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.