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Redox Biology 51 (2022) 102274
Available online 24 February 2022
2213-2317/© 2022 Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Mulberrin confers protection against hepatic brosis by Trim31/
Nrf2 signaling
Chenxu Ge
a
,
c
,
1
, Jun Tan
a
,
c
,
*
,
1
, Deshuai Lou
a
,
c
,
**
, Liancai Zhu
b
,
1
, Zixuan Zhong
a
,
c
,
1
,
Xianling Dai
a
,
b
,
1
, Yan Sun
a
,
b
,
1
, Qin Kuang
a
,
b
,
1
, Junjie Zhao
a
,
c
, Longyan Wang
a
,
c
, Jin Liu
a
,
c
,
Bochu Wang
b
,
****
, Minxuan Xu
a
,
b
,
c
,
***
a
Chongqing Key Laboratory of Medicinal Resources in the Three Gorges Reservoir Region, School of Biological and Chemical Engineering, Chongqing University of
Education, Chongqing, 400067, PR China
b
Key Laboratory of Biorheological Science and Technology (Chongqing University), Ministry of Education, College of Bioengineering, Chongqing University, Chongqing,
400030, PR China
c
Research Center of Brain Intellectual Promotion and Development for Children Aged 0-6 Years, Chongqing University of Education, Chongqing, 400067, PR China
ARTICLE INFO
Keywords:
Liver brosis
Mulberrin (Mul)
TRIM31-Nrf2 axis
Hepatocyte injury
HSCs activation
ABSTRACT
Mulberrin (Mul) is a key component of the traditional Chinese medicine Romulus Mori with various biological
functions. However, the effects of Mul on liver brosis have not been addressed, and thus were investigated in
our present study, as well as the underlying mechanisms. Here, we found that Mul administration signicantly
ameliorated carbon tetrachloride (CCl
4
)-induced liver injury and dysfunction in mice. Furthermore, CCl
4
-triggerd
collagen deposition and liver brosis were remarkably attenuated in mice with Mul supplementation through
suppressing transforming growth factor β1 (TGF-β1)/SMAD2/3 signaling pathway. Additionally, Mul treatments
strongly restrained the hepatic inammation in CCl
4
-challenged mice via blocking nuclear factor-κB (NF-κB)
signaling. Importantly, we found that Mul markedly increased liver TRIM31 expression in CCl
4
-treated mice,
accompanied with the inactivation of NOD-like receptor protein 3 (NLRP3) inammasome. CCl
4
-triggered he-
patic oxidative stress was also efciently mitigated by Mul consumption via improving nuclear factor E2-related
factor 2 (Nrf2) activation. Our in vitro studies conrmed that Mul reduced the activation of human and mouse
primary hepatic stellate cells (HSCs) stimulated by TGF-β1. Consistently, Mul remarkably retarded the inam-
matory response and reactive oxygen species (ROS) accumulation both in human and murine hepatocytes. More
importantly, by using hepatocyte-specic TRIM31 knockout mice (TRIM31
Hep-cKO
) and mouse primary hepato-
cytes with Nrf2-knockout (Nrf2
KO
), we identied that the anti-brotic and hepatic protective effects of Mul were
TRIM31/Nrf2 signaling-dependent, relieving HSCs activation and liver brosis. Therefore, Mul-ameliorated
hepatocyte injury contributed to the suppression of HSCs activation by improving TRIM31/Nrf2 axis, thus
providing a novel therapeutic strategy for hepatic brosis treatment.
1. Introduction
Liver brosis occurs in many types of liver disease, such as hepatitis
and chronic alcoholism, and leads to scarring and injury to the liver [1].
Liver brosis is characterized by excessive accumulation of extracellular
matrix (ECM) proteins, hepatocyte damage, distortion of the hepatic
* Corresponding author. Chongqing Key Laboratory of Medicinal Resources in the Three Gorges Reservoir Region, School of Biological and Chemical Engineering,
Chongqing University of Education, Chongqing, 400067, PR China.
** Corresponding author. Chongqing Key Laboratory of Medicinal Resources in the Three Gorges Reservoir Region, School of Biological and Chemical Engineering,
Chongqing University of Education, Chongqing, 400067, PR China.
*** Corresponding authors. Chongqing Key Laboratory of Medicinal Resources in the Three Gorges Reservoir Region, School of Biological and Chemical Engi-
neering, Chongqing University of Education, Chongqing, 400067, PR China.
**** Corresponding author. Key Laboratory of Biorheological Science and Technology (Chongqing University), Ministry of Education, College of Bioengineering,
Chongqing University, Chongqing, 400030, China.
E-mail addresses: tanjun@cque.edu.cn (J. Tan), wangbc2000@126.com (B. Wang), minxuanxu@foxmail.com (M. Xu).
1
These authors contributed equally to this work.
Contents lists available at ScienceDirect
Redox Biology
journal homepage: www.elsevier.com/locate/redox
https://doi.org/10.1016/j.redox.2022.102274
Received 20 January 2022; Received in revised form 14 February 2022; Accepted 22 February 2022
Redox Biology 51 (2022) 102274
2
lobules, and changes in the vascular architecture [2,3]. HSCs activation
plays an essential role in the pathogenesis of liver brosis. Activated
HSCs can transform to myobroblast-like cells, expressing
α
-SMA and
secreting ECM that consists of various proteoglycans and proteins [4].
Presently, the most effective therapeutic approach for hepatic cirrhosis
is liver transplantation, but its clinical application is limited due to the
shortage of donor material, the prerequisite for expert technical support,
and the high hospital costs [5]. If left untreated or without effective
treatment, liver brosis can progress to cirrhosis or even hepatocellular
carcinoma (HCC) [6]. Herein, exploring the mechanisms and nding
promising therapeutic strategies are urgently necessary for liver brosis
management.
Hepatic brosis is generally prolonged by chronic inammatory
response, and persistent inammation is involved in hepatic brosis
progression and cirrhosis development [7]. Continuous hepatic injury
leads to inammation pathology and inammatory cells inltration,
such as macrophages and lymphocytes [8]. A key factor of
hepatocyte-driven liver brosis is the activation of the pro-inammatory
NF-κB signaling in hepatocytes. The family of NF-κB transcription fac-
tors are crucial regulators of inammatory processes [9]. NF-κB acti-
vation in injured hepatocytes results in the secretion of various
pro-inammatory cytokines and chemokines including interleukin 1β
(IL-1β), tumor necrosis factor-
α
(TNF-ɑ), IL-6, and monocyte chemo-
attractant protein-1 (MCP-1) [10]. Chronic hepatic inammation,
derived from liver injury or infection, is a major driving force for liver
brosis. Although previous work has reported that activated NF-κB is
involved in brosis development, the exact contribution process still
remains enigmatic.
