榆黄菇多糖对体外诱导炎症模型细胞因子的分泌及一氧化氮合酶（NOS）的影响

Effects of Pleurotus citrinipileatus Polysaccharides on the of Secretion Cytokines and Nitric Oxide Synthase (NOS) in An in Vitro Induced Inflammation Model

摘要:

以榆黄菇为原料，采用水提醇沉法提取榆黄菇粗多糖，Sevage法去除蛋白，利用DEAE-Cellulose离子交换和Superdex-75凝胶层析方法纯化获得PSI和PSII两个组分；通过脂多糖（LPS）刺激建立炎症模型；利用MTT法筛选出两个多糖的优势浓度，采用吸光度分析法测定一氧化氮合酶（NOS）的活力、酶联免疫吸附试剂盒测定细胞因子IL-1β、IL-6和TNF-α的分泌水平来评价多糖对RAW264.7细胞的抗炎作用。研究结果显示，LPS浓度为5 μg/mL时，细胞炎症水平达到最高，在模型的基础上筛选出PSI的优势浓度为500 μg/mL、PSII的优势浓度为200 μg/mL；与LPS模型组相比较，PSI对NOS活性、IL-1β、IL-6和TNF-α的分泌水平抑制率分别为37.54%、28.84%、28.27%和30.25%，PSII对NOS活性、IL-1β、IL-6和TNF-α的分泌水平抑制率分别为51.24%、37.56%、31.35%和32.67%。研究证实，榆黄菇多糖PSI和PSII对LPS诱导RAW264.7炎症细胞具有缓解作用，主要表现在抑制炎症细胞因子的分泌水平、促进细胞增殖、降低巨噬细胞的吞噬能力等方面，而对比PSI来说，PSII对于一氧化氮合酶（NOS）以及细胞因子IL-1β、IL-6和TNF-α的分泌水平的抑制力要更强，具有更好的抗炎效果。

Abstract:

Pleurotus citrinipileatus was used as the raw material, and crude polysaccharides were extracted by water extraction and alcohol precipitation from its fruiting bodies of. The Sevage method was used to remove proteins, and two components, PSI and PSII were obtained after purification by DEAE-Cellulose ion exchange and Superdex-75 gel chromatography. The in vitro inflammation model was established by lipopolysaccharide (LPS) stimulation. The optimal doses of the two polysaccharides were screened by the MTT method. The activity of nitric oxide synthase (NOS) was determined by the absorbance assay, and the levels of secreted cytokines, IL-1β, IL-6 and TNF-α, were determined by enzyme linked immunosorbent assay kits to evaluate the anti-inflammatory effect of the polysaccharides on RAW264.7 cells. The results showed that the cellular inflammation level reached the highest when the concentration of LPS was 5 μg/mL. On the basis of the cell model, the optimal dose of PSI and PSII were 500 μg/mL and 200 μg/mL, respectively. Compared with the LPS model group, the inhibition rates of PSI on NOS activity, IL-1β, IL-6 and TNF-α were 37.54%, 28.84%, 28.27% and 30.25%, respectively. The inhibitory rates of PSII on NOS activity, IL-1β, IL-6 and TNF-α were 51.24%, 37.56%, 31.35 % and 32.67%, respectively. The research confirmed that PSI and PSII had the alleviating effects on LPS-induced inflammatory cells, which were mainly manifested in inhibiting the level of secreted inflammatory cytokines, promoting cell proliferation, and reducing the phagocytic ability of macrophages. Compared with PSI, PSII had a greater inhibitory effect on the secretion levels of nitric oxide synthase (NOS) and cytokines, IL-1β, IL-6 and TNF-α, thereby exhibiting a better anti-inflammatory effect. Key words: