Materials and Methods
Chemicals and reagents
Dios (Fig. S1) was from the College of Pharmaceutical Sciences,
Southwest University, P. R. China. The standard diet (SD, HD005) and
high-fat diet (HFD, HD001+2% cholesterol, containing 45% fat and 2%
cholesterol) were purchased from BiotechHD Co.Ltd. (Beijing, China). The
aspartate aminotransferase (AST), alanine aminotransferase (ALT), total
cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol
(LDL-C) and high-density lipoprotein cholesterol (HDL-C) commercial kits
were obtained from Nanjing Jiancheng Bioengineering Institute (Jiangsu,
China) and the triglyceride (TG) commercial kit was purchased from
Applygen Technologies Inc. (Beijing, China). The Oil Red O and
Nile Red staining kits were obtained slarbio (Beijing, China).
Dulbecco’s modified Eagle medium (DMEM) was purchased from KeyGen
Biotech (Jiangsu, China), and fetal bovine serum (FBS) was obtained from
Tianhang (Zhejiang, China). Palmitic acid (PA) was purchased from
Sigma-Aldrich (St. Louis, MO, USA). Fludarabine (HY-B0069, STAT1
inhibitor) was purchased from MedChemExpress (NJ, USA). siRNA for CXCL10
(human) and the control siRNA (human) were purchased from Tsingke
Biological Technology (Beijing, China). The STAT1 overexpression plasmid
STAT1-pcDNA3.1 and the control plasmid pcDNA3.1 were obtained from
Pulateze (Hunan, China). Lipo6000™ transfection reagent was obtained
from
Beyotime
Biotechnology (Shanghai, China). Antibodies against STAT1 (1:5000),
CXCL10 (1:500), sterol regulatory element-binding proteins-1c (SREBP-1c)
(1:1000), carbohydrate response element-binding protein (CHREBP)
(1:1000), liver X receptor-alpha (LXRα) (1:1000), liver X receptor-beta
(LXRβ) (1:1000), nuclear factor-κB p65 (p65) (1:1000),
phosphorylated-p65 (p-p65) (1:1000), tumor necrosis factor-α (TNF-α)
(1:1000), interleukin-6 (IL-6) (1:1000), β-actin (1:2000) and Lamin B
(1:2500) were obtained from Proteintech Group, Inc. (Hubei, China).
Antibody against STAT1Y701 (1:1000) was purchased from
Affinity Biosciences (Jiangsu, China). Antibody against
STAT1S727 (1:1000) was obtained from Beyotime
Biotechnology (Shanghai, China).
Animals experiments
Animal studies are reported in compliance with the ARRIVE guidelines
(Kilkenny, Browne, Cuthill, Emerson & Altman, 2010) and with the
recommendations made by the British Journal of Pharmacology (Curtis et
al., 2018). All animal experiments were performed in accordance with the
National Institutes of Health Guide for the Care and Use of Laboratory
Animals. The animal studies were performed after receiving approval of
the Institutional Animal Care and Use Committee (IACUC) in Southwest
University, P.R. China (IACUC approval No. yxy202007). All the animals
were housed under specific pathogen-free (SPF) conditions at a
controlled temperature (22-25 °C) and humidity (50 ± 5%), and
alternating 12-hour light / dark cycles. The animals used in this study
were housed in individually ventilated cages with natural soft sawdust
as bedding up to five per cage and maintained on a normal chow diet
with clean water ad libitum.
Eight-week-old male C57BL/6J mice (18-22g) were obtained from Beijing
Huafukang Biotechnology Co., Ltd. (SCXK-Jing-2019-0008, Beijing, China).
Mice were fed with either SD or HFD for 14 weeks to induce NASH model.
Thereafter, the HFD-fed NASH mice were randomly divided into HFD group
and HFD + Dios group (n = 5 per group) and administered intragastrically
either
Dios of 60 mg·kg-1·day or saline for four weeks (Yang
et al., 2017). Mice were euthanized by cervical dislocation under
anesthesia, and the livers were removed by two independent individuals
who were blinded to the treatments and either snap-frozen or fixed in
buffered 4% formalin for RNA-Seq and followed experiments.