The tripartite motif (TRIM) family consists of a RING domain, one or
two B-box domains, and a coiled-coil domain, which contributes to a
wide range of biological processes [11]. TRIM31, a member of the TRIM
protein family, can mediate various pathological conditions including
inammatory diseases, viral infection and tumor progression [12].
Recently, TRIM31 was shown to attenuate NLRP3 inammasome acti-
vation, subsequently accelerating IL-1β releases and alum-induced
peritonitis in vivo [13]. More recently, TRIM31 over-expression was
reported to reduce the effects of oxidized low-density lipoprotein
(ox-LDL) on NLRP3 expression, pyroptosis, and inammatory cytokine
levels in vitro [14]. Under stimuli conditions, NLRP3 assembles into a
large cytoplasmic complex through recruiting apoptosis associated
speck-like protein (ASC) and Caspase-1, thereafter, causing the cleavage
of pro-IL-1β, which enables its maturation and releases from cells
depending on NF-κB activation [15]. Increasing studies have demon-
strated that excessive NLRP3 inammasome activation plays an essen-
tial role in the regulation of liver inammation and brosis [16,17].
Therefore, we hypothesized that TRIM31/NLRP3 signaling pathway
might be involved in liver brosis. However, as for this, little has been
investigated and reported, and thus was explored in our study to disclose
whether it could be a therapeutic target for liver brosis treatment.
Oxidative stress is another predominant pro-brogenic factor
involved in liver brosis progression [18]. Liver brosis-associated
oxidative stress is largely attributed to the abundant ROS production
and weakened antioxidant capacity. Nrf2, as a key transcription factor,
crucially protects cells against oxidative stress by promoting the
expression of numerous antioxidative genes, such as heme oxygenase-1
(HO-1), NAD(P)H: quinone oxidoreductase (NQO1), glutamate-cysteine
ligase modier subunit (GCLM) and glutamate-cysteine ligase catalytic
subunit (GCLC) [19,20]. Nrf2 signaling inactivation is intimately asso-
ciated with the progression of liver brosis [21]. Compounds or ap-
proaches that can better Nrf2 activation have been reported to
efciently ameliorate hepatic brosis development [20,22,23]. Mul-
berrin (Mul) is a key component of the traditional Chinese medicine
Romulus Mori and has been demonstrated to exert anti-inammatory and
antioxidant biological activities [24]. Mul could meliorate spinal cord
injury by suppressing neuroinammation, oxidative stress and neuronal
death in vivo and in vitro [25]. Recently, Mul was shown to attenuate
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkin-
son’s disease through inhibiting microglial activation and inammatory
response [26]. Given these effects of Mul, we supposed that Mul might
have therapeutic potential against liver brosis.
In the present work, we found that Mul treatments signicantly
ameliorated CCl
4
-induced hepatic brosis in mice via depressing
inammation and oxidative stress. The anti-brotic, anti-inammatory
and antioxidant biofunctions of Mul were validated in HSCs and hepa-
tocytes in vitro. An interaction between TRIM31 and Nrf2 was detected.
Importantly, we identied that the protective effects of Mul against
hepatic injury were TRIM31/Nrf2 signaling-dependent in hepatocytes,
contributing to the suppression of HSCs activation and liver brosis.
Together, Mul may be a promising therapeutic agent for the manage-
ment of liver brosis.
2. Materials and methods
For extended Material and Methods, see online Supplementary ma-
terials and methods.
2.1. Animals and treatments
The male wild type (WT) C57BL/6 N mice (6- to 8-week-old; 22–25 g
body weight) used in the current study were purchased from Beijing
Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). All
mice were housed in a constant temperature, humidity (controlled by
GREE central air-conditioner, #GMV-Pd250W/NaB-N1, China) and
pathogen-free-controlled environment (23 ±25 ◦C, 50–60%) cage with
a standard 12 h light/12 h dark cycle, plenty of water and food (path-
ogen-free) in their cages. All animal experimental procedures were
approved by the Institutional Animal Care and Use Committee in
Chongqing Key Laboratory of Medicinal Resources in the Three Gorges
Reservoir Region, School of Biological and Chemical Engineering,
Chongqing University of Education (Chongqing, China). Mice received
humane care according to the criteria outlined in the Guide for the Care
and Use of Laboratory Animals prepared by the National Academy of
Sciences and published by the National Institutes of Health (NIH) in
1996.
2.1.1. Animal model 1#
WT mice were allowed to adapt to their living environment for 1
week before all experiment’s proper starts. All mice were then randomly
divided into 6 groups, including the Oil/Veh group (n =20), Oil/Mul-H
(high dosage 60 mg/kg; n =20) group, CCl
4
/Veh group (n =25), CCl
4
/
Mul-L (low dosage 15 mg/kg; n =25) group, CCl
4
/Mul-M (moderate
dosage 30 mg/kg; n =25) group, and CCl
4
/Mul-H (high dosage 60 mg/
kg; n =20) group.
2.1.2. Animal model 2#
To generate mice with a conditional knockout allele of TRIM31, the
TRIM31
Flox/Flox
mice with C57BL/6 N background were constructed
using CRISPR/Cas9-regulated genome engineering system. The exon 4/
5 of TRIM31 was selected as conditional knockout region (cKO). Briey,
the selected exons of TRIM31 were anked by two loxP sites, and
therefore two single guide RNAs (gRNA1# and gRNA2#) targeting
TRIM31 introns were designed. The targeting vector containing TRIM31
exon 4/5 anked by two loxP sites and the two homology arms was used
as the template. The targeting vector, gRNA1# and gRNA2#, and
together with Cas9 were co-injected into fertilized eggs for cKO mouse
production. The obtained mice, which had exon 4/5 anked by two loxP
sites on one allele, were used to establish TRIM31
Flox/Flox
mice.
Hepatocyte-specic TRIM31 deletion (TRIM31
Hep-cKO
) mice were pro-
duced by mating TRIM31
Flox/Flox
mice with albumin-Cre (Alb-Cre) mice
(Jackson Laboratory, Bar Harbor, Maine, USA). A simple schematic di-
agram has been indicated in Supplementary Fig. S13A. TRIM31
Flox/Flox
(Flox) mice littermates were used in the work as controls for the
C. Ge et al.
Redox Biology 51 (2022) 102274
3
obtained TRIM31
Hep-cKO
mice. All TRIM31
Flox/Flox
and TRIM31
Hep-cKO
mice were then randomly divided into three subgroups, including the
Oil/Veh group (n =15), CCl
4
/Veh group (n =20), and CCl
4
/Mul-H
(high dosage 60 mg/kg; n =15) group.
