Results
Dios promotes STAT1 and CXCL10 molecular phenotype in livers
of NASH mice
A liver-transcriptome analysis by RNA sequencing (RNA-Seq) was adopted
to evaluate the key targets 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 Fig. 1A and 1B (A,
HFD vs SD; B, HFD + Dios vs HFD). The DEGs of SD, HFD, and HFD + Dios
group were compared with Hierarchical cluster analysis (Fig.
1C) . 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) (Fig. 1D) . 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 (Tab. 2) suggested that changes in BP of DEGs were
enriched in the immune system process, inflammatory response,
chemotaxis, and positive regulation of T cell activation. Changes in CC
were mainly enriched in the membrane, plasma membrane, and cytoplasm.
Changes in MF of DEGs were enriched in GTP binding, chemokine activity,
and protein kinase binding. Besides, 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-κB signaling pathway (Tab. 2) . PPI
network consisting of 408 nodes and 3294 edged (Fig. 1E) was
also established by STRING database to reveal the interactive relation
of 456 DEGs. The clusters of 456-shared DEGs were examined with MCODE, a
plug-in Cytoscape, resulting in four clusters and the most significant
cluster scored 32.33 (Fig. 1F) . The top 10 hub genes in the
DEGs were confirmed by Cytohubba including TNF, CCL5, CXCL10, STAT1,
CXCL9, ITGAX, CD274, IFNG, PSMB8, and CD40 (Fig. 1G) . The
abbreviations, official names, and functions of these hub genes were
shown in Tab. 3 . Ultimately, the qRT-PCR analysis was applied
to examine the expression of six genes from DEGs (Fig. 1H) . The
levels of those tested genes were aligned with that of RNA-Seq analysis.
Dios reduces the indicators of lipogenesis and inflammation
in HepG2
cells
PA was utilized to establish a NASH model in vitro. The cytotoxicity of
PA was measured (Fig. 2A) , and there were no apparent changes
in HepG2 cells exposed to PA at concentrations of less than 0.2 mM.Fig. 2B and 2C 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
μM in HepG2 cells (Fig. 2D) . Dios dose-dependently enhanced the
viability of PA-induced HepG2 cells at a concentration of greater than 5
μM (Fig. 2E) . 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 (Fig. 2F-H) . Dios
significantly reduced the expression of STAT1Y701, STAT1S727, and CXCL10
protein after PA treatment (Fig. 2I, J) . Dios also suppressed
expressions of the lipogenic
proteins LXRα, LXRβ, CHREBP, and SREBP-1c (Fig. 2K, L) and
decreased that of the inflammatory proteins p-p65 and IL-6 (Fig.
2M, N) induced by PA in HepG2 cells.
Inhibition and overexpression of STAT1 modulate the
expressions of relative proteins in HepG2 cells via STAT1/CXCL10
pathway
A STAT1 inhibitor, fludarabine (Fig. S2) , was utilized to
recognize the role of STAT1 in the hepatoprotective effects of Dios. The
TG content and lipid accumulation induced by PA were depressed by
pretreatment with fludarabine in HepG2 cells (Fig. 3A, B) ,
implying a critical role for STAT1 in the amelioration of hepatocyte
steatosis. As shown in Fig. 3C , the inhibition of STAT1
apparently decreased the levels of p-STAT1Y701,
p-STAT1S727, and CXCL10 proteins in HepG2 cells.
Additionally, the inhibition of STAT1 significantly reduced the
expression of lipogenic proteins (LXRα, LXRβ, CHREBP, and SREBP-1c)(Fig. 3D) and downregulated inflammatory indicators p-p65(Fig. 3E) at the protein level. To further confirm our
findings, we transfected HepG2 cells with STAT1 overexpression plasmid.Fig. 3F displayed the transfection efficiency of STAT1
overexpression plasmid at the mRNA level. STAT1 overexpression
aggravated PA-induced lipid accumulation as observed with O Red O(Fig. 3G) and upregulated the amounts of
p-STAT1Y701, p-STAT1S727, and CXCL10
protein.
