Study On The Immunomodulatory Activity Of The Triple Helical Polysaccharide From Dictyophora Indusiata

Dictyophora indusiata(D. Indusiata) is well known as “queen of the mushrooms” in China, due to its distinctive flavor, high nutritive value and attractive appearance. Recent studies have revealed that a triple helical polysaccharide from D. indusiata(DIP) have many biological activities, including antitumor activity, anti-inflammatory activity, antioxidant activity as well as immunomodulatory activity. This study focused on the immunomodulatory activity of DIP and aimed to explore its mechanism in vitro.The main results of this work are listed as follows:(1) The effects of DIP on macrophages, including cell morphology, proliferation, phagocytosis activity, NO and cytokine levels, were studied by fluorescence microscopy, MTT assay, neutral red test, flow cytometry, Griess method as well as ELISA, respectively. The results demonstrated that DIP could significantly activate macrophages, promote cell proliferation and increase its phagocytic activity. Further study indicated that the secretion of NO and cytokine in macrophages was increased. RT-PCR experiment revealed the expression levels of iNOS and cytokine mRNAs were elevated.(2) The interaction between DIP and macrophages was studied by laser-scanning microscope, flow cytometry and ELISA. And then, the effects of DIP on MAPK and NF-κB expression were studied by western blotting. The results showed that 2-AMAC-DIP could specifically bind to macrophage cells, and this binding was significantly inhibited by unlabled DIP, anti-TLR4 or anti-Dectin-1, indicating TLR4 and Dectin-1 are potential receptors for DIP. Furthe study revealed that anti-TLR4 or anti-Dectin-1 could inhibit the secretion of NO and cytokine from macrophages induced by DIP. In addition, DIP could induce higher expression of TLR4. We found that DIP could significantly elevate the expression levels of phospho-MAPK and phospho-NF-κB p65, suggesting that they were activated by DIP. Immunofluorescence assay found that activated NF-κB was translocated to cell nuclear.Taken together, DIP showed great immune enhancing acticity by interact with macrophages through either TLR4 or Dectin-1 on the cell surface. This interaction induces the phosphyration of MAPK and NF-κB, and triggers these signaling pathways, then elevating the secretion of NO and cytokine. Our results indicated that DIP has great potential to be explored as immune enhancing agent.