虫草素对脂多糖诱导巨噬细胞过度活化的抑制作用研究  

Inhibitory effect of cordycepin on macrophage hyperactivation induced by lipopolysaccharide

作　　者：孟雪莲[1] 刘佳[1] 刘莹莹[1] 郑良超 高程程[1] 王丹[1] 吕晶[1] 陈长兰[1] 

机构地区：[1]辽宁大学药学院,辽宁沈阳110036

出　　处：《食品工业科技》2017年第13期297-301,306,共6页Science and Technology of Food Industry

基　　金：国家自然科学基金项目(81503085;31371085)

摘　　要：目的:研究虫草素对脂多糖(LPS)诱导巨噬细胞过度活化的抑制作用。方法:培养RAW264.7小鼠巨噬细胞,采用LPS(1μg/m L)激活巨噬细胞,同时给予0.1~10μmol/L的虫草素处理细胞,采用四甲基偶氮唑盐(MTT)法测定细胞存活率,Griess法检测细胞一氧化氮(NO)释放量,酶联免疫吸附分析实验(ELISA)分别检测三种细胞炎症因子肿瘤坏死因子α(TNF-α)、白介素-1β(IL-1β)和白介素-6(IL-6)的释放水平,采用Fluo-3/AM染色法以流式细胞仪测定胞浆游离钙离子浓度([Ca^(2+)]i)的变化,采用水杨酸法考察虫草素对羟自由基(·OH)清除作用,L-酪氨酸法检测其对过氧亚硝酸根离子自由基(ONOO-)清除作用,邻苯三酚自氧化法检测其对超氧阴离子自由基(O_2^-·)清除作用。结果:虫草素在0.1~10μmol/L浓度范围内可显著抑制LPS激活的RAW264.7巨噬细胞释放NO及3种炎症因子(IL-1β、IL-6和TNF-α),同时对细胞存活率无显著影响;虫草素(10μmol/L)可显著抑制LPS诱导的巨噬细胞内[Ca^(2+)]i水平异常升高;此外,虫草素对·OH、ONOO-和O_2^-·自由基具有显著清除作用。结论:虫草素可抑制LPS激活的RAW264.7巨噬细胞释放NO及炎症因子TNF-α、IL-1β和IL-6,这可能与其抑制了细胞内[Ca^(2+)]i的增高及清除·OH、ONOO-和O_2^-·自由基有关。虫草素可能对巨噬细胞过度激活引起的炎症相关疾病具有一定的防治作用。Objective：To study the inhibitiory effect of cordycepin on macrophage hyperactivation induced by lipopolysaccharide （LPS）.Methods： Mouse RAW264.7 macrophages were cultured with LPS（ 1μg/mL）and different concentrations of eordyeepin （0.1- 10 μmoL/L）.The cell viability was evaluated by MTT assay.Nitrite levels in culture supernatant fluids were examined by Griess assay.The release of eytokines （ TNF-α, IL-1β, and IL- 6 ） was examined by enzyme- linked immunosorbent assay （ELISA） .The cytosolie free Ca^2＋ level（ [ Ca^2＋]i ） of macrophages was measured by staining the cells with Fluo-3/AM followed by flow cytometry analysis.The scavenging activity of cordycepin against hydroxy radical（·OH）, peroxynitrite（ ONOO- ）, and superoxide anion （O2^-· ） was examined by salicylic acid, L - tyrosine, and pyrogallie acid methods, respectively. Results ： Cordyeepin （0.1-10 txmoL/L）could inhibit the release of NO and cytokines（ TNF-α, IL-1β, and IL-6）without the influence of cell viability in LPS- activated maerophages. Cordyeepin （ 10 μmoL/L） could suppress the [ Ca2＋ ]i level in LPS- activated macrophages.In addition, eordyeepin showed direct scavenging activity towards · OH, ONOO-, and O2^-· free radicals, significantly.Conclusion：It is suggested that cordycepin can inhibit LPS-induced release of NO and cytokines （TNF-α, IL-1β, and IL-6）by suppressing the [ Ca^2＋]i level and directly scavenging · OH, ONOO- , and O2^- · free radicals. Cordyeepin may have therapeutic potential in the treatment of inflammation-related diseases accompanied by macrophage activation.

关 键 词：虫草素 脂多糖 巨噬细胞 过度活化 

分 类 号：TS201.4[轻工技术与工程—食品科学]