Dios attenuates expressions of STAT1 and CXCL10 in
HFD-induced NASH
mice
The inhibitory effect of Dios against NASH and the changes of key
targets (STAT1 and CXCL10) were studied in HFD-induced NASH mice. As
depicted in Fig. 4A, Dios sharply reversed lipid accumulation
in livers of HFD-induced NASH mice from Oil Red O staining. Furthermore,
as shown in HE staining (Fig. 4B) , the liver tissues exhibited
histological changes including steatosis, hepatocyte ballooning, and
lobular inflammation in the HFD group, but those changes were reduced
after Dios treatment. From the hub genes combined with the literature,
we selected STAT1 and CXCL10 as key targets for further research. The
qRT-PCR result told that Dios dramatically decreased the mRNA levels of
STAT1 and CXCL10 induced by HFD (Fig. 4C) . Moreover, the levels
of p-STAT1Y701, p-STAT1S727, and
CXCL10 protein were up- and downregulated in the HFD and HFD + Dios
group, respectively (Fig. 4D) . In the immunofluorescence assay,
the nuclear localization of p-STAT1Tyr701(Fig. 4E) and p-STAT1Ser727 (Fig.
4F) 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(Fig. 4G) . Lipid metabolism played a critical role in the
progress of NASH, the expressions of LXRα, LXRβ, CHREBP, and SREBP-1c
protein relevant with lipogenesis were downregulated after Dios
treatment (Fig. 4H) . As illustrated in Fig. 4I , the
amount of p65 phosphorylation and the release of inflammatory factors
(TNFα and IL-6) were repressed by Dios.
Inhibition of STAT1 improves functional parameters and liver
morphological in HFD-induced NASH
mice
To investigate the contribution of STAT1 in the protective effect of
Dios on NASH, 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(Fig. 5A, B), with no effect on food consumption was found
among all groups (Fig. S3A, B). Fig. 5C 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 the livers(Fig. 5D) and abdominal fat pad, without a significant
difference in the ration of kidney and spleen (Fig. S3C, D) .
The results of Oil Red O staining and the contents of liver TG and TC
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 (Fig. 5E-H) . 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(Fig. 5I-N) . Meanwhile, the obvious pathological changes of
livers were observed including increased hepatic steatosis, hepatocyte
ballooning, and lobular inflammation, but no fibrosis. These changes
were remarkably ameliorated by Dios and fludarabine in HFD-fed mice(Fig. 5O, P) . 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 (Fig.
S4A-D) .
Interference with STAT1/CXCL10 pathway mediates
Dios-conferred hepatoprotective
effect
STAT1/CXCL10 was regarded as a crucial pathway relevant to lipogenesis
and inflammation in NASH mice. Fig. 6A revealed that the levels
of p-STAT1Y701, p-STAT1S727, and
CXCL10 protein significantly were eliminated in the HFD + Dios group.
Additionally, in comparison with the HFD group, the expressions of these
proteins were inhibited in the HFD-treated group with the STAT1
inhibitor. As depicted in Fig. 6B, C , Dios-treated mice
displayed decreased protein expression levels of LXRα, LXRβ, CHREBP,
SREBP-1c, and p-p65. The protein levels of all of these markers were
completely normalized in the fludarabine-treated mice. Because STAT1 was
reported to be an upstream regulator of CXCL10, siCXCL10 was used to
further investigate the relation between them. Fig. 6D showed
the transfection efficiency of CXCL10 siRNA at the mRNA level. Compared
with siNull treatment, siCXCL10-treated HepG2 cells displayed
down-regulated expression levels of CXCL10, LXRα, LXRβ, and p-p65
proteins, but no effects on the levels of STAT1,
p-STAT1Y701, and p-STAT1S727 protein(Fig. 6E, F) , which indicated that CXCL10 was the downstream
targeted gene of STAT1 and could regulate lipogenesis and inflammatory
response by LXRα / β and p65, respectively.