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\begin{document}

\title{Diosmetin ameliorates~ nonalcoholic steatohepatitis through modulating
lipogenesis and inflammatory response~ in STAT1/CXCL10-dependent
manner~~~~~~~~~~~~}



\author[1]{Nanxuan Luo}%
\author[1]{Changqing Yang}%
\author[1]{Yurong Zhu}%
\author[1]{Baoshun Zhang}%
\affil[1]{Southwest University}%


\vspace{-1em}



  
  \date{May 14, 2024}


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\selectlanguage{english}
\begin{abstract}
{}{Nonalcoholic steatohepatitis (NASH) is an inflammatory lipotoxic
disorder, characterized by lipid accumulation and inflammation. This
study investigated the effects of the flavonoid diosmetin (Dios) on
lipogenesis and inflammatory response and the underlying molecular
mechanisms in NASH. Here, high-fat diet (HFD) and palmitic acid (PA)
were used to induced NASH model in mice and HepG2 cells, respectively.
After Dios supplementation, the functional parameters, morphological
changes, lipogenesis and inflammatory response in HepG2 cells and NASH
mice were reduced. The liver RNA-Seq analysis revealed the key targets
CXCL10 and STAT1 interfered by Dios.}{}{Interestingly, Dios prevented
CXCL10- and STAT1-mediated lipogenesis and inflammatory response through
siCXCL10, STAT1 inhibitor and STAT1 overexpression in HepG2 cells. In
conclusion, Dios alleviated NASH through suppression of lipogenesis and
inflammatory response via STAT1/CXCL10-dependent pathway. ~}{}%
\end{abstract}%



\sloppy


\par\null

\section*{Keywords}\label{keywords}

Nonalcoholic steatohepatitis (NASH) ; Diosmetin (Dios); Lipogenesis;
Inflammation; Macrophage chemotactic ligand 10 (CXCL10); Signal
transducers and activators of transcription 1 (STAT1).

~

~

\section*{Introduction}\label{introduction}

Nonalcoholic fatty liver disease (NAFLD) has become one of the most
important causes of liver disease with an estimated global prevalence of
24\%,\textsuperscript{1} encompassing a wide histological spectrum rang
from steatosis to nonalcoholic steatohepatitis (NASH), advanced
fibrosis, and even hepatocellular carcinoma.\textsuperscript{2, 3}
{}{Given the worldwide epidemic, NASH has caused an enormous clinical
and economic burden.\textsuperscript{4}} Some investigators predict that
NASH may become the leading factor of liver transplantation in the next
about 10 years.\textsuperscript{5} For NASH prevention, until now, there
is no approved therapy aside from addressing
lifestyle.\textsuperscript{6} The occurrence of NASH is characterized by
hepatic steatosis {}{and inflammation} injury.\textsuperscript{7, 8}
Excessive amounts of lipid accumulation cause toxicity to
cells.\textsuperscript{9} Hepatocytes with {}{lipotoxicity} are likely
to be more vulnerable to give rise to hepatic inflammatory
response.\textsuperscript{10} {The} manipulation of lipogenesis and
inflammatory response is a promising strategy to prevent NASH.
Therefore, it will dramatically be crucial to identify new active
compounds for treating NASH.{}

Diosmetin (3', 5, 7-trihydroxy-4'-methoxy flavone, Dios), a natural
flavonoid isolated from citrus fruits,\textsuperscript{11} has a variety
of favorable pharmacological activity in the treatment of diabetes,
cancer and oxidative-damage diseases.\textsuperscript{12-15} The
benefits of citrus fruit have been known to improve blood lipid
profiles.\textsuperscript{16} Recent studies demonstrate that Dios
inhibits the activation of NLRP3 inflammasome in LPS-induced acute lung
injury.\textsuperscript{17} What's more, our previous study reveals that
Dios exhibits a supplementary effect on promoting fatty acid oxidation
in diabetes-mice liver.\textsuperscript{18} Many compounds in foods of
plant origin, particularly dietary flavonoids, exerts the protective
effects through regulating lipid metabolism and inflammation against
NASH.\textsuperscript{19-21} According to these results, we reasonably
hypothesized that Dios exerted a hepatoprotective effect against NASH.

In the present study, we utilized the established models that HepG2
cells and C57BL/6J mice respectively induced by palmitic acid (PA) and
high-fat diet (HFD) to explore the protective effects and the underlying
molecular mechanism of Dios against NASH.

\section*{Materials and Methods}\label{materials-and-methods}

\subsection*{Chemicals and reagents}\label{chemicals-and-reagents}

Diosmetin (Dios, purity [?] 98\%, \textbf{Fig. 1A}) was purchased from
Chengdu Herbpurify CO., LTD (Sichuan, 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\emph{.}
(Beijing, China). The Oil Red O and Nile Red staining kits were obtained
solarbio (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, CXCL10, sterol regulatory element-binding
proteins-1c (SREBP-1c), carbohydrate response element-binding protein
(CHREBP), liver X receptor-alpha (LXR\selectlanguage{greek}α\selectlanguage{english}), liver X receptor-beta (LXR\selectlanguage{greek}β\selectlanguage{english}),
nuclear factor-\selectlanguage{greek}κ\selectlanguage{english}B p65 (p65), phosphorylated-p65 (p-p65), tumor necrosis
factor-\selectlanguage{greek}α \selectlanguage{english}(TNF-\selectlanguage{greek}α\selectlanguage{english}), interleukin-6 (IL-6), \selectlanguage{greek}β-\selectlanguage{english}actin and Lamin B were
obtained from Proteintech Group, Inc. (Hubei, China). Antibody against
STAT1\textsuperscript{Y701} was purchased from Affinity Biosciences
(Jiangsu, China). Antibody against STAT1\textsuperscript{S727} was
obtained from Beyotime Biotechnology (Shanghai, China).

