Figure 3.

Shiitake mushroom-derived exosome-like nanoparticles (S-ELNs) prevented inflammasome formation and inhibited both protein and mRNA levels of the Il1b gene. (A) Representative pictures of immunofluorescence staining in macrophages. Magnification: 60×. Cells were treated with S-ELNs (3 × 10¹⁰/mL) for 16 h. Afterward, Caspase 1 (Casp1) inhibitor VX765 (10 µM) was added to the cells for 30 min, followed by lipopolysaccharide (LPS) and free fatty acid (FFA) treatment. Macrophages were subjected to immunofluorescence staining using a primary antibody (1st Ab) and secondary antibody (2nd Ab). The nucleotide-binding domain and leucine-rich repeat related (NLR) family, pyrin domain containing 3 (NLRP3) inflammasome was visualized as red specks (indicated by yellow arrows). 4′,6-Diamidino-2-phenylindole (DAPI) was used to stain nuclei. (B) The percentage of speck positive cells was decreased by the pre-treatment of S-ELNs. ** p < 0.01 relative to cells treated with LPS+FFA (black bar). (C) Protein levels of NLPR3, apoptotic speck protein containing a caspase recruitment domain (ASC), Casp1, and never in mitosis gene a (NIMA)-related expressed kinase 7 (Nek7) were not significantly altered by S-ELN treatment. (D) S-ELNs decreased the expression of the Il1b gene. * p < 0.05, ** p < 0.01 relative to cells primed with LPS alone (black bar). un: un-treated naïve macrophages.