2.1.3. Establishments of hepatic brosis murine model in vivo
Toxic liver brosis was established by intraperitoneal (i.p.) injections
of CCl
4
(1 ml/kg, dissolved in corn oil at a ratio of 1:4) twice a week for 6
weeks. The mice were sacriced 24 h after the nal CCl
4
(Aladdin,
Shanghai, China) injection. Same volume of corn oil was subjected to the
normal control group. Mul (Catalog number: #S-119; HPLC>98%;
Chengdu Herbpurify CO., LTD, Chengdu, China) at 15, 30 or 60 mg/kg
was administered to each group of mice by gavage daily. The dosages of
Mul for animal treatment were referred to previous studies and our
preliminary experiments [25,26]. Vehicle groups of mice received 0.9%
saline. Animal experimental protocols were presented in Fig. 1A. During
the treatments, body weights of mice were recorded weekly. Survival
rates of mice were measured. Blood samples were taken either from the
tail vein or via the cardiac puncture. The sampled blood was collected
into EDTA-coated tubes through retro-orbital bleeding, and centrifuged
at 5000 rpm for 10 min at 4 ◦C. The supernatant was then collected and
stored at −80 ◦C for further biological analysis. The liver samples were
either immediately frozen in liquid nitrogen and kept at −80 ◦C or xed
with 4% paraformaldehyde for histological analysis.
2.2. In vitro experiments
2.2.1. Cells treatment
Human hepatocyte cell line L02 was purchased from the Type Cul-
ture Collection of the Chinese Academy of Sciences (Shanghai, China).
Human hepatic stellate cell (HSC) line LX2 was obtained from American
Type Culture Collection (ATCC; Manassas, VA, USA). All cells were
maintained in Dulbecco’s Modied Eagle Medium (DMEM; Gibco, USA)
with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin-
streptomycin in a 5% CO
2
incubator at 37 ◦C. To imitate the in vivo
hepatic injury, cells were incubated with recombinant TGF-β1 (#240-B-
002 and #7666-MB; R&D system, USA), recombinant TNF-
α
(#210-TA;
R&D system), recombinant IL-1β (#201-LB; R&D system), LPS (derived
from Escherichia coli (055:B5); #L2880, Sigma-Aldrich, St. Louis, USA)
or H
2
O
2
(Sigma-Aldrich) as demonstrated in gure legends to investi-
gate the effects of Mul on liver brosis in vitro.
2.2.2. Primary hepatocytes isolation and culture
Mouse primary hepatocytes used in the study were isolated from
wild type or Nrf2-knockout (Nrf2
KO
) mice with C57BL/6J background
(Jackson Laboratory, Bar Harbor, ME) using liver perfusion method as
described previously [27,28]. Briey, mice abdominal cavity was
opened under a painless anesthesia condition. Thereafter, the liver tissue
was carefully perfused with 1×liver perfusion medium (#17701-038,
Gibco™) and 1×liver digest medium (#17703-034, Gibco™) via the
portal vein. Subsequently, 100
μ
m steel mesh was used to grind and lter
the digested liver samples. The mice primary hepatocytes were then
collected through centrifuging the lter liquor at 800 rpm, 4 ◦C for 5
min, and were further puried using 50% percoll solution
(#17-0891-01, GE Healthcare Life Sciences). The obtained hepatocytes
were maintained in DMEM medium containing 10% FBS and 1%
penicillin-streptomycin and cultured at 37 ◦C in a cell incubator with 5%
CO
2
.
2.2.3. Primary HSCs isolation
Primary HSCs were isolated from wild type C57BL/6 N mice. Briey,
HSCs were isolated through collagenase-pronase perfusion of livers as
described previously with the minor modications [29,30]. Liver of
mice was digested using Collagenase IV (Sigma-Aldrich, USA) and Pro-
nase E (Sigma-Aldrich) dissolved in PB buffer. Suspension of dispersed
cells was layered through gradient centrifugation in Nycodenz (Sig-
ma-Aldrich) according to manufacturer’s instructions. Isolated HSCs
were cultured in DMEM (Gibco) containing 10% FBS and 1%
penicillin-streptomycin at 37 ◦C in a humidied atmosphere containing
5% CO
2
. The medium was changed 24 h after seeding to remove dead
Fig. 1. Mulberrin ameliorates hepatic dysfunction in CCl
4
-induced mice. (A) The experiment design scheme. (B) Body weights of each group of mice. (C)
Survival rate of mice from all groups was quantied. Serum (D) TBIL, (E) ALT, AST, ALP, and (F) LDH levels were examined. Representative data were expressed as
mean ±SEM (n =10 per group). **P <0.01 and ***P <0.001 vs the Oil/Veh group; +P <0.05 and ++P <0.01 vs the CCl
4
/Veh group.
C. Ge et al.
Redox Biology 51 (2022) 102274
4
cells and debris.
2.2.4. Vectors establishment and transfection
For in vitro transfection, TRIM31 si-RNAs (siTRIM31), Nrf2 siRNAs
(siNrf2), and the corresponding negative control siRNAs (Ctrl/siRNAs)
were obtained from Generay Biotechnology (Shanghai, China). Trans-
fection for TRIM31 or Nrf2 knockdown was performed using Lipofect-
amine 3000 reagent (Invitrogen Life Technologies, Carlsbad, USA)
according to the provider’s instructions. To overexpress TRIM31, the
entire coding region of human TRIM31 was introduced to a replication-
defective adenoviral vector under the control of the cytomegalovirus
promoter. Recombinant adenoviruses expressing Flag tagged TRIM31
protein were puried. Adenovirus was infected at a multiplicity of
infection of 50 in cells for 24 h.
2.3. Histological and immunohistochemical (IHC) analysis
To explore histopathologic changes, the liver tissues were xed with
10% neutral formalin, embedded in parafn, and then sectioned trans-
versely (5-
μ
m-thick). The thin liver tissue sections were then stained
with hematoxylin and eosin (H&E). The deposition of collagen bers in
hepatic sections was measured using Masson staining and Sirius red
staining by the conventional protocols. Fibrosis stage was assessed ac-
cording to the Ishak score [31]. As for IHC staining, embedded liver
sections were dewaxed, and antigens were retrieved via sodium citrate
heating. Endogenous peroxidase was removed by adding 30% H
2
O
2
, and
an immunohistochemical pen was used to draw a circle around the tis-
sue. Then, 5% goat serum (#C0265, Beyotime Biotechnology) was
added to block the liver tissues. The liver sections were then incubated
with primary antibodies (Supplementary Table 1) at 4 ◦C overnight.