\subsection*{Animals experiments}\label{animals-experiments}

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 \selectlanguage{ngerman}°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 six 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 = 6 per group), and administered
intragastrically either Dios or not Dios of 60
mg·kg\textsuperscript{-1}·day for four weeks \textsuperscript{12}. Mice
were euthanized by a cervical dislocation under anesthesia, and the
livers were removed 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, STAT1 inhibitor, 0.8\selectlanguage{english} mg*kg\textsuperscript{-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 = 6 per group) including HFD, HFD + Dios, Flu-HFD,
and Flu-HFD + Dios, and those mice were either administered or not
administered intragastrically Dios (60 mg*kg\textsuperscript{-1}*day)
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 a cervical dislocation under anesthesia, and the
livers, kidneys, spleens and abdominal fat pads were removed and
weighted. Serum was prepared by solidification and centrifugation (4 degC,
12000 x g, 10 min) and stored at -80 degC 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.

\subsection*{Cell culture and
treatment}\label{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\% CO\textsubscript{2}
humidified incubator at 37 degC. 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 \selectlanguage{greek}μ\selectlanguage{english}M, \textbf{Fig. S1}) was used to pretreat HepG2 cells for 1 h
before Dios and PA.

\subsection*{Transfection of overexpression plasmid and
siRNA}\label{transfection-of-overexpression-plasmid-and-sirna}

HepG2 cells (3\selectlanguage{ngerman}× 10\textsuperscript{5} 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, the control vector pcDNA3.1 and siCXCL10
(5'-GGUCUUUAGAAAAACUUGATT-3', 3'-UCAAGUUUUUUCUAA-AGACCTT-5') mixed in
Lipo6000\selectlanguage{english} 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 \selectlanguage{greek}μ\selectlanguage{english}M) for 24 hours. After treatment, cells were
harvested for further experiments.

\subsection*{RNA Sequencing (RNA-Seq)
assay}\label{rna-sequencing-rna-seq-assay}

Total RNA isolated from liver of C57BL/6J mice were used to construct
high throughput sequencing libraries using NEBNext\selectlanguage{ngerman}® UltraTM RNA Library
Prep Ki. 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
log\textsubscript{2} ratio was greater than 1 (two-fold change), the
unigenes were considered to be differentially expressed. The enrichment
analysis of GO (Gene Ontology) and KEGG (Kyoto encyclopedia of genes and
genomes) pathway was performed for DEGs using the David which is an
online biological information database for annotation, visualization,
and integrated discovery \textsuperscript{22}. Protein-protein
interaction (PPI) analysis of differentially expressed genes was based
on the STRING database. Hub genes were identified using Cytohubba (a
plug-in of Cytoscape software) filtered with the criterion of degrees
\textgreater{} 10 criteria (each node had more than 10 interactions).

\subsection*{Biochemical assessment of serum and
liver}\label{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\textsuperscript{-1}). All procedures were carried out according
to the manufacturer's instructions.

\subsection*{Histological assessment}\label{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 \selectlanguage{greek}μ\selectlanguage{english}m sections,
deparaffinized, rehydrated, then stained with hematoxylin eosin or mason
trichrome. Images of liver sections were captured with light microscope.
The amount of steatosis, activity, and fibrosis were scored using the
Fatty Liver Inhibition of Progression (FLIP)
Algorithm.\textsuperscript{23} 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.

\subsection*{Immunofluorescence
analysis}\label{immunofluorescence-analysis}

Liver samples embedded with paraffin were dewaxed, blocked and then
incubated with phosphorylated STAT1 \textsuperscript{Tyr701} and
phosphorylated STAT1 \textsuperscript{Ser727} antibodies overnight at 4
\selectlanguage{ngerman}°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\selectlanguage{english}', 6-diamidino-2-phenylindole (DAPI) for 5 min
at room temperature. Images were captured using fluorescent microscopy.

\subsection*{Lipid content analysis}\label{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 \selectlanguage{greek}μ\selectlanguage{english}g\selectlanguage{ngerman}·mL\textsuperscript{-1} Nile red for 30 min at
room temperature. Lipid-bound Nile red fluorescence was detected using
fluorescence microscopy.

\selectlanguage{english}\subsection*{\texorpdfstring{Quantitative real-time polymerase chain
reaction(qRT-PCR){}}{Quantitative real-time polymerase chain reaction(qRT-PCR)}}\label{quantitative-real-time-polymerase-chain-reactionqrt-pcr}

To verify the quantification of gene expression levels, qRT-PCR was
performed as previously described \textsuperscript{21}. 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 \textbf{Tab.
1}. Relative mRNA expression was determined by a comparative method
(2\selectlanguage{greek}\textsuperscript{-ΔΔCt}\selectlanguage{english}) using GAPDH as a reference gene.

