Aim of the study: 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).
Materials and methods: 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.
Results: 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.
Conclusions: Therefore, our data suggest that PPS may exert its immunostimulating potency via TLR-4 activation of signaling pathway.
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