Sections were then washed, followed by incubation with secondary
antibodies (Supplementary Table 1) for 1 h at room temperature. IHC
staining was observed using 3,3′-diaminobenzidine (DAB) substrate kit
(#ab64238, Abcam, USA) and were counterstained with hematoxylin.
All images were captured under a microscope. The percentage of
collagen areas or positive signal expression elds was measured by
Image Pro Plus software (Media Cybernetics, USA).
2.4. Examination for oxidative stress markers
Commercial assay kits for the examination of hepatic malondialde-
hyde (MDA; #A003-1-2), catalase (CAT; #A007-1-1), superoxide dis-
mutase (SOD; #A001-3-2) and glutathione (GSH; #A006-2-1) were
obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing,
China). Hydrogen peroxide (H
2
O
2
) levels in liver were tested using
commercially available kit (#S0038; Beyotime Biotechnology) in line
with the provider suggested. Hepatic lipid peroxidation was examined
by thiobarbituric acid reactive substances (TBARS) formation [32].
TBARS Assay Kit (#801192, ZeptoMetrix Corporation, USA) was used
for the measurements of liver TBARS contents following the manufac-
turer’s protocols.
2.5. Immunouorescence (IF) staining
For IF analysis, the frozen liver sections or cells after treatments were
washed with PBS and were then blocked in 10% goat serum (#C0265,
Beyotime Biotechnology) containing 0.3% Triton X-100 (#ST797,
Beyotime Biotechnology) for 1 h at room temperature and incubated
with primary antibodies (Supplementary Table 1) at 4 ◦C overnight.
Samples were then washed, and secondary uorescent antibodies
(Supplementary Table 1) were prepared for incubation at room tem-
perature in dark for 45 min. After washing, 2-(4-Amidinophenyl)-6-
indolecarbamidine dihydrochloride solution (DAPI; #C1006, Beyotime
Biotechnology) was added to the samples for nuclei staining. Images
were captured under a uorescence microscopy.
2.6. Statistical analysis
Statistical analysis was conducted using GraphPad Prism 8.0 (San
Diego, CA, USA). Data represented as mean ±standard error of the
mean (SEM) unless otherwise indicated. All analysis were repeated
independently with similar results at least three times. Differences be-
tween two groups were analyzed using Student’s t-test. One-way anal-
ysis of variance (ANOVA) with Tukey’s post hoc tests were performed
for comparisons between multiple groups. P value <0.05 was consid-
ered indicative of statistical signicance. The experimenters were blin-
ded to the animal grouping information.
3. Results
3.1. Mulberrin ameliorates hepatic dysfunction and brosis in CCl
4
-
induced mice
In the present study, a mouse model with hepatic brosis was
established using CCl
4
to explore the regulatory effects of Mul on liver
injury (Fig. 1A). As displayed in Fig. 1B, CCl
4
injection signicantly
reduced the body weights of mice, but were moderately rescued by Mul
at higher dosages (30 and 60 mg/kg). CCl
4
treatment led to poorer
survival rates of mice over the course of the experiment compared with
Oil/Veh group, while Mul treatments reduced the mortality of CCl
4
-
challenged mice (Fig. 1C). CCl
4
injection signicantly induced the he-
patic dysfunction and injury in mice, as evidenced by the increased
serum TBIL, ALT, AST, ALP and LDH contents; however, all these results
caused by CCl
4
were efciently reversed by Mul treatments in a dose-
dependent manner (Fig. 1D–F). These ndings initially revealed the
protective effects of Mul against CCl
4
-triggered hepatic damage.
Furthermore, we showed that under normal physiological conditions,
Mul at 60 mg/kg had no signicant inuences on the changes of body
weights, animal survival and hepatic functions, indicating the safe use of
Mul in vivo.
3.2. Mulberrin inhibits CCl
4
-induced activation of TGF-β1/SMADs
signaling in liver of mice
H&E staining showed that CCl
4
led to evident histological changes in
liver tissues, conrming the hepatic injury. Sirius red and Masson’s
Trichrome staining demonstrated that CCl
4
-challenged mice exhibited
severer collagen deposition and brosis in liver sections than that of the
Oil/Veh group. Notably, these histological alterations and hepatic
brosis were signicantly mitigated by Mul treatments via a
concentration-dependent fashion (Fig. 2A–D). Furthermore, the levels of
three key liver brosis hallmarks including hyaluronic acid, PC III and
Collagen IV were strongly promoted by CCl
4
, while being strongly
ameliorated in Mul-treated mice compared with the model group
(Fig. 2E–G). These results demonstrated that Mul could mitigate liver
brosis progression in vivo.
To further reveal the protective effect of Mul in the mouse model
with liver brosis,
α
-SMA, Col-I and TGF-β1/SMADs signaling that plays
a contributory role in brosis, were then explored. IF staining showed
that Mul signicantly reduced the positive expression of
α
-SMA and Col-
I in liver samples of CCl
4
-challenged mice compared with CCl
4
/Veh
group (Fig. 3A–D). Consistently, CCl
4
-enhanced mRNA expression levels
of brotic genes including
α
-SMA, collagen types I (Col1a1), collagen
types III (Col3a1) and TGF-β1, were markedly reversed by Mul treat-
ments in a dose-dependent manner (Fig. 3E). Western blotting demon-
strated that the protein expression levels of TGF-β1, p-SMAD2/3 and
α
-SMA in liver were strongly down-regulated by Mul in CCl
4
-treated
mice compared with the model group (Fig. 3F). Therefore, Mul could
restrain TGF-β1/SMADs signaling to meliorate hepatic brosis.
C. Ge et al.
Redox Biology 51 (2022) 102274
5
3.3. Mulberrin ameliorates hepatic inammation in CCl
4
-challenged mice
Inammatory response was then investigated to reveal whether it
might be a therapeutic target for Mul to perform its protective function.