\subsection*{Western blotting analysis}\label{western-blotting-analysis}

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-STAT1\textsuperscript{Y701}
(1:1000), p-STAT1\textsuperscript{S727} (1:1000), CXCL10 (1:500), p65
(1:1000), p-p65 (1:1000), IL-6 (1:1000), TNF\selectlanguage{greek}α \selectlanguage{english}(1:1000), LXR\selectlanguage{greek}α \selectlanguage{english}(1:1000),
LXR\selectlanguage{greek}β \selectlanguage{english}(1:1000), CHREBP (1:1000), SREBP-1c (1:1000), \selectlanguage{greek}β-\selectlanguage{english}actin (1:2000) and
Lamin B (1:2500) overnight at 4 \selectlanguage{ngerman}°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 \selectlanguage{greek}β-\selectlanguage{english}actin or Lamin B.

\subsection*{Statistical analysis}\label{statistical-analysis}

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 \selectlanguage{ngerman}±
SD. Difference among groups were 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
\textless{} 0.05 was considered statistically significant.

\section*{Results}\label{results}

\subsection*{Dios decreases lipogenesis and inflammatory response in
HFD-induced NASH
mice}\label{dios-decreases-lipogenesis-and-inflammatory-response-in-hfd-induced-nash-mice}

In order to identify the effect of Dios against through lipogenesis and
inflammatory response, a NASH mice model induced by HFD was established
and treated by Dios. As depicted in \textbf{Fig. 1B,} Dios sharply
reversed lipid accumulation in livers of HFD-induced NASH mice from Oil
Red O staining. Furthermore, as shown in HE and Masson staining
\textbf{(Fig. 1C and 1D)}, the liver tissues exhibited obvious
histological changes including steatosis, hepatocyte ballooning, and
lobular inflammation, but no fibrosis in the HFD group, and those
changes were reduced after Dios treatment. Lipid metabolism plays a
critical role in the progress of NASH, the expressions of LXR\selectlanguage{greek}α, \selectlanguage{english}LXR\selectlanguage{greek}β,
\selectlanguage{english}CHREBP, and SREBP-1c protein relevant with lipogenesis were
downregulated after Dios treatment \textbf{(Fig. 1E)}. As illustrated in
\textbf{Fig. 1F}, the amount of p65 phosphorylation and the release of
inflammatory factors (TNF\selectlanguage{greek}α \selectlanguage{english}and IL-6) were repressed by Dios.{}

PA was utilized to establish a NASH model in vitro. The cytotoxicity of
PA was measured \textbf{(Fig. S2A)}, and there were no apparent changes
in HepG2 cells exposed to PA at concentrations of less than 0.2 mM.
\textbf{Fig. S2B and S2C} showed that PA treatment for 24 h increased
intracellular lipid accumulation and TG content in HepG2 cells in a
dose-dependent manner. Thus, the concentration of PA at 0.2 mM was used
for achieving maximal lipid accumulation without cytotoxicity in
experiments. Dios exhibited no cytotoxicity at the lesser dose than 320
\selectlanguage{greek}μ\selectlanguage{english}M in HepG2 cells \textbf{(Fig. S2D)}. Dios dose-dependently enhanced
the viability of PA-induced HepG2 cells at a concentration of greater
than 5 \selectlanguage{greek}μ\selectlanguage{english}M \textbf{(Fig. S2E)}. Compared with the PA group, both the
lipid accumulation and intracellular TG content were reduced
significantly by co-treated with Dios and PA in HepG2 cells
\textbf{(Fig. 2A-C)}. Dios also suppressed expressions of the lipogenic
proteins LXR\selectlanguage{greek}α, \selectlanguage{english}LXR\selectlanguage{greek}β, \selectlanguage{english}CHREBP, and SREBP-1c \textbf{(Fig. 2D)} and
decreased that of the inflammatory proteins p-p65 and IL-6 \textbf{(Fig.
2E)} induced by PA in HepG2 cells. Summarizing the above results, we
concluded that Dios conferred protection on NASH in vivo and in vitro.

\subsection*{Dios promotes STAT1 and CXCL10 molecular phenotype in livers
of NASH
mice}\label{dios-promotes-stat1-and-cxcl10-molecular-phenotype-in-livers-of-nash-mice}

Considering the remarkable effects of Dios in vivo and vitro, a
liver-transcriptome analysis by RNA sequencing (RNA-Seq) was adopted to
evaluate the key genes involved in the blockage of NASH disease by Dios.
After normalization and analysis of the sequencing data, the DEGs from
three sets of the samples were shown in \textbf{Fig. 3A and B} (A, HFD
vs SD; B, HFD + Dios vs HFD). The DEGs of SD, HFD, and HFD + Dios group
were compared with Hierarchical cluster analysis \textbf{(Fig. 3C)}.
From the Venn diagram, the result illustrated 456-shared DEGs were
discovered between HFD vs SD and HFD + Dios vs HFD (HFD vs SD: 2236
genes; HFD + Dios vs HFD: 566 genes) \textbf{(Fig. 3D)}. To identify the
biological features and explore the{}{enrichment-pathways}of 456-shared
DEGs, the enrichment analysis of GO and KEGG pathway was accomplished by
DAVID online tools. The result of GO analysis \textbf{(Tab. 2)}
suggested that changes in biological processes (BP) of DEGs were
enriched in the immune system process, inflammatory response,
chemotaxis, and positive regulation of T cell activation. Changes in
cellular components (CC) were mainly enriched in the membrane, plasma
membrane, and cytoplasm. Changes in molecular functions (MF) of DEGs
were enriched in GTP binding, chemokine activity, and protein kinase
binding. Beside, we found that DEGs were enriched in different pathways,
such as antigen processing and presentation, type I diabetes mellitus,
cytokine-cytokine receptor interaction, cell adhesion molecules (CAMs)
and NF-\selectlanguage{greek}κ\selectlanguage{english}B signaling pathway \textbf{(Tab. 2)}. PPI network consisting of
408 nodes and 3294 edged \textbf{(Fig. 3E)} was also established by
STRING database to reveal the interactive relation of 456 DEGs. The top
10 hub genes in the DEGs were confirmed by Cytohubba including TNF,
CCL5, CXCL10, STAT1, CXCL9, ITGAX, CD274, IFNG, PSMB8, and CD40
\textbf{(Fig. 3F)}. The abbreviations, official names, and functions of
these hub genes were shown in \textbf{Tab. 3}. Ultimately, qRT-PCR
analysis was applied to examine the expression of six genes from DEGs
\textbf{(Fig. 3G)} and the levels of those tested genes were aligned
with that of RNA-Seq analysis. From the hub genes combined with the
literatures, we selected STAT1 and CXCL10 as key genes for further
research. The qRT-PCR result told that Dios dramatically decreased the
mRNA levels of STAT1 and CXCL10 induced by HFD \textbf{(Fig. 3H)}.
Moreover, the levels of p-STAT1\textsuperscript{Y701},
p-STAT1\textsuperscript{S727}, and CXCL10 protein were up- and
downregulated in the HFD and HFD + Dios group, respectively
\textbf{(Fig. 3I)}. {}{Therefore, in the following research, we focused
on the functional fraction of STAT1 and CXCL10 and investigate their
underlying mechanism in the treatment of NASH by Dios.}

