银杏内酯B对小鼠腹膜巨噬细胞体外增殖、吞噬及产生NO和ROS的影响

Effects of ginkgolide B on proliferation,phagocytosis,NO and ROS production of murine peritoneal macrophages in vitro

目的:银杏内酯B(GB)是已知的天然而强效的血小板激活因子(PAF)受体(PAFR)拮抗剂,本研究中GB对小鼠腹膜巨噬细胞的增殖、吞噬及产生NO和ROS的影响,初步探讨其潜在的免疫调节作用与机制,从而为临床应用提供可靠的实验依据。方法:分离纯化小鼠腹膜巨噬细胞(PM),以不同浓度的GB(1.25～20μmol/L)预孵后加以脂多糖(LPS)刺激并继续培养至相应时间点;以MTT法检测GB对PM活力及LPS诱导的增殖的影响;以荧光微球的摄入结合流式细胞术评价PM的吞噬作用;以Griess法检测GB对LPS诱导的NO释放的影响;以H2DCFDA标记结合流式细胞术检测PM的ROS水平。结果:LPS刺激后24 h时PM吞噬能力增强,ROS水平显著上升,并释放大量NO,至48 h时PM增殖明显,而经终浓度为5、10、20μmol/L GB在LPS刺激前对PM进行4 h预孵处理可以大幅下调PM对荧光微球的吞噬作用,显著降低PM的ROS水平及NO释放量,并能有效抑制PM的增殖。结论:GB能有效抑制LPS诱导的细胞增殖、吞噬作用、产生NO和ROS的水平,凭借GB对巨噬细胞的体外行为和功能的出色调节作用,理应将其作为天然的免疫抑制剂候选者进行更深入的研究。

AIM： To explore the potential immunomodulatory effects and related mechanisms of ginkgolide B （GB）, a known potent antagonist of platelet-activating factor receptor, we investigated the proliferation, phagocytosis, NO and ROS production of macrophage. METHODS： After murine peritoneal macrophages （PMs） preparation, PMs were treated with different concentrations of GB before culture time and then activated by LPS. Drug toxicology and PM proliferation were measured by MTT assays. Fluorescent beads ingestion and flow cytometry were used to assess phagocytosis of LPS-activated PMs. Griess reagent system was used to determine the amount of LPS-induced NO production. H2DCFDA labeling and flow cytometry were used to trace ROS level of both rest and LPS-activated PMs. RESULTS： In a dose-dependent manner, GB （5, 10, and 20 umoVL） significantly suppressed the phagocytosis as well as NO and ROS production at 24 h and inhibited cell proliferation at 48 h after LPS stimulation. CONCLUSION： According to these interesting effects of GB on macrophage behaving and functioning, it＇s quite reasonable to do further studies of GB as a nature occurring immunomodulator candidate.