Abstract
Ganoderma sinense is a Chinese unique medicinal fungus that has been used in folk medicine for thousands of years. Polysaccharides are considered to be biologically active ingredients due to their immune-modulating functions. Previously we found that GSP-2, a new polysaccharide isolated from Ganoderma sinense, exerts an immunomodulatory effect in human peripheral blood mononuclear cells but the underlying mechanism is unclear. The present study aimed to investigate how GSP-2 triggers immunologic responses and the implicated signaling pathways. GSP-2 effects were investigated both in a macrophagic cell line, RAW264.7, and in primary macrophages. Moreover, the molecular basis of GSP-2 recognition by immune cells, and the consequent activation of signaling cascades, were explored by employing recombinant human HEK293-TLR-Blue clones, individually overexpressing various Toll-like receptors. GSP-2 dose-dependently induced the overexpression of Toll-like receptor 4 (TLR4) but did not affect the expression of other TLRs. Moreover, GSP-2 induced TNFα secretion in primary macrophages from wild-type, but not TLR4-knockout mice. In addition, GSP-2 upregulated TLR4 protein expression and activated the MAPK pathway in RAW246.7 macrophages. Finally, GSP-2 induced the production of the cytokines TNFα, IL1β, and IL6. Our data demonstrated that GSP-2 was specifically recognized by TLR4, promoting cytokine secretion and immune modulation in macrophages.
Highlights
Ganoderma sinense (GS) is a Chinese unique medicinal fungus that has a long history of use in traditional Chinese medicine [1]
RAW264.7 cells (4×105 cells/ml) were treated with GSP-2 or LPS in polymyxin B (PMB, 10 μg/ml) for 18 h in Dulbecco’s Modified Eagle’s medium (DMEM), and the nitrite levels were determined in the supernatant
The results showed that GSP-2 induced nitric oxide production in a dose-dependent manner in RAW264.7 cells and that LPS can be blocked by PMB (Fig 1B), indicating that the effect of LPS can be excluded in the following studies
Summary
Ganoderma sinense (GS) is a Chinese unique medicinal fungus that has a long history of use in traditional Chinese medicine [1]. GSP-2 is a novel toll-like receptor 4 agonist decision to publish, or preparation of the manuscript
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