\subsection*{Dios prevents CXCL10-mediated lipogenesis and inflammatory
response}\label{dios-prevents-cxcl10-mediated-lipogenesis-and-inflammatory-response}

To further identify the mediation of Dios on CXCL10, we applied siCXCL10
to make CXCL10 silence in HepG2 cells. \textbf{Fig. 4A} showed the
transfection efficiency of CXCL10 siRNA at the mRNA level. And
siCXCL10-treated HepG2 cells displayed downregulated expression levels
of CXCL10 protein \textbf{(Fig. 4B)}. As seen in \textbf{Fig. 4C} and
\textbf{4D}, the TG content and lipid accumulation level were
significantly decreased after siCXCL10 treatment. Compared with siNull
treatment, siCXCL10-treated HepG2 cells displayed downregulated
expression levels of LXR\selectlanguage{greek}α, \selectlanguage{english}LXR\selectlanguage{greek}β, \selectlanguage{english}and p-p65 proteins \textbf{(Fig. 4E)}.
Thus, lipid accumulation and inflammation caused by PA were efficiently
inhibited by siCXCL10, suggesting that CXCL10 was critical to mediate
lipogenesis and inflammatory response.

\subsection*{Dios inhibits the nuclear localization of phosphorylated
STAT1 to prevent~ lipogenesis and inflammatory
response}\label{dios-inhibits-the-nuclear-localization-of-phosphorylated-stat1-to-prevent-lipogenesis-and-inflammatory-response}

The regulation of Dios on STAT1 was explored in our study. In the
immunofluorescence assay, the nuclear localization of
p-STAT1\textsuperscript{Tyr701} \textbf{(Fig. 5A)} and
p-STAT1\textsuperscript{Ser727} \textbf{(Fig. 5B)} in the HFD group was
as well decreased in the presence of Dios. Correspondingly, the result
obtained from the detection of western blot was consistent with that of
{}{immunofluorescence} \textbf{(Fig. 5C)}. To further recognize the role
of STAT1 in the hepatoprotective effects of Dios identify, the specific
STAT1 inhibitor (fludarabine) was utilized to inhibit the activation of
STAT1. The TG content and lipid accumulation induced by PA were
depressed by pretreatment with fludarabine in HepG2 cells \textbf{(Fig.
6A and 6B)}, implying a critical role for STAT1 in the amelioration of
hepatocyte steatosis. As shown in \textbf{Fig. 6C}, the inhibition of
STAT1 apparently decreased the levels of p-STAT1\textsuperscript{Y701}
and p-STAT1\textsuperscript{S72} proteins in HepG2 cells. Additionally,
the inhibition of STAT1 significantly reduced the expression of
lipogenic proteins (LXR\selectlanguage{greek}α, \selectlanguage{english}LXR\selectlanguage{greek}β, \selectlanguage{english}CHREBP, and SREBP-1c) \textbf{(Fig. 6D)}
and downregulated inflammatory indicators p-p65 \textbf{(Fig. 6E)} at
the protein level. Therefore, we further verified that Dios mitigated
the lipogenesis and inflammation were STAT1-dependent.

\subsection*{Dios interferes with STAT1/CXCL10 pathway to ameliorate
NASH}\label{dios-interferes-with-stat1cxcl10-pathway-to-ameliorate-nash}

As shown in \textbf{Fig. 7A}, the inhibition of STAT1 apparently
decreased the levels of CXCL10 protein. Compared with siNull treatment,
siCXCL10-treated HepG2 cells displayed no effects on the levels of
STAT1, p-STAT1\textsuperscript{Y701}, and p-STAT1\textsuperscript{S727}
protein \textbf{(Fig. 7B)}, which indicated that CXCL10 was the
downstream gene of STAT1. To further confirm our finding, we transfected
HepG2 cells with STAT1 overexpression plasmid. \textbf{Fig. 7C}
displayed the transfection efficiency of STAT1 plasmid at the mRNA
level. STAT1 overexpression aggravated PA-induced lipid accumulation as
observed with O Red O \textbf{(Fig. 7D)} and upregulated the amounts of
p-STAT1\textsuperscript{Y701}, p-STAT1\textsuperscript{S727}, and CXCL10
protein \textbf{(Fig. 7E)}. {}

