Materials and Methods
Experimental animals. Specific pathogen-free C57BL/6J (6-8 weeks, 18-23g) female mice were purchased from Charles River Laboratories (Beijing, China) and maintained in controlled conditions (22℃, 50% humidity, 12 h light/dark cycle, with lights on at 7:00 AM). The mouse care and experimental protocols conducted in this study were approved by the Huazhong University of Science and Technology Animal Care and Use Committee.
Isolation and identification of secoemestrin C. The fungusAspergillus nidulans was growing on rice culture medium (40kg) at room temperature for 21 days. The fermented medium was then extracted with 95% ethanol, and the extract was suspended in water and further treated with ethyl acetate (AcOEt) to obtain a dark brown extract (300g). The AcOEt extract was chromatographed using silica gel (100-200 mesh) and eluted with petroleum ether/AcOEt (50:1-0:100) to give six fractions (Fr1-Fr6). Secoemestrin C (5.2g) was isolated from Fraction 4, and the purity (≥ 99.0%) was determined via HPLC with a UV detector. The structure of secoemestrin C was elucidated based on NMR and HR-ESI-MS (Supplemental Table 1 and Supplemental Fig. 1) and found to be identical to that reported previously (Ooike et al. , 1997).
Reagents and antibodies. Carboxy-fluorescein diacetate succinimidyl ester (CFSE) was purchased from Invitrogen (ThermoFisher Scientific). Concanavalin A type IV from Canavalia ensiformis(C2010) was obtained from Sigma-Aldrich. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH) were measured with an automated biochemical analyzer BS-200 (Mindray, China). GolgiStop brefeldin A solution and Fix/Perm Buffer Set were obtained from Biolegend. Flow cytometry antibodies: antibodies against mouse TCRβ (H57-597), NK1.1 (PK136), CD4 (GK1.5), CD8 (53-6.7), CD69 (H1.2F3), and IFN-γ (XMG1.2) were purchased from BD Biosciences and Biolegend, with fluorescence conjugation. Cytokines in serum or in culture supernatants were determined by using a cytometric bead array-based mouse Th1/Th2/Th17 Cytokine Kit (BD Biosciences).
Isolation of hepatic mononuclear cells and splenocytes. Hepatic mononuclear cells (MNCs) and splenocytes were isolated as previously described (Zhou et al. , 2017). In brief, each mouse liver was perfused with phosphate buffer saline to eliminate blood, and the liver tissue was removed and pressed through a 70-μm cell strainer (BD Biosciences). The liver cell suspension was then collected. Hepatic MNCs were purified by density gradient centrifugation in a 38% Percoll solution (GE Healthcare). Purified splenocytes were prepared by homogenizing with a syringe, followed by passage through a 0.1-mm sterile nylon mesh and erythrocyte depletion.
Hepatitis induction and treatment. C67BL/6J mice in the Con A-challenged and secoemestrin C-treated groups were injected intravenously with a single dose of Con A (12 mg·kg-1) to simulate autoimmune hepatitis. In the case of the secoemestrin C-treated group, secoemestrin C (1 mg·kg-1 or 10 mg·kg-1) was intraperitoneally injected for 3 days, and then the mice were injected with Con A 1 h after the last secoemestrin C injection.
Blood samples were collected from the orbital venous plexus under mild pentobarbital anesthesia 8 h after the Con A injection, and the serum was separated by centrifugation. Later, mice were sacrificed by cervical dislocation under mild pentobarbital anesthesia, and livers were harvested.
Cytokine assay. The levels of IL-2, IL-4, IL-6, IL-10, IL-17, IFN-γ, and TNF-α in the serum 8 h after Con A challenge or in culture supernatants 72 h after Con A stimulation were measured with a CBA kit for mouse Th1/Th2/Th17 cytokines.
Flow cytometry. Procedures for surface staining, intracellular cytokine staining, and CBA kit use were performed according to the manufacturer’s instructions. Flow cytometry data were acquired on a BD FACSCelesta and analyzed with FlowJo software (TreeStar).
Histological examination. Liver samples were fixed in 4% formaldehyde and dehydrated with xylene, absolute ethyl alcohol, and 75% alcohol, then embedded in paraffin wax. Four micron-thick sections were cut and stained with hematoxylin-eosin (H&E) and observed by three pathologists in a blinded manner. The extent of the pathology was scored from 0 (no pathology) to 3 (severe pathology) as previously described (Yan et al. , 2011).
Immunohistochemistry and immunofluorescent staining. Liver sections were stained with primary antibody (anti-Fas or anti-cleaved caspase-3) according to a standard protocol (Servicebio, China). Apoptotic cells were evaluated using a TUNEL assay kit (Roche Applied Science) according to the manufacturer’s instructions.
Statistical analysis. Graphs were generated and analyzed by GraphPad Prism 6.0 (GraphPad Software). Data were analyzed by Student’st -test for comparisons of groups with normal distribution and by equal variance or Mann-Whitney U test for non-normally distributed variables among groups. Data are presented as means ± standard error of the mean (SEM). P<0.05 was considered significant.