Cell experiment:

RBL-2H3 cells are seeded into 24-well plates containing gelatin-coated cover glasses at 0.75 × 105 cells/well and sensitized with anti-DNP IgE. After culture for 24 hours, the cells cultured on cover glasses are pretreated with or without 50 μM Ergosterol or 1 mM methyl beta cyclodextrin (MβCD) in PIPES buffer for 20 minutes. The cells are then challenged with DNP-HSA (50 ng/mL) for 20 minutes. After washing with ice-cold PBS immediately, the cells are fixed with 3.7% formaldehyde in PBS for 20 minutes and blocked with 1% BSA in PBS. The IgE/α-chain of FcεRI complexes on cell surfaces are detected using goat anti-mouse IgE antiserum, and Alexa Fluor 488-conjugated anti-goat IgG. Fluorescence images are acquired using a laser scanning confocal microscope with Zen software. The data are quantified by counting the aggregation number of FcεRI positive cells and presented as aggregation positive cells/total cells. The cells are counted in six independent micrographs for each sample[1].

Animal experiment:

Rats[2]Experimental myocardial injury in rats is performed by LPS injection (15 mg/kg). Dexmedetomidine (Dex) is used as a positive control. The experimental animals are randomly divided into five groups (n = 10) as follows: Control group, rats receive 2% gum acacia suspension orally at a dose of 2 mL/kg for 5 days, followed by normal saline injected intraperitoneally on day 5; LPS group, rats receive 2% gum acacia suspension at dose of 2 mL/kg for 5 days with LPS simultaneously injected intraperitoneally day 5; LPS+ Dex group, rats are treated with 2 mg/kg Dex suspension followed by LPS injection on day 5; LPS + Ergosterol (25 mg/kg, 50 mg/kg) groups, 25 or 50 mg/kg Ergosterol are given to rats orally for 5 consecutive days, and LPS is injected on day 5. Twelve hours after LPS treatment, blood samples are collected through the retro-orbital plexus. The serum specimens are centrifugated at 4, 000 × g for 15 min and stored at –80℃ until needed. Thereafter, rats are anesthetized and sacrificed. Heart tissues are removed and homogenized in ice-cold phosphate buffered saline (50 mM, pH 7.4). Heart tissue homogenates from different groups are centrifuged at 12, 000 × g for 45 min at 4℃ and the supernatants retained for further biochemical evaluations[2].

Ergosterol is the primary sterol found in fungi, with antioxidative, anti-proliferative, and anti-inflammatory effects.

Ergosterol is a sterol isolated from Grifola frondosa, which can be used in the research of mast cell-dependent allergic diseases. Ergosterol (10, 20, 50 μM) inhibits the antigen-induced release of β-hexosaminidase and histamine in antigen-stimulated RBL-2H3 cells. Ergosterol (20 and 50 μM) significantly reduces the mRNA levels of of IL-4 and TNF-α. Ergosterol (50 μM) inhibits the antigen-induced aggregation of FcεRI[1].

Ergosterol (25, 50 mg/kg, p.o.) significantly mitigates the reduced cardiac performance in rats induced by LPS, increases SOD activity and decreases the formation of MDA, CK-MB, and LDH in LPS-induced sepsis rats[2].

References:
[1]. Kawai J, et al. Ergosterol and its derivatives from Grifola frondosa inhibit antigen-induced degranulation of RBL-2H3 cells by suppressing the aggregation of high affinity IgE receptors. Biosci Biotechnol Biochem. 2018 Jul 2:1-9.
[2]. Xu J, et al. Ergosterol Attenuates LPS-Induced Myocardial Injury by Modulating Oxidative Stress and Apoptosis in Rats. Cell Physiol Biochem. 2018;48(2):583-592.