To investigate the contribution of STAT1 in the protective effect of
Dios on NASH, a STAT1 inhibitor {}{fludarabine} was pretreated to induce
low expression of STAT1 in mice.{A substantial body-weight reduction was
found in NASH mice treated with Dios and fludarabine \textbf{(Fig. 8A)},
with no effect on food consumption was found among all groups
\textbf{(Fig. S3B)}. \textbf{Fig. 8B} showed that Dios and fludarabine
markedly alleviated and even reverse pathological changes in liver
morphology of NASH mice. Additionally, STAT1-inhibited mice treated with
Dios exhibited a decreased index of liver \textbf{(Fig. 8C)} and
\textbf{} abdominal fat pad \textbf{(Fig. S3C and S3D)}.}Furthermore,
the results of the biochemical analysis showed that Dios also reduced
the levels of serum TC, LDL-C, ALT, and AST, but no difference was seen
for serum TG and serum HDL-C \textbf{(Fig. 8D-I, Fig. S3D-F)}. The
results of the contents of liver TG \textbf{(Fig. 8E)} and TC
\textbf{(Fig. 8F)} and Oil Red O staining \textbf{(Fig. 8A)} revealed
that there was massive lipid accumulation in the livers of NASH mice,
which was fully alleviated by Dios in mice and STAT1-inhibited mice.
Meanwhile, the obvious pathological changes of livers was observed
including increased hepatic steatosis, hepatocyte ballooning, and
lobular inflammation \textbf{(Fig. 8J)}. These changes were remarkably
ameliorated by Dios and fludarabine in HFD-fed mice. SAF score was
performed by histological analysis according to a grading system
established for mouse models of NAFLD/NASH, showing that the majority of
HFD-fed mice displayed a pathophysiological characteristic of NASH,
whereas both Dios and fludarabine dramatically attenuated that
characteristic \textbf{(Fig. S4A-D)}.

STAT1/CXCL10 was regarded as an crucial pathway relevant with
lipogenesis and inflammation in NASH mice. \textbf{Fig. 9A} revealed
that the levels of p-STAT1\textsuperscript{Y701},
p-STAT1\textsuperscript{S727}, and CXCL10 protein significantly were
eliminated in the HFD + Dios group. Additionally, in comparison with HFD
group, the expressions of these proteins were inhibited in the
HFD-treated group with STAT1 inhibitor. As depicted in \textbf{Fig. 9B
and 9C}, Dios-treated mice displayed decreased protein expression levels
of LXR\selectlanguage{greek}α, \selectlanguage{english}LXR\selectlanguage{greek}β, \selectlanguage{english}CHREBP, SREBP-1c, and p-p65. The protein levels of all of
these markers were completely normalized in the fludarabine-treated
mice. These indicated that Dios could regulate lipogenesis and
inflammatory response by inhibiting STAT1/CXCL10- LXR\selectlanguage{greek}α/β-\selectlanguage{english}p65 pathway.

\section*{Discussion}\label{discussion}

{}{Emerging evidence}suggest that histopathological evaluation of biopsy
specimens is a gold standard for diagnosing NASH.\textsuperscript{24}
The typical characteristic such as steatosis, lobular inflammation, and
hepatocellular ballooning are all observed in NASH model liver but
fibrosis is not uncertain.\textsuperscript{25} Actually, by Oil Red O,
HE and Mason trichrome staining in the livers of NASH mice in our study,
liver tissues occurred these histological changes without fibrosis.
Meanwhile, the SAF scores of liver histology assisted us to recognize
that the establishment of NASH mice model was
successful.\textsuperscript{23} As for the impact of Dios on NASH mice,
Dios distinctly blocked the pathological changes of liver. In vitro
HepG2 cells, Dios eliminated lipid deposition and TG content induced by
PA. Our finding was consistent with Zeng's report.\textsuperscript{4} In
the pathway of lipogenesis, LXR\selectlanguage{greek}α/β, \selectlanguage{english}SREBP-1c and ChREBP are the
important transcription factors to regulate hepatic lipogenesis in
NAFLD,\textsuperscript{26, 27} and LXR\selectlanguage{greek}α/β \selectlanguage{english}can activate SREBP-1c and
ChREBP to mediate the metabolism of cholesterol and fatty
acids.\textsuperscript{28, 29} Those proteins mediated lipogenesis were
inhibited by Dios in NASH mice and HepG2 cells. In the pathway of
inflammation, the important marker NF-\selectlanguage{greek}κ\selectlanguage{english}B (p65) and the inflammatory
factors including TNF\selectlanguage{greek}α \selectlanguage{english}and IL-6 are regarded as the indicator of hepatic
inflammation.\textsuperscript{30, 31} And Dios inhibited the levels of~
the inflammatory protein and factors in NASH mice and HepG2 cells. Our
study showed that Dios exerted a hepatoprotective effect against NASH,
but the key target genes and proteins interfered by Dios in NASH mice
were unclear and should be explored. Hence, the RNA-Seq analysis was
employed in the further seeking of the novel key targets.