To explore the potential mechanism of STAT1 in the beneficial effect of
Dios in NASH, another animal experiment was carried out ( Eight-week-old
Male C57BL/6J, SCXK-Jing-2019-0010) that mice were injected with
fludarabine (Flu, a STAT1 inhibitor, 0.8 mg·kg-1·day)
by the abdominal cavity two cycles for 5 days every 2 weeks in the whole
experiment, and mice were fed with either SD or HFD for 14 weeks to
induce NASH. Thereafter, the HFD-fed mice were randomly divided into
different groups (n = 5 per group) including HFD, HFD + Dios, Flu-HFD,
and Flu-HFD + Dios, and those mice were either administered
intragastrically Dios (60 mg·kg-1·day) or saline for
four weeks for four weeks. The diet intake of the mouse was monitored
per day and the body weight was recorded weekly. After the last
administration, all the mice euthanized by cervical dislocation under
anesthesia, and the livers, kidneys, spleens, and abdominal fat pads
were removed and weighed by two independent individuals who were blinded
to the treatments. Serum was prepared by solidification and
centrifugation (4 °C, 12000 × g, 10 min) and stored at -80 °C until the
analysis of biochemical parameters. Liver samples were either
snap-frozen or fixed in buffered 4% formalin for histological staining,
hepatic triglyceride content measurement, quantitative real-time PCR,
and western blot assay.
Cell culture and treatment
HepG2 cells were obtained from the Cell Bank at the Chinese Academy of
Sciences (Shanghai, China) and cultured in DMEM supplemented with 10%
FBS and 1% penicillin/streptomycin in a 5 % CO2humidified incubator at 37 °C. HepG2 cells were treated with different
doses (0.10, 0.15, 0.20, 0.25, 0.30, 0.35 and 0.40 mM) of PA for 24 h to
induce NASH cell model . To detect the effect of Dios, HepG2 cells were
treated with or without Dios under the indicated concentrations combined
with PA treatment for 24 h. To study the importance of STAT1 in
Dios-mediated hepatoprotective effect, the STAT1 inhibitor fludarabine
(10 μM) was used to pretreat HepG2 cells for 1 h before Dios and PA.
Transfection of overexpression plasmid and
siRNA
HepG2 cells (3× 105 cells per well) were seeded in
6-well plates and incubated until the cells reached 70-80% confluence.
Cells were then transfected with the STAT1 reporter vector
STAT1-pcDNA3.1 (Fig. S5) , the control vector pcDNA3.1 and
siCXCL10 (5’-GGUCUUUAGAAAAACUUGATT-3’, 3’-UCAAGUUUUUUCUAA-AGACCTT-5’)
mixed in Lipo6000™ reagent according to the manufacturer’s guidelines.
Then, the medium was exchanged with fresh complete medium after 6 h.
After 24 h transfection, cells were incubated with 0.2 mM PA in the
presence or absence of Dios (80 μM) for 24 h. After treatment, cells
were harvested for further experiments.
RNA Sequencing (RNA-Seq)
Total RNA isolated from the livers of C57BL/6J mice was used to
construct high throughput sequencing libraries using NEBNext® UltraTM
RNA Library Prep Kit. High throughput RNA-sequencing was performed using
a HiSeq 4000 instrument (Illumina) at Novogene (Beijing, China). Adaptor
sequences and low-quality reads were initially filtered from the raw
data. Then the remaining ones, called clean reads, were aligned to the
reference genome of a mouse, using the HISAT2 v2.0.5 program.
Subsequently, unigene expression was calculated as the FPKM (fragments
per kilobases of exons for per million mapped reads) with featureCounts
v1.5.0-p3. Differential expression genes (DEGs) of two groups were
performed using the DESeq2 R package, and when the P-value was less than
0.05 and the log2 ratio was greater than 1 (two-fold
change), the unigenes were considered to be differentially expressed.
GO and pathway enrichment analyses of
DEGs
The enrichment analysis of GO (Gene Ontology) and KEGG (Kyoto
encyclopedia of genes and genomes) pathway was performed for DEGs using
David which is an online biological information database for annotation,
visualization, and integrated discovery (Huang, Sherman & Lempicki,
2009). GO consists of biological processes (BP), cellular components
(CC), and molecular functions (MF). P-value < 0.05 was chosen
as the cut-off point for GO and KEGG analyses.
PPI network, module analysis, and hub genes
identification
Protein-protein interaction (PPI) analysis of differentially expressed
genes was based on the STRING database. The significant module in the
PPI network was identified by molecular complex detection (MCODE). The
parameters of DEGs clustering and scoring were set as follows: MCODE
score ≥ 4, degree cut-off = 2, node score cut-off = 0.2, max depth =
100, and k-score = 2. Hub genes were identified using Cytohubba (a
plug-in of Cytoscape software) filtered with the criterion of degrees
> 10 criteria (each node had more than 10 interactions).
Biochemical assessment of serum and
liver
Serum triglyceride (TG), total cholesterol (TC), low-density lipoprotein
cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C),
alanine transaminase (ALT) and aspartate aminotransferase (AST) were
quantified using enzymatic assays. The content of TC and TG of liver
tissues and HepG2 cells were determined with an enzymatic assay kit and
the protein concentrations were determined with a BCA protein assay kit.
