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FIP-SJ75, a novel fungal immunomodulatory protein gene, was shuffled from the genes of three different mushroom species: Ganoderma lucidum, Flammulina velutipes, and Volvariella volvacea. Based on the expression of FIP-SJ75 gene in Escherichia coli, recombinant FIP-SJ75 (rFIP-SJ75) was routinely confirmed, consisting of 115 amino acids, and six peptides were identified by LC/Q-TOF MS with a coverage rate of 84.3%. Bioactivity assay in vitro indicated that rFIP-SJ75 promoted the proliferation of RAW264.7 cells at a range of 1-8 μg/mL, and significantly activated RAW264.7 cells in a dose-dependent manner. Real-time PCR revealed rFIP-SJ75 obviously promoted pro-inflammatory genes (TNF-α and IL-6), but reduced the expression of anti-inflammatory genes (IL-10 and TGF-β1) at the transcriptional level. These data implied that rFIP-SJ75 possessed immunomodulatory activity in macrophages by promoting macrophage M1 polarization and initiating pro-inflammatory responses, which could lay the foundation for rFIP-SJ75 to become a stable immunomodulator resource for further research and potential applications.

Ganoderma lucidum, a traditional edible and medicinal fungus, holds an important status in health care systems in China and other Asian countries. Fungal immunomodulatory protein (FIP), one of the active ingredients isolated from G. lucidum, is a class of naturally occurring proteins and possesses potential biological functions. This study was conducted to explore the molecular mechanism of its immunomodulatory potency in immune responses of macrophages. In vitro assays of biological activity indicated that rFIP-glu significantly activated macrophage RAW264.7 cells, and possessed the ability of pro- and anti-inflammation the cells. RNA sequencing analysis showed that macrophage activation involved Toll-like receptors and mitogen-activated protein kinases pathways. Furthermore, qRT-PCR indicated that phosphoinositide 3 kinase inhibitor LY294002 blocked the mRNA levels of MCP-1, MEK1/2 inhibitor U0126 reduced the mRNA levels of TNF-α and MCP-1, and JNK inhibitor SP600125 prevented the up-regulation of iNOS mRNA in the rFIP-glu-induced cells. FIP-glu mediated these inflammatory effects not through a general pathway, instead through a different pathway for different inflammatory mediator. These data indicate the possibility that rFIP-glu has an important immune-regulation function and thus has potential therapeutic uses.