Here a total of 456 mutual DEGs were identified using liver RNA-Seq in
our experiment. After the enrichment analysis of those DEGs, most of
them were focused on the fields of inflammatory and immune response.
Specially, CXCL10 (macrophage chemotactic ligand 10) and STAT1 (signal
transducers and activators of transcription 1) were the hub genes among
the DEGs, and were likely the major regulated targets for Dios.
Hepatocytic lipotoxicity promotes the release of CXCL10, inducing
macrophage chemotaxis, increased inflammation, and liver
injury.\textsuperscript{32} STAT1 is known to be a transcription factor
that mediates immune and pro-inflammatory responses\textsuperscript{33}
and modulates lipid metabolism, energy expenditure, and mitochondrial
biogenesis.\textsuperscript{34} In the liver of NASH mice and patients,
{}{the levels of STAT1 phosphorylation and CXCL10 are
increased}.\textsuperscript{33, 35} Similarly, our data showed that the
mRNA and protein expressions of STAT1 and CXCL10 were significantly
upregulated both in NASH mice and HepG2 cells, however, Dios exhibited
the repression on those expressions. In a word, CXCL10 and STAT1 were
considered as key targets had a pivotal role for the attenuation of Dios
on NASH. However, whether STAT1 and CXCL10 mediated related proteins
involved lipogenesis and inflammatory response needed to be discussed.
{}

NASH displays the disorder in lipid metabolism and
inflammation,\textsuperscript{36} while the elevation of CXCL10 and
STAT1 expression may contribute to that disorder.\textsuperscript{37,
38} In other words, if the disorder were mediated by CXCL10 and STAT1,
the down-stream proteins involved in the pathways of lipogenesis and
inflammation should be interfered. The lipogenic factor LXR\selectlanguage{greek}α/β \selectlanguage{english}and
inflammatory protein NF-\selectlanguage{greek}κ\selectlanguage{english}B (p65) are diminished in CXCL10-deficient
mice.\textsuperscript{35} Our finding equally manifested that when HepG2
cells were administrated with CXCL10 siRNA, the content of TG and lipid
accumulation and the expressions of LXR\selectlanguage{greek}α/β \selectlanguage{english}and p-p65 protein were
reduced. Meanwhile, our results indicated that Dios reduced the nuclear
localization of p-STAT1\textsuperscript{Y701} and
p-STAT1\textsuperscript{S727} in livers of NASH mice induced by HFD. In
terms of the related reference, saturated free fatty acids induced
hepatocyte lipotoxicity in NASH could cause the nuclear localization of
p-STAT1\textsuperscript{Y701},\textsuperscript{39} and this conclusion
supported our results appropriately. ~The pretreatment of
STAT1-inhibitor fludarabine decreased the lipid accumulation,
downregulated the level of STAT1 phosphorylation, and induced the
elimination of relative protein levels of lipogenesis and inflammatory
response in HepG2 cells. And all inflammatory indicators were inhibited
as well after the reduction of STAT1 in our result. Thus, Dios could
prevent CXCL10- and STAT1-mediated lipogenesis and inflammatory
response. Furthermore, the hepatoprotective mechanism of Dios against
NASH through STAT1-CXCL10 was need to be elucidated.

For better identification of the relation between the downstream
proteins and STAT1-CXCL10, the STAT1-inhibitor, overexpression vector of
STAT1, and CXCL10 siRNA were used here. Combined our study and the
numerous evidences, the expression of CXCL10 was positively correlated
with the STAT1 phosphorylation.\textsuperscript{39, 40} However, the
levels of STAT1 and p-STAT1 after siCXCL10 treatment in HepG2 cells
showed no differences in our result. According to the reference, lipid
accumulation and hepatic TG level are increased in the livers of
STAT1\textsuperscript{+/+} mice.\textsuperscript{38} In our result, the
hepatocyte steatosis induced by PA were aggravated due to
over-expression of STAT1, but that was overwhelmed by Dios. The
inhibition or over-expression of STAT1 led to a decrease or increase of
CXCL10 protein expression. Therefore, STAT1 was the upstream of CXCL10.
Besides, the STAT1-inhibitor restrained the changes of biochemistry
indicators, hepatic pathological parameters and the expression of
lipogenesis and inflammation associated proteins in NASH mice, which
further revealed that the whole potential hepatoprotective mechanism of
Dios on lipogenesis and inflammatory response were most likely through
the STAT1/CXCL10 signaling pathway in NASH.

Taken together, the protective mechanism of Dios against NASH was
through the modulation of lipogenesis and the alleviation of
inflammatory response via STAT1/CXCL10-dependent pathway. The present
observations may provide a promising candidate for the treatment of
NASH, and Dios can be developed as functional foods or therapeutic
medicines for application.

{}

\section*{Author contributions}\label{author-contributions}

B. Z. designed the research. N. L. performed the experiments, with the
assistance of C. Y. and Y. Z. analyzed the data. N. L. wrote the initial
manuscript and B. Z. revised the manuscript.

\section*{Conflicts of interest}\label{conflicts-of-interest}

The authors state that they have no conflicts of interest.

\section*{Acknowledgments}\label{acknowledgments}

This work was supported by the Fundamental Research Funds for the
Central Universities (XDJK2020B056), and the National Key Research and
Development Program of China for Traditional Chinese Medicine
Modernization (2017YFC1702605, 2017YFC1702606).

