First Affiliated Hospital, Wenzhou Medical College, Wenzhou 325035, PR China.
J Ethnopharmacol. 2011 Apr 26;135(1):1-6. doi: 10.1016/j.jep.2010.06.028. Epub 2010 Jul 1.
Zhu Ling (Polyporus umbellatus) is well-known to reduce the risk of a variety of diseases. In this study, we explored the molecular mechanism of its immunostimulatory potency in immune responses of macrophages, using polysaccharides prepared from Polyporus umbellatus (PPS).
Splenocyte proliferation was analyzed with (3)H-TdR incorporation method. Nitric oxide (NO) was measured by Griess method and cytokines of culture supernatants was 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 confocal microscopy. NF-κB activity was measured by ELISA assay.
We found that PPS is able to strongly upregulate the functions of macrophages such as Nitric oxide (NO) production and cytokine expression. Compared with C3H/HeJ group, PPS significantly stimulated the proliferation of splenocytes and the production of TNF-α, IL-1β and NO of peritoneal macrophages from C3H/HeN mice. The function blocking antibodies to TLR-4, but not TLR-2 and CR3, markedly suppressed PPS-mediated TNF-α and IL-1β production. Flow cytometric and confocal laser-scanning microscopy analysis shown that fluorescence-labeled PPS (f-PPS) can bind specifically to the target cells, and the binding can blocked by unlabeled PPS and anti-TLR4, but not anti-TLR2 and CR3 monoclonal antibodies. Nuclear translocation and DNA binding activity of NF-κB was significantly induced by PPS.
Therefore, our data suggest that PPS may exert its immunostimulating potency via TLR-4 activation of signaling pathway.
灵芝（多孔菌科）被广泛认为可以降低多种疾病的风险。在这项研究中，我们使用从灵芝（多孔菌科）中提取的多糖（PPS）来探索其在巨噬细胞免疫反应中的免疫刺激作用的分子机制。
采用（3）H-TdR 掺入法分析脾细胞增殖。采用 Griess 法测定一氧化氮（NO），采用酶联免疫吸附试验（ELISA）检测培养上清液中的细胞因子。采用氰基溴化法制备荧光胺标记的 PPS（Flu-PPS）和葡聚糖（Flu-dextran）。采用 FACS 和共聚焦显微镜分析 Flu-PPS 的细胞结合活性。采用 ELISA 法测定 NF-κB 活性。
我们发现 PPS 能够强烈上调巨噬细胞的功能，如一氧化氮（NO）的产生和细胞因子的表达。与 C3H/HeJ 组相比，PPS 显著刺激了 C3H/HeN 小鼠腹腔巨噬细胞的脾细胞增殖和 TNF-α、IL-1β和 NO 的产生。TLR-4 功能阻断抗体，但不是 TLR-2 和 CR3 阻断抗体，显著抑制了 PPS 介导的 TNF-α 和 IL-1β 的产生。流式细胞术和共聚焦激光扫描显微镜分析表明，荧光标记的 PPS（f-PPS）可以特异性地与靶细胞结合，这种结合可以被未标记的 PPS 和抗 TLR-4 抗体阻断，但不能被抗 TLR-2 和 CR3 单克隆抗体阻断。PPS 显著诱导 NF-κB 的核转位和 DNA 结合活性。
因此，我们的数据表明，PPS 可能通过 TLR-4 激活信号通路发挥其免疫刺激作用。