F4/80 and CD68 are macrophage markers and indicate the inltration of
macrophages, which is a sign of inammation [33]. IHC staining showed
that CCl
4
injection signicantly up-regulated the positive expression of
F4/80 and CD68 in liver sections, and these effects were remarkably
ameliorated in mice co-treated with Mul (Fig. 4A–C). By ELISA analysis,
we found that Mul treatments strongly reduced the systematic
inammatory cytokines and chemokine in CCl
4
-challenged mice, as
proved by the decreased serum IL-1β, TNF-
α
, IL-6 and CXCL-10 contents
(Fig. 4D). Consistently, CCl
4
-elevated expression levels of inammatory
genes such as IL-1β, TNF-
α
, IL-6, IL-18, MCP-1 and CXCL-10 were
signicantly abolished in mice with Mul administration (Fig. 4E). NF-κB
signaling is a classic pathway for inammatory response induction
through promoting the releases of pro-inammatory factors [9]. West-
ern blotting analysis subsequently showed that CCl
4
strongly promoted
the phosphorylation of IKK
α
, IκB
α
, and NF-κB/P65 in liver tissues, which
were, however, efciently abolished by Mul treatments. Meanwhile,
Fig. 2. Mulberrin reduces liver brosis in CCl
4
-treated mice. (A) H&E, Sirius red and Masson’s Trichrome staining of liver sections. (B) Quantication for brosis
score. (C,D) Quantication for brotic areas by Sirius red and Masson’s Trichrome staining, respectively. Serum contents of (E) hyaluronic acid, (F) procollagen III
(PC III) (G) and Collagen IV were measured. Representative data were expressed as mean ±SEM (n =5 for histological analysis, or 10 for biological analysis per
group). Magnication: ×100. **P <0.01 and ***P <0.001 vs the Oil/Veh group; +P <0.05, ++P <0.01 and +++P <0.001 vs the CCl
4
/Veh group. (For
interpretation of the references to color in this gure legend, the reader is referred to the Web version of this article.)
C. Ge et al.
Redox Biology 51 (2022) 102274
6
Mul restored total IκB
α
expression in liver of CCl
4
-treated mice (Fig. 4F),
indicating the suppressed activation of NF-κB signaling pathway.
Together, these ndings illustrated that Mul could repress inammatory
response induced by CCl
4
through retarding NF-κB signaling.
3.4. Mulberrin mediates TRIM31/NLRP3 signaling pathway in liver of
CCl
4
-treated mice
TRIM31/NLRP3 signaling is involved in the mediation of
Fig. 3. Mulberrin inhibits CCl
4
-induced activation of TGF-β1/SMADs signaling in liver of mice. IF staining for (A)
α
-SMA and (B) Col-I expression in liver
sections from each group of mice. Quantication for positive uorescent intensity of (C)
α
-SMA and (D) Col-I following IF assays. (E) RT-qPCR results for brogenic
genes including
α
-SMA, Col1a1, Col3a1 and TGF-β1 in liver of all groups of mice. (F) Western blotting analysis for TGF-β1, p-SMAD2/3 and
α
-SMA protein expression
in liver tissues of all groups of mice. Representative data were expressed as mean ±SEM (n =3 per group). Magnication: ×200. ***P <0.001 vs the Oil/Veh group;
+P <0.05 and ++P <0.01 vs the CCl
4
/Veh group.
C. Ge et al.
Redox Biology 51 (2022) 102274
7
inammation under various stimuli [13,14], and was previously re-
ported to regulate NF-κB pathway to subsequently control various
cellular events, particularly inammatory response [9]. Here, we
notably found that CCl
4
challenge signicantly reduced the expression
of TRIM31 in liver samples, evidenced by the weakened uorescent
intensity; however, such effect was greatly abolished by Mul treatments
(Fig. 5A and B). Consistently, RT-qPCR results conrmed that Mul
administration markedly up-regulated TRIM31 expression levels in liver
of CCl
4
-challenged mice. On the contrary, hepatic NLRP3 and its
down-streaming signal ASC stimulated by CCl
4
were strongly decreased
by Mul (Fig. 5C). As expected, the protein expression levels of TRIM31
restrained by CCl
4
were efciently restored in mice with Mul adminis-
tration. However, the expression levels of NLRP3 and its associated
inammasome were greatly diminished by Mul in liver of CCl
4
-chal-
lenged mice, as evidenced by the markedly decreased NLRP3, ASC,
Caspase-1, mIL-1β and mIL-18, which were all comparable with the
brosis model group (Fig. 5D and E). These ndings indicated that Mul
could mediate TRIM31/NLRP3 signaling to mitigate hepatic brosis.
3.5. Mulberrin restrains hepatic oxidative stress in CCl
4
-challenged mice
Oxidative stress is another key for the progression of liver brosis
[18] and was thus investigated. As shown in Fig. 6A–C, CCl
4
challenge
signicantly increased the oxidative stress hallmarks including H
2
O
2
,
MDA and TBARS in liver of brotic mice, while being dose-dependently
reduced by Mul treatments. In contrast, antioxidants such as CAT, GSH
and SOD were strongly restored in liver of Mul-treated mice after CCl
4
challenge (Fig. 6D–F). IF staining conrmed that hepatic lipid peroxi-
dation product 4-HNE was highly induced by CCl
4
, whereas being
greatly abrogated in mice with Mul supplementation (Fig. 6G and H).
Fig. 4. Mulberrin ameliorates hepatic inammation in CCl
4
-challenged mice. (A) IHC staining for F4/80 and CD68 expression in hepatic sections. (B) F4/80-
and (C) CD68-positive areas were quantied after IHC assays. (D) ELISA analysis for serum contents of IL-1β, TNF-
α
, IL-6 and CXCL-10 from the shown groups of
mice. (E) RT-qPCR analysis for inammatory factors including IL-1β, TNF-
α
, IL-6, IL-18, MCP-1 and CXCL-10 in liver tissues from the shown groups of mice. (F)
Western blotting analysis for hepatic p-IKK
α
, p-IκB
α
, IκB
α
and p-NF-κB/p65 protein expression levels. Representative data were expressed as mean ±SEM (n =3 for
IHC, RT-qPCR and western blotting assays, or 8 for biological analysis per group). Magnication: ×200. **P <0.01 and ***P <0.001 vs the Oil/Veh group; +P <
0.05 and ++P <0.01 vs the CCl
4
/Veh group.
C. Ge et al.
Redox Biology 51 (2022) 102274
8
What’s more, compared with CCl
4
/Veh group, mice co-treated with Mul
exhibited higher expression levels of antioxidant genes including HO-1,
NQO1, GCLC and GCLM (Fig. 6I). NAD(P)H oxidase is a key source of
ROS production and plays an essential role in liver injury and brosis
[34]. The mRNA expression levels of NAD(P)H oxidase subunits
including NOX1, NOX2, NOX4 and p22
phox
were highly up-regulated in
liver of CCl
4
-challenged mice, which were, however, efciently miti-
gated by Mul treatments (Fig. 6J). IHC and western blotting assays
showed that CCl
4
injection clearly reduced Nrf2 nuclear translocation in
liver tissues compared with the Oil/Veh group. Of note, Mul markedly
rescued nuclear Nrf2 expression in hepatic samples compared with
CCl
4
/Veh group (Fig. 6K-M), indicating the improved activation of Nrf2
signaling. Cytochrome P450 2E1 (CYP2E1) aggravates oxidative stress
and ROS generation, leading to various kinds of liver cell damage and
death and brotic liver progression [35]. As expected, we found that
CCl
4
-treated mice exhibited higher CYP2E1 expression levels than that
of the Oil/Veh group, while being signicantly abrogated by Mul
consumption via a dose-dependent manner (Fig. 6N). These results
elucidated that Mul could facilitate Nrf2 activation to attenuate hepatic
oxidative stress under brotic pressure.