\section*{Abbreviations}\label{abbreviations}

ALT, alanine aminotransferase; AST, aspartate aminotransferase; CHREBP,
carbohydrate response element-binding protein; CXCL10, macrophage
chemotactic ligand 10; DEGs, different expression genes; D-GaIN,
D-galactosamine; HDL-C, high density lipoprotein cholesterol; HFD,
high-fat diet; IL-6, interleukin-6; LDL-C, high density lipoprotein
cholesterol; LPS, lipopolysaccharide; LXR\selectlanguage{greek}α/β, \selectlanguage{english}liver X receptor
alpha/beta; MCODE, molecular complex detection; NASH, nonalcoholic
steatohepatitis; NF-\selectlanguage{greek}κ\selectlanguage{english}B, nuclear factor kappa B; PA, palmitic acid; PPI,
protein-protein interaction; SREBP-1c, sterol regulatory element-binding
proteins-1c; STAT1, signal transducers and activators of transcription
1; TC, total cholesterol; TG, triglyceride; TNF-\selectlanguage{greek}α, \selectlanguage{english}tumor necrosis
factor-alpha.

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~

~

\section*{Figure Legends}\label{figure-legends}

\textbf{~}

\textbf{Figure 1} \textbf{Effect of Dios on the indicators of
lipogenesis and inflammation in HFD-induced NASH mice.} (A) The chemical
structure of diosmetin (Dios). Histological evaluations of mice liver
tissues in different groups were through Oil Red O staining (B), HE
staining (C) and mason trichrome staining (D), respectively. Scale bar
100 \selectlanguage{greek}μ\selectlanguage{english}m. (1, steatosis; 2, hepatocyte ballooning; 3, lobular
inflammation). (E) The determination of relative proteins expression of
lipid metabolism were used Western blot. (F) The expression of IL-6,
TNF\selectlanguage{greek}α, \selectlanguage{english}p65 and p-p65 proteins. \selectlanguage{greek}β-\selectlanguage{english}actin was used as an internal control.
Data are expressed as mean \selectlanguage{ngerman}± SD (n = 6). *P \textless{} 0.05, vs. the SD
group; \#P \textless{} 0.05, vs. the HFD group.

\textbf{~}

\textbf{Figure 2 Impact of Dios on the indicators of lipogenesis and
inflammation induced by PA in HepG2 cells.} (A) Intracellular TG content
of was detected by using commercial kit after the treatment of 0.2 mM PA
in the presence or absence of Dios. (B) Representative images of
hepatocellular lipid accumulation which were treated with 0.2 mM PA in
the presence or absence of Dios by Oil Red O staining. Scale bar, 20 \selectlanguage{greek}μ\selectlanguage{english}m.
(C) Representative fluorescence of HepG2 cells was shown after PA and
Dios treatment with Nile red staining, and the mean fluorescent
intensity was measured as well. Scale bar, 20 \selectlanguage{greek}μ\selectlanguage{english}m. (D) The levels of
LXR\selectlanguage{greek}α, \selectlanguage{english}LXR\selectlanguage{greek}β, \selectlanguage{english}CHREBP, SREBP-1c protein expression after the same treatment
as above. (E) The levels of IL-6, p65 and p-p65 protein expression after
the same treatment as above. Data are presented as mean \selectlanguage{ngerman}± SD (n = 3).
*P\textless{}0.05, vs. the Control group; \#P\textless{}0.05, vs. the PA
group.

\textbf{Figure 3} \textbf{Effect of Dios on molecular phenotype of STAT1
and CXCL10 in mice liver by using RNA-Seq.} (A-B) Identification of DEGs
in different groups. (A) HFD vs SD group. (B) HFD + Dios vs HFD group.
The red points represent upregulated genes. The green points represent
downregulated genes. The blue points represent genes with no significant
difference. DEGs were screened on P \textless{} 0.05 and \textbar{} log2
(Fold change) \textbar{} \textgreater{} 1. (C) Heat map analysis was
employed to the discrimination of expression profile of DEGs across the
samples. Red and blue areas separately represent highly and lowly
expressed genes in mice livers among SD, HFD and HFD+Dios groups. (D)
Venn diagram of DEGs among different groups. 456 shared DEGs had
obtained from this diagram. (E) The PPI network of 456 shared DEGs was
analysized through STING database. There were 408 nodes and 3294 edged
in the PPI network. (F) The ten hub genes including CXCL10 and STAT1
were confirmed by Cytohubba. (G) The amount of DEGs mRNA expression
(FPR2, PSMB8, PTAFR, CCR7, CXCR4, LCK) was measured under the method of
quantitative RT-PCR. (H) The mRNA expressions of STAT1 and CXCL10 in
mice livers by qRT-PCR assay. (I) The expression of CXCL10, total STAT1,
p-STAT1Y701 and p-STAT1S727 proteins were examined through western blot
assay. \selectlanguage{greek}β-\selectlanguage{english}actin was used as an internal control. Data are expressed as
mean \selectlanguage{ngerman}± SD (n = 3). *P \textless{} 0.05, vs. the SD group; \#P
\textless{} 0.05, vs. the HFD group.

~

\textbf{Figure 4} \textbf{Dios prevents lipogenesis and inflammatory
response, which is CXCL10-dependent.} HepG2 cells treated with siRNA
either siNull or siCXCL10 for 24h before the stimulation of Dios and PA.
The mRNA (A) and protein (B) expression of CXCL10 in HepG2 cells
transfected with siCXCL10. (C) Intracellular TG content of HepG2 cells
after treatment. (D) Lipid accumulation by Oil Red O staining. Scale
bar, 20 \selectlanguage{greek}μ\selectlanguage{english}m. (E) The amount of LXR\selectlanguage{greek}α, \selectlanguage{english}LXR\selectlanguage{greek}β, \selectlanguage{english}p65, and p-p65 expression, and
\selectlanguage{greek}β-\selectlanguage{english}actin was used as an internal control. Data are presented as mean \selectlanguage{ngerman}± SD
(n = 3). *P\textless{}0.05, compared between the marked groups.

