Oral bioavailability of polysaccharides: an exploration of TLRs/Dectin-mediated phagocytosis to Ganoderma glucan GSP-2 by intestinal macrophages in vitro and in vivo

Are polysaccharides orally “absorbable”? This project aims to give a definite answer through an exploration of TLRs/Dectin-mediated phagocytosis to Ganoderma glucan GSP-2 by intestinal macrophages in vitro and in vivo.

Very few reports have demonstrated the pharmacokinetics of orally administered polysaccharides. It is commonly accepted that oral bioavailability of polysaccharides is poor, although it has been reported that fluorescence-labeled beta-glucans can translocate into blood after gavage. The question arises as to whether the labeling reagent itself played the crucial role in the absorption procedure, and the underlying mechanism remains unclear.

In order to clarify the effect of labeling reagents, we have developed a new analytical method, namely high-performance gel-permeation chromatography coupled with charged aerosol detection (HPGPC-CAD). The chromatography can check if the absorbed polysaccharide remains intact, while CAD is very sensitive, comparable to fluorescent detector (FD), yet is free of fluorescence-labeling.

It has been reported that gut-associated lymphoid tissue (GALT) cells, isolated from Peyer’s patches of mice gavaged with fluorescently labeled glucans, can recognize and bind these glucans, and expression of receptor Dectin-1 on the cell surface of macrophages in these GALT cells significantly increased. In our preliminary studies, gavage of crude polysaccharides of Ganoderma sinense (GSP) significantly suppressed the growth of carcinoma 4T1 in mice. Further studies on a beta-glucan GSP-2 isolated from GSP revealed that RAW 264.7 cells exhibited potent efficiency of phagocytosis to GSP-2, while GSP-2 in turn significantly induced the production of NO, TNF-α, and IL-6 in RAW cells and enhanced their suppression of 4T1 cells in vitro. Preliminary screening of receptors of RAW cells indicated that GSP-2 might interact with multiple receptors (TLR-1, 7 and Dectin-1). Based on these results, we hypothesize that polysaccharides can be absorbed through phagocytosis by intestinal phagocytes via TLRs/Dectins.

This project aims: 1) to establish HPGPC-CAD/FD-based pharmacokinetic research platform, and to clarify the effect of labeling reagents by comparing both the oral bioavailability and RAW cell’s phagocytosis amount/rate of labeled/unlabeled GSP-2; 2) to determine the role of TLRs/Dectins in macrophage’s phagocytosis by checking changes in the phagocytosis amount/rate caused by specific antibodies and small interfering RNA (siRNA); 3) to confirm the results using specific gene knock-off mice and cells.

If successful, this project will provide solid evidence for the oral availability of polysaccharides and it will elucidate the underlying mechanism. This new knowledge will help to break the current bottleneck in research on polysaccharides and will greatly push forward the R&D of orally used polysaccharides, especially those from sugar-rich Chinese medicines.