元蘑多糖的结构特征及其调节巨噬细胞免疫活性机制

Structural Characteristics of Sarcomyxa edulis Polysaccharide and Its Mechanism of Regulating Macrophage Immunomodulatory Activity

【目的】为明确元蘑多糖的结构特征,并探究其调节巨噬细胞免疫活性机制。【方法】采用水提醇沉法获得元蘑多糖粗提物,并通过DEAE-52离子交换层析及Sephacryl S-400葡聚糖凝胶过滤层析对该多糖粗提物进行分离纯化,获得多糖组分SEP-0a。分别采用高效凝胶渗透色谱法、离子交换色谱法、傅立叶红外光谱法检测了SEP-0a的理化性质。进一步采用CCK-8法、ELISA法、qPCR法、Western blot等方法考察了元蘑多糖SEP-0a对巨噬细胞RAW264.7的免疫调节作用及机制。【结果】元蘑多糖SEP-0a由半乳糖、甘露糖、葡萄糖和岩藻糖组成,相对分子量为4.23×10^(4)Da,并且具有α构型与β构型。体外免疫活性结果表明,元蘑多糖SEP-0a可显著增强RAW264.7细胞增殖和吞噬能力,促进该细胞活性氧(reactive oxygen species,ROS)、一氧化氮(nitric oxide,NO)的分泌,提高肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素(interleukin,IL)-1β、IL-6的表达,Western blot检测发现,该多糖可显著提高核转录因子-κB(nuclear factor-κB,NF-κB)信号通路p65蛋白磷酸化表达。【结论】元蘑多糖SEP-0a可通过NF-κB信号通路调控巨噬细胞的免疫活性。

【Objective】In order to clarify the structural characteristics of Sarcomyxa edulis polysaccharides and explore its mechanism of regulating macrophage immunomodulatory activity.【Method】The crude polysaccharide of S.edulis was obtained by water extraction and ethanol precipitation,and the purified polysaccharide component SEP-0a was obtained by DEAE-52 ion exchange column chromatography and Sephacryl S-400 glucan gel column chromatography.The physicochemical properties of SEP-0a were investigated by high performance gel permeation chromatography evaporative light-scattering detector,ion exchange chromatography high performance liquid chromatography and Fourier transform infrared spectrometer.On this basis,the immunomodulatory effect of polysaccharide SEP-0a from S.edulis on macrophage cell RAW264.7 was further studied by CCK-8,ELISA,qPCR,Western Blot and other methods.【Result】The S.edulis polysaccharide SEP-0a was composed of galactose,mannose,glucose and fucose with a relative molecular weight of 4.23×10^(4) Da,and hadαandβconfigurations.The results of in vitro immunocompetence showed that S.edulis polysaccharide SEP-0a significantly increased the proliferation and phagocytic ability of RAW264.7 cells,promoted the secretion of reactive oxygen species(ROS),nitric oxide(NO)and increased the expressions of tumor necrosis factor-α(TNF-α),interleukin(IL)-1β,and IL-6 in these cells.Western blot results showed that this polysaccharide significantly increased p65 protein phosphorylation expression in the nuclear factor-κB(NF-κB)signaling pathway.【Conclusion】The polysaccharide SEP-0a of S.edulis may regulates the immune activity of macrophages by NF-κB signal pathway.