~

\textbf{Figure 5} \textbf{Dios prevents the nuclear localization of
phosphorylated STAT1 in vivo.} Representative immunofluorescence of mice
liver sections were stained with (A) phospho-STAT1 (Tyr701) and (B)
phospho-STAT1 (Ser727) antibodies. The staining was observed following
the addition of DAPI by fluorescence microscope. Scale bar, 20\selectlanguage{greek}μ\selectlanguage{english}m. (C)
The levels of p-STAT1\textsuperscript{Y701},
p-STAT1\textsuperscript{S727}, STAT1, \selectlanguage{greek}β-\selectlanguage{english}actin and LaminB protein
expression from the cytosol into the nucleus were analyzed by Western
blot. Data are expressed as mean \selectlanguage{ngerman}± SD (n = 3). *P\textless{}0.05, vs.
the SD group; \#P\textless{}0.05, vs. the HFD group.\textbf{{}}

~

\textbf{Figure 6} \textbf{Dios prevents lipogenesis and inflammatory
response, which is STAT1-dependent.} HepG2 cells pretreated with or
without fludarabine inhibitor (10 \selectlanguage{greek}μ\selectlanguage{english}M) before the stimulation of Dios and
PA. (A) Intracellular TG content of HepG2 cells after treatment. (B)
Lipid accumulation by Oil Red O staining. The expression of (C) STAT1,
p-STAT1\textsuperscript{Y701}, p-STAT1\textsuperscript{S727}, (D) LXR\selectlanguage{greek}α,
\selectlanguage{english}LXR\selectlanguage{greek}β, \selectlanguage{english}CHREBP and SREBP-1c, (D) p65 and p-p65 were examined by Western
blot assay and quantified by densitometric analysis. \selectlanguage{greek}β-\selectlanguage{english}actin was used as
an internal control. Data are presented as mean \selectlanguage{ngerman}± SD (n = 3).
*P\textless{}0.05, compared between the marked groups.

~

\textbf{Figure 7 Dios inhibits CXCL10 by decreasing phosphorylation of
STAT1.} (A) The level of CXCL10 protein expression in HepG2 cells, which
were pretreated with or without fludarabine inhibitor (10 \selectlanguage{greek}μ\selectlanguage{english}M) before the
stimulation of Dios and PA. (B) The level of STAT1,
pSTAT1\textsuperscript{Y701} and pSTAT1\textsuperscript{S727} protein
expression in HepG2 cells, which were treated with siRNA either siNull
or siCXCL10 for 24h before the stimulation of Dios and PA. From C to E,
HepG2 cells treated with plasmid either pcDNA3.1 or STAT1-pcDNA3.1 for
24h before the stimulation of Dios and PA. (C) The mRNA expression of
STAT1. (D) Lipid accumulation by Oil Red O staining. (E) The levels of
STAT1, p-STAT1\textsuperscript{Y701}, p-STAT1\textsuperscript{S727} and
CXCL10 protein, and \selectlanguage{greek}β-\selectlanguage{english}actin was used as an internal control. Data are
presented as mean \selectlanguage{ngerman}± SD (n = 3). *P\textless{}0.05, compared between the
marked groups.{}

~

\textbf{Figure 8} \textbf{Inhibition of STAT1 improves lipid
accumulation and inflammation in HFD-induced NASH mice.} (A) Weekly body
weights of mice and STAT1-inhibited mice were recorded during 18 weeks.
(B) Gross examination of liver. (C) Liver index. The contents of liver
TG (D) and TC (E) were detected using a commercial kit. The levels of
serum TG (F), TC (G), ALT (H) and AST (I) were tested via corresponding
kits. (J) Histological analysis of mice livers by Oil Red O staining
Scale bars, 100 \selectlanguage{greek}μ\selectlanguage{english}m. (K) Histological changes of liver sections was
respectively evaluated with hematoxylin eosin staining. Scale bar, 100
\selectlanguage{greek}μ\selectlanguage{english}m. Data are presented as mean \selectlanguage{ngerman}± SD (n = 6). *P \textless{} 0.05,
compared between the marked groups.\textbf{{}}

~

\textbf{Figure 9} \textbf{Interference with STAT1/CXCL10 pathway
mediates Dios-conferred hepatoprotective effect.} (A) The levels of
p-STAT1\textsuperscript{Y701}, p-STAT1\textsuperscript{S727}, STAT1, and
CXCL10 protein in the livers of STAT1-inhibited or non-inhibited mice.
(B) The levels of LXR\selectlanguage{greek}α, \selectlanguage{english}LXR\selectlanguage{greek}β, \selectlanguage{english}CHREBP, and SREBP-1C protein in the livers
of STAT1-inhibited or non-inhibited mice. (C) The levels of p65 and
p-p65 protein in the livers of STAT1-inhibited or non-inhibited mice.
\selectlanguage{greek}β-\selectlanguage{english}actin was used as an internal control. Data are presented as mean \selectlanguage{ngerman}± SD
(n = 3). *P \textless{} 0.05, compared between the marked groups.

~

\section*{Table Legends}\label{table-legends}

\textbf{Table 1} Primers used for real-time RT-PCR analysis.

\textbf{Table 2} GO and KEGG pathway enrichment analysis of DEGs.

\textbf{Table 3} Functional roles of 10 hub genes.

\par\null

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