3.6. Mulberrin reduces the expression of brosis markers in TGF-β1-
activated LX-2 cells
Our in vivo results indicated the protective effects of Mul against
brosis, inammation and oxidative stress in CCl
4
-treated mice. To
conrm the function of Mul, in vitro experiments were subsequently
performed using human or mouse hepatocyte and HSC cell lines. At rst,
CCK-8 analysis was used to examine the safe use of Mul. As displayed in
Supplementary Figs. 1A and B, Mul treatments at various concentrations
had no signicant inuences on the survival of human HSC cell line LX-2
and hepatocyte L02, respectively, indicating the non-cytotoxicity of
Mul. Considering that TGF-β1 plays an important role in hepatic brosis
and is an inducer of the brotic response [36], TGF-β1 was then exposed
Fig. 5. Mulberrin mediates TRIM31/NLRP3 signaling pathway in liver of CCl
4
-treated mice. (A) IF staining for TRIM31 in hepatic sections from all groups of
mice. (B) TRIM31-positive uorescent intensity was quantied following IF staining. (C) RT-qPCR analysis for hepatic TRIM31, NLRP3 and ASC gene expression
levels. (D,E) Western blotting analysis for TRIM31, NLRP3, ASC, Caspase-1, mature IL-1β (mIL-1β) and mature IL-18 (mIL-18) protein expression levels in liver tissues
of all groups of mice. Representative data were expressed as mean ±SEM (n =3 per group). Magnication: ×200. **P <0.01 and ***P <0.001 vs the Oil/Veh
group; +P <0.05, ++P <0.01 and +++P <0.001 vs the CCl
4
/Veh group.
C. Ge et al.
Redox Biology 51 (2022) 102274
9
Fig. 6. Mulberrin restrains hepatic oxidative stress in CCl
4
-challenged mice. Examination for (A) H
2
O
2
, (B) MDA, (C) TBARS, (D) CAT, (E) GSH and (F) SOD in
liver tissues of all groups of mice. (G) IF staining for 4-HNE expression in hepatic sections. (H) Positive uorescent intensity of 4-HNE was quantied after IF analysis.
(I) RT-qPCR analysis for antioxidants including HO-1, NQO1, GCLC and GCLM in liver tissues. (J) RT-qPCR results for oxidative stress markers NOX1, NOX2, NOX4
and p22
phox
gene expression levels in liver samples. (K) IHC staining for hepatic Nrf2 expression levels. (L) Nrf2-positive nucleus was quantied following IHC assay.
(M) Nuclear and cytoplastic Nrf2 protein expression levels were measured using western blotting analysis. (N) Western blotting results for hepatic CYP2E1 protein
expression levels. Representative data were expressed as mean ±SEM (n =3 for IHC, IF, RT-qPCR and western blotting assays, or 8 for biological analysis per group).
Magnication: ×200. **P <0.01 and ***P <0.001 vs the Oil/Veh group;
+
P <0.05 and
++
P <0.01 vs the CCl
4
/Veh group.
C. Ge et al.
Redox Biology 51 (2022) 102274
10
to HSCs for its stimulation. RT-qPCR results showed that the expression
levels of brosis markers
α
-SMA, Col1a1, Col3a1 and Fibronectin were
markedly up-regulated by TGF-β1 in LX-2 cells, which were signicantly
and dose-dependently decreased by Mul, particularly starting from 10
μ
M (Fig. 7A). Thereafter, 25
μ
M (low dosage), 50
μ
M (moderate con-
centration) and 100
μ
M (high dosage) of Mul were used for further in
vitro studies. CCK-8 results conrmed the safe use of 100
μ
M Mul
treatment from 0 to 72 h both in LX-2 and L02 cells (Supplementary
Figs. 1C and D). IF analysis then validated that Mul treatments reduced
α
-SMA expression in LX-2 cells after TGF-β1 stimulation (Fig. 7B and C).
Finally, western blotting results demonstrated that compared with
TGF-β1/Veh group, Mul-treated cells exhibited lower protein expression
levels of
α
-SMA and p-SMAD2/3 in LX-2 cells following TGF-β1 stimu-
lation (Fig. 7D). To verify the suppressive effects of Mul on brosis in
vitro, primary mouse HSCs were isolated and employed. Morphology of
the isolated primary mouse HSCs was displayed in Supplementary
Fig. 1E. CCK-8 results conrmed the safety of Mul (100
μ
M) exposure to
primary HSCs for 24 h (Supplementary Figs. 1F and G). As expected,
TGF-β1 stimulation signicantly increased the expression of
α
-SMA,
Col1a1, Col3a1 and Fibronectin in primary HSCs, while being strongly
abolished upon Mul co-culture (Supplementary Fig. 2A). IF staining and
western blotting results conrmed that TGF-β1-induced elevation of
α
-SMA and p-SMAD2/3 in primary mouse HSCs was highly eliminated
after Mul incubation (Supplementary Figs. 2B–D).
Inammatory response and oxidative stress in HSCs are involved in
brosis progression [37,38], and thus were investigated in LX-2 cells or
primary HSCs under TGF-β1 stimuli. As shown in Fig. 7E, TGF-β1
stimulation led to inammatory response in LX-2 cells, proved by the
markedly enhanced gene expression levels of TNF-
α
, IL-1β, IL-6, IL-18,
MCP-1 and CXCL10; however, such event caused by TGF-β1 was
signicantly abrogated following Mul exposure. Such anti-inammatory
effects mediated by Mul were validated in mouse primary HSCs upon
TGF-β1 stimulation (Supplementary Fig. 2E). Moreover, HO-1 and
NQO1 gene expression levels were highly decreased in LX-2 cells and
mouse primary HSCs stimulated by TGF-β1, while being efciently
reversed upon Mul co-treatment. In contrast, NOX2 and NOX4 expres-
sion levels were greatly abolished by Mul in a dose-dependent fashion
(Fig. 7F and Supplementary Fig. 2F). In response to TGF-β1, nuclear Nrf2
protein expression levels were signicantly weakened, whereas being
markedly rescued after Mul exposure. Opposite prole was detected in
the expression of cytoplastic Nrf2 both in LX-2 and primary mouse HSCs
(Fig. 7G and Supplementary Fig. 2G). These in vitro ndings suggested
that Mul could restrain the activation, inammation and oxidative stress
in HSCs.