The results were expressed as mmol per g protein
(mmol·g-1). All procedures were carried out according
to the manufacturer’s instructions.
Histological assessment
Histologic examination of the liver was performed by hematoxylin-eosin
staining. Liver samples were fixed briefly in 4% paraformaldehyde and
embedded with paraffin. The samples were cut into 5 μm sections,
deparaffinized, rehydrated, then stained with hematoxylin-eosin or mason
trichrome. Images of liver sections were captured with light microscope.
The amounts of steatosis, activity, and fibrosis were scored using the
Fatty Liver Inhibition of Progression (FLIP) Algorithm (Bedossa &
Consortium, 2014). The SAF score was defined as the total of scores for
steatosis (S0-S3), activity including lobular inflammation (0-2) and
hepatocellular ballooning (0-2) scores, and the fibrosis (F0-F4) score.
Immunofluorescence
analysis
Liver samples embedded with paraffin were dewaxed, blocked, and then
incubated with phosphorylated STAT1Tyr701 and
phosphorylated STAT1Ser727 antibodies overnight at 4
°C. Slides were washed three times with PBS and then incubated with
secondary antibody labeled fluorescence for 30 min at 37 °C. The nuclei
were counterstained with 4′, 6-diamidino-2-phenylindole (DAPI) for 5 min
at room temperature. Images were captured using fluorescent microscopy.
Lipid content analysis
Hepatic lipid accumulation was detected by Oil Red O staining. Liver
sample cryosections and hepatocytes were prepared for staining with
pre-warmed Oil Red O working solution for 20 min, being rinsed with 60%
isopropanol 3 times. And then counterstained with hematoxylin for 3 min,
gently washed with 60% isopropanol. The images were captured using
light microscopy. HepG2 cells were seeded in a 24-well plate and treated
with 0.2 mM PA and indicated concentrations of Dios for 24 h. The cells
were washed twice with PBS and fixed with 4% formaldehyde for 20 min
and then stained with 1 μg·mL-1 Nile red for 30 min at
room temperature. Lipid-bound Nile red fluorescence was detected using
fluorescence microscopy.
Quantitative real-time polymerase chain
reaction(qRT-PCR)
To verify the quantification of gene expression levels, qRT-PCR was
performed as previously described (Zhou et al., 2018). Total RNA was
extracted from livers and HepG2 cells using Trizol reagent (Sangon
Biotech Co., Ltd., Shanghai, China), and RNA was reverse-transcribed
using the Fastingking RT kit (TiangenBiotech CO. LTD., Beijing, China).
Quantitative real-time PCR was carried out with SYBR Premix Plus
(TiangenBiotech CO. LTD., Beijing, China) according to the
manufacturer’s instructions, and the gene primers listed in Tab.
1 . Relative mRNA expression was determined by a comparative method
(2-ΔΔCt) using GAPDH as a reference gene.
Western blotting analysis
The antibody-based procedures used in this study comply with the
recommendations made by the British Journal of Pharmacology (Alexander
et al., 2018). The proteins were extracted from mouse liver and HepG2
cells, then equal amounts of protein extract were denatured. After
separated by electrophoresis in 12% SDS–PAGE, the protein samples were
transferred onto Polyvinylidene Fluoride (PVDF) membranes. The membranes
were blocked with 5% skimmed milk for 2 h, subsequently incubated with
primary antibodies against STAT1 (1:5000), p-STAT1Y701(1:1000), p-STAT1S727 (1:1000), CXCL10 (1:500), p65
(1:1000), p-p65 (1:1000), IL-6 (1:1000), TNFα (1:1000), LXRα (1:1000),
LXRβ (1:1000), CHREBP (1:1000), SREBP-1c (1:1000), β-actin (1:2000) and
Lamin B (1:2500) overnight at 4 °C. After three times washing, the
membranes were incubated with an HRP-conjugated secondary antibody at
room temperature for 2 h. Antibody binding was detected by enhanced
chemiluminescence detection kit (Affinity Biosciences, Jiangsu, China)
and the digital images were analyzed by Image J. The relative protein
levels were normalized to β-actin or Lamin B.
Data and statistical
analysis
The data and statistical analysis comply with the recommendations on
experimental design and analysis in British Journal of Pharmacology
(Curtis et al., 2018). Randomization was used to assign samples to the
experimental groups and treatment conditions for all in vivo studies.
Data collection and acquisition of all in vivo and in vitro experimental
paradigms were performed in a blinded manner. All results are expressed
as the mean ± SEM (n = 5). Difference among groups was analyzed with
one-way analysis of variance (ANOVA) followed by a Bonferroni post hoc
analysis using GraphPad Prism 5 software. Post hoc tests were conducted
only if F was significant, and there was no variance inhomogeneity. A
value of p < 0.05 was considered statistically significant.