量依赖性。Western Blot法测定结果显示LBP1和LBP2诱导RAW264.7细胞产生免疫应答与特异性激活JNK MAPK通路相关。此外,在对RAW264.7细胞的刺激作用基础上,LBP1的调节作用明显优于LBP2。结论:LBP对RAW264.7巨噬细胞的免疫调节作用可能与其特异性激活JNK MAPK信号转导通路相关,且其活性强弱与LBP分子量表现出较大的关联性。

Objective:To investigate the immunoregulatory effect of Lycium barbarum polysaccharides(LBP)of different molecular weights(Mw)on RAW264.7 macrophages.Methods:LBP with different Mw was obtained by water extraction,alcohol precipitation and chromatographic column separation.CCK8 method was used to detect the effect of LBP on proliferation of RAW 264.7 cells.The effect of LBP on cell phagocytosis was determined by neutral red method.Real-time quantitative PCR was applied to detect the mRNA expression of TNF-α,IL-6,IL-1βand iNOS.Western blot was used to detect the phosphorylation level of proteins in MAPK signaling pathway.Results:Two high purity homogeneous components,LBPl(Mw=1207 kDa)and LBP2(Mw=125 kDa)were obtained by separation and purification.Compared with the control group,LBP1 and LBP2 at different concentrations significantly promoted the proliferation and phagocytosis of macrophages(P<0.05).LBP1 and LBP2 in the concentration range of 50~200μg·mL-1 could promote the expression of TNF-α,IL-1β,IL-6 and iNOS mRNA in macrophages in a dose-dependent manner.Western blot analysis showed that LBP1 and LBP2 induced immune response in RAW264.7 cells,which was related to the specific activation of JNK MAPK pathway.In addition,the regulatory effect of LBP1 was better than that of LBP2 on RAW264.7 cells.Conclusion:The immunoregulatory effect of LBP on RAW264.7 macrophages may be related to its specific activation of JNK MAPK signal transduction pathway,and its activity is closely related to the Mw of LBP.