Given the crucial role of inammatory response in the modulation of
HSCs activation, LX-2 cells were then exposed to recombinant human
TNF-
α
(rTNF-
α
) and IL-1β (rIL-1β) to further examine the effects of Mul
on inammation-caused brosis in vitro. As shown in Supplementary
Figs. 3A–D, we found that rTNF-
α
and rIL-1β treatment signicantly
increased
α
-SMA expression in LX-2 cells, proved by the stronger uo-
rescent intensity, which were, however, markedly abolished after Mul
treatment. Consistently, in response to rTNF-
α
and rIL-1β stimulation,
brosis markers
α
-SMA, Col1a1, Col3a1 and Fibronectin were greatly
induced, whereas being remarkably diminished in LX-2 cells with Mul
exposure (Supplementary Figs. 3E and F). As expected, the protein
expression levels of
α
-SMA, p-SMAD2 and p-SMAD3 enhanced by rTNF-
α
and rIL-1β were dramatically ameliorated upon Mul treatment (Sup-
plementary Figs. 3G and H). These in vitro data suggested that inam-
matory factors contributed to HSCs activation, which could be
attenuated by Mul.
3.7. Mulberrin mitigates inammatory response through mediating
TRIM31/NLRP3 signaling in L02 cells
Inammatory response in hepatocytes participates in HSCs activa-
tion and liver brosis [3,17,39], and thus was explored by the use of L02
cells and the isolated primary mouse hepatocytes (Supplementary
Fig. 4A). CCK-8 results suggested that 100
μ
M of Mul treatment for 24 h
was safe for primary mouse hepatocytes culture (Supplementary Figs. 4B
and C). We found that TGF-β1 stimulation signicantly increased the
expression of IL-1β, TNF-
α
, IL-6, IL-18, MCP-1 and CXCL-10 in L02 cells
and the mouse primary hepatocytes, and such effect was remarkably
abolished upon Mul co-incubation (Fig. 8A and Supplementary Fig. 5A).
Consistently, lower contents of IL-1β and TNF-
α
in supernatants were
detected collected from Mul-exposed L02 cells and the mouse primary
hepatocytes after TGF-β1 treatment, which was comparable with
TGF-β1/Veh group (Fig. 8B and Supplementary Fig. 5B). LPS is
frequently used to induce inammatory response [40]. As expected,
LPS-enhanced IL-1β, TNF-
α
, IL-6, IL-18, MCP-1 and CXCL-10 in L02 cells
or supernatants were considerably mitigated by Mul (Fig. 8C and D),
conrming the anti-inammatory bioactivity of Mul in vitro. We also
found that Mul-treated L02 cells exhibited markedly decreased TGF-β1
both in the collected supernatants and cells compared with the LPS
group (Supplementary Figs. 6A and B). IF results showed that
TRIM31-positive expression was markedly restrained by TGF-β1 or LPS
in L02 cells and the isolated primary murine hepatocytes, which were,
however, strongly restored by Mul treatments (Fig. 8E–G, and Supple-
mentary Figs. 5C and D). In line with in vivo results, RT-qPCR and
western blotting analysis veried that in response to TGF-β1 or LPS, Mul
treatments signicantly improved TRIM31 and total IκB
α
expression
levels, while decreased NLRP3, ASC, Caspase-1, p-IκB
α
, p-NF-κB, mIL-1β
and mIL-18 expression levels in L02 cells and mouse primary hepato-
cytes (Fig. 8H and I, and Supplementary Figs. 5E–G). These results
illustrated that Mul suppressed inammatory response by mediating
TRIM31/NLRP3 signaling in vitro.
To explore whether improving TRIM31 was required for Mul to
suppress inammation, TRIM31 was then silenced in L02 cells by
transfecting with its siRNAs. As shown in Supplementary Figs. 7A and B,
transfection efcacy of siTRIM31 was conrmed by RT-qPCR and
western blotting assays. Because siTRIM31-1# exerted the strongest
inhibitory effect on TRIM31, and thus was selected for subsequent in
vitro analysis. RT-qPCR results indicated that TGF-β1-increased expres-
sion of IL-1β, TNF-
α
, IL-6, IL-18, MCP-1 and CXCL-10 was further
accelerated upon TRIM31 knockdown. Importantly, Mul-ameliorated
expression of these inammatory factors was completely abolished by
siTRIM31 (Supplementary Fig. 8A). IF and western blotting results
conrmed that after TGF-β1 stimulation, Mul-improved TRIM31
expression was strongly abolished in L02 cells with TRIM31 silence
(Supplementary Figs. 8B–D). As expected, under TGF-β1 stimuli,
TRIM31 knockdown signicantly abrogated the capacity of Mul to
suppress NLRP3, ASC, Caspase-1, p-NF-κB, mIL-1β and mIL-18 protein
expression levels in L02 cells (Supplementary Fig. 8D). These supporting
data demonstrated that Mul-inhibited hepatocyte inammation was
largely through increasing TRIM31 expression.
3.8. Mulberrin meliorates oxidative stress via improving Nrf2 signaling in
L02 cells
The regulatory role of Mul on oxidative stress and Nrf2 signaling was
further explored in human hepatocytes and the isolated mouse primary
hepatocytes. DCF-DA staining showed that TGF-β1 exposure clearly
elevated ROS production in L02 cells and the mouse primary hepato-
cytes, which was strongly abrogated by Mul treatments. Furthermore,
H
2
O
2
-induced ROS generation was also diminished by Mul (Fig. 9A and
Supplementary Figs. 9A and B), conrming the antioxidant bioactivity
of Mul. Moreover, Mul-incubated L02 cells and primary murine hepa-
tocytes showed lower H
2
O
2
and MDA contents after TGF-β1 or H
2
O
2
stimulation (Fig. 9B and C, and Supplementary Figs. 9C and D). On the
contrary, SOD, CAT and GSH activities were greatly reduced in TGF-β1-
or H
2
O
2
-exposed L02 cells and primary murine hepatocytes, whereas
being efciently rescued by Mul (Fig. 9D–F, and Supplementary
Figs. 9E–G). HO-1, NQO1, GCLC and GCLM gene expression levels
C. Ge et al.
Redox Biology 51 (2022) 102274
11
Fig. 7. Mulberrin reduces the expression of brosis markers in TGF-β1-activated LX-2 cells. (A) After stimulation with TGF-β1 (10 ng/ml) for 24 h in the
absence or presence of the indicated concentrations of Mul, LX-2 cells were collected for RT-qPCR analysis of
α
-SMA, Col1a1, Col3a1 and Fibronectin. (B–G) LX-2
cells were subjected to TGF-β1 (10 ng/ml) treatment for 24 h with or without Mul treatments (25, 50 or 100
μ
M). Then, all cells were harvested for studies as follows.
