摘要
目的观察猪苓多糖(PPS)对小鼠腹腔巨噬细胞的活化作用,并研究Toll样受体4(TLR4)在其中的作用。方法用PPS12.5,25,50及100mg·L-1刺激C3H/HeN小鼠和TLR4缺陷的C3H/HeJ小鼠脾细胞72h或腹腔巨噬细胞24h,以[3H]TdR掺入法检测脾细胞增殖反应;以Griess法测定腹腔巨噬细胞培养上清一氧化氮(NO)水平,ELISA检测白细胞介素1β(IL-1β)和肿瘤坏死因子α(TNF-α)含量;以溴化氰活化多糖的方法制备荧光胺(Flu)标记的PPS(Flu-PPS)及Flu-葡聚糖,应用流式细胞仪及激光共聚焦显微镜检测Flu-PPS与小鼠腹腔巨噬细胞的结合。结果 PPS可明显促进C3H/HeN小鼠脾细胞增殖并诱导腹腔巨噬细胞分泌NO,IL-1β和TNF-α,且具有浓度依赖性(r=0.999,P<0.01)。用抗TLR4单抗20mg·L-1与C3H/HeN小鼠腹腔巨噬细胞预孵育1h后加入PPS50mg·L-1,与单独PPS50mg·L-1孵育比较,巨噬细胞培养上清IL-1β和TNF-α浓度分别由(0.38±0.06)μg·L-1和(0.29±0.05)μg·L-1降至(0.18±0.01)μg·L-1和(0.12±0.02)μg·L-1,降幅达52.6%和58.6%(P<0.01)。PPS对C3H/HeN小鼠的脾细胞增殖、腹腔巨噬细胞产生IL-1β和TNF-α的作用明显强于C3H/HeJ小鼠:PPS12.5～100mg·L-1组脾细胞增殖分别增加了2.4,1.7,1.5和22.2倍,IL-1β增加了0.9,1.3,1.2和1.1倍,TNF-α增加了1.3,0.9,0.6和0.6倍,差异均有统计学意义(P<0.01)。流式细胞仪及激光共聚焦显微镜分析结果表明,Flu-PPS1mg·L-1与小鼠腹腔巨噬细胞结合的荧光强度显著高于Flu-葡聚糖,增加Flu-PPS浓度至5mg·L-1,荧光强度并未相应增强,表明Flu-PPS与小鼠腹腔巨噬细胞的结合具有饱和性;未标记的PPS100mg·L-1及抗TLR4单抗200mg·L-1可明显阻断Flu-PPS与巨噬细胞的结合。结论 PPS可能通过TLR4活化小鼠腹腔巨噬细胞。
OBJECTIVE To observe the effect of polysaccharides from Polyporus umbellatus(Pers.) Fries(PPS) on mouse peritoneal macrophages and evaluate the role of the Toll-like receptor(TLR) in the process.METHODS Splenocytes and peritoneal macrophages from C3H/HeN or C3H/HeJ mice were incubated with different concentrations of PPS(12.5,25,50 and 100 mg·L-1) for 72 h or 24 h.Splenocyte proliferation was analyzed with [3H]TdR incorporation method.Nitric oxide(NO) was measured by Griess method and cytokines of culture supernatants were detected by enzyme linked immunosorbent assay(ELISA).The fluoresceinamine-labeled PPS(Flu-PPS) and dextran(Flu-dextran) were prepared by the cyanogen bromide activation method.The cell-binding activity of Flu-PPS was analyzed with FACS and the confocal microscopy.RESULTS PPS significantly stimulated the proliferation of splenocytes from C3H/HeN mice and induced the production of NO,interleukin-1β(IL-1β) and tumor necrosis factor-α(TNF-α) of peritoneal macrophages in a concentration-dependent manner(r = 0.999,P < 0.01).Compared with the PPS 50 mg·L-1 group,the levels of IL-1β and TNF-α decreased from(0.38 ± 0.06) μg·L-1 and(0.29 ±0.05) μg·L-1 to(0.18 ±0.01) μg·L-1 and(0.12 ±0.02) μg·L-1 when macrophages from C3H/HeN mice were pretreated with anti-mouse TLR4 20 mg·L-1 monoclonal antibody for 1 h before adding PPS;the inhibitory rate was 52.6% and 58.6%(P < 0.05),respectively.The effect of PPS on splenocyte proliferation,NO,IL-1β and TNF-α production in C3H/HeN mice were stronger than in C3H/HeJ mice:splenocyte proliferation was increased 2.4,1.7,1.5 and 22.2 times,and IL-1β production increased 0.9,1.3,1.2 and 1.1 times,TNF-α production increased 1.3,0.9,0.6 and 0.6 times in PPS 12.5-100 mg·L-1 groups,respectively.The Flu intensity of murine macrophages staining with Flu-PPS(1 mg·L-1) was higher than that of Flu-dextran group in FACS and confocal laser scanning microscopy analysis.The Flu intensity did not change while the concentration of PPS was increased to 5 mg·L-1
出处
《中国药理学与毒理学杂志》
CAS
CSCD
北大核心
2010年第4期266-273,共8页
Chinese Journal of Pharmacology and Toxicology
基金
温州市科学技术局资助项目(Y20080126)
温州医学院科研启动基金资助项目(QTJ07021)~~
关键词
猪苓多糖
腹腔巨噬细胞
TOLL样受体4
polyporus polysaccharides
peritoneal macrophages
Toll-like receptor 4