(B) IF staining for
α
-SMA expression in LX-2 cells. (C) Quantication for
α
-SMA positive uorescent intensity was exhibited. (D) Western blotting results for
α
-SMA, p-
SMAD2/3 protein expression levels in LX-2 cells. (E) RT-qPCR analysis for inammatory factors including TNF-
α
, IL-1β, IL-6, IL-18, MCP-1 and CXCL10, and (F)
oxidative stress related molecules such as HO-1, NQO1, NOX2 and NOX4 in LX-2 cells. (G) Western blotting results for nuclear and cytoplastic Nrf2 protein expression
levels in LX-2 cells. Representative data were expressed as mean ±SEM (n =3 per group). Scale bar, 10
μ
m. ***P <0.001 vs the Ctrl/Veh group;
+
P <0.05 and
++
P
<0.01 vs the TGF-β1/Veh group. Mul-L, 25
μ
M; Mul-M, 50
μ
M; and Mul-H, 100
μ
M.
C. Ge et al.
Redox Biology 51 (2022) 102274
12
Fig. 8. Mulberrin mitigates inammatory response through mediating TRIM31/NLRP3 signaling in L02 cells. L02 cells were incubated with TGF-β1 (10 ng/
ml) or LPS (100 ng/ml) for 24 h in the presence or absence of Mul (25, 50 or 100
μ
M). Subsequently, all cells and supernatants were harvested for experiments as
follows. (A) RT-qPCR analysis for inammatory factors including TNF-
α
, IL-1β, IL-6, IL-18, MCP-1 and CXCL10 in L02 cells. (B) ELISA analysis for TNF-
α
and IL-1β in
the collected supernatants. (C) Inammatory gene markers as shown were measured using RT-qPCR assay. (D) TNF-
α
and IL-1β contents in the collected supernatants
were examined using ELISA analysis. (E) TRIM31 expression in L02 cells by IF staining. (F) TRIM31-positive uorescent intensity was quantied following IF assay.
Rt-qPCR results for (G) TRIM31, (H) NLRP3 and ASC gene expression levels. (I) Western blotting results for the protein expression levels of TRIM31, NLRP3, ASC,
Caspase-1, p-IκB
α
, IκB
α
, p-NF-κB/P65, mIL-1β and mIL-18 in L02 cells. Representative data were expressed as mean ±SEM (n =3 for IF, RT-qPCR and western
blotting assays, or 6 for ELISA analysis per group). Scale bar, 10
μ
m. **P <0.01 and ***P <0.001 vs the Ctrl/Veh group;
+
P <0.05 and
++
P <0.01 vs the TGF-β1/
Veh or LPS/Veh group;
#
P <0.05,
##
P <0.01 and
###
P <0.001.
C. Ge et al.
Redox Biology 51 (2022) 102274
13
decreased by TGF-β1 or H
2
O
2
were considerably abolished by Mul co-
incubation in L02 cells or primary murine hepatocytes (Fig. 9G and
Supplementary Fig. 9H). Nevertheless, NOX1, NOX2, NOX4 and p22
phox
stimulated by TGF-β1 or H
2
O
2
were highly down-regulated by Mul in
L02 cells and primary murine hepatocytes (Fig. 9H and Supplementary
Fig. 9I). IF staining thereafter showed that nuclear Nrf2 was highly
decreased in L02 cells and primary murine hepatocytes after TGF-β1 or
H
2
O
2
exposure, which was reversed upon Mul co-treatment (Fig. 9I and
J, and Supplementary Figs. 9J and K). Western blotting results
conrmed the capacity of Mul to improve Nrf2 nuclear translocation
both in L02 cells and primary murine hepatocytes under brotic or
oxidative stresses (Fig. 9K and Supplementary g. 9L).
To further investigate whether Nrf2 was required for Mul to perform
its antioxidant function, Nrf2 was then knocked down in L02 cells. RT-
qPCR and western blotting results showed that siNrf2-2# showed the
highest transfection efcacy (Supplementary Figs. 7C and D), and thus
Fig. 9. Mulberrin meliorates oxidative stress via improving Nrf2 signaling in L02 cells. L02 cells were subjected to 24 h of TGF-β1 (10 ng/ml) or H
2
O
2
(100
μ
M)
incubation combined with or without Mul (25, 50 or 100
μ
M). Next, all cells were harvested for studies as follows. (A) DCF-DA staining for ROS production in L02
cells. Scale bar, 50
μ
m. Cellular (B) H
2
O
2
, (C) MDA, (D) SOD, (E) CAT and (F) GSH contents or activities were examined. RT-qPCR results for (G) antioxidants
including HO-1, NQO1, GCLC and GCLM, and (H) oxidative stress hallmarks NOX1, NOX2, NOX4 and p22
phox
gene expression levels in L02 cells. (I) IF staining for
Nrf2 expression in L02 cells. Scale bar, 10
μ
m. (J) Quantication for nuclear Nrf2-positive uorescent intensity was exhibited. (K) Western blotting analysis for
nuclear and cytoplastic Nrf2 protein expression levels in L02 cells. Representative data were expressed as mean ±SEM (n =3 for DCF-DA, IF, RT-qPCR and western
blotting assays, or 6 for biological analysis per group). **P <0.01 and ***P <0.001 vs the Ctrl/Veh group;
#
P <0.05 and
##
P <0.01.
C. Ge et al.
Redox Biology 51 (2022) 102274
14
was chosen for subsequent analysis. DCF-DA staining indicated that in
TGF-β1-stimulated L02 cells, Mul-ameliorated ROS generation was
signicantly diminished upon Nrf2 silence (Supplementary Fig. 10A).
Consistently, H
2
O
2
and MDA levels restrained by Mul were also
restrengthened by siNrf2 in TGF-β1-exposed L02 cells (Supplementary
Figs. 10B and C). In contrast, siNrf2 markedly eliminated the capacity of
Mul to improve SOD and GSH levels in L02 cells under TGF-β1 stimuli
(Supplementary Figs. 10D and E), along with decreased HO-1 and NQO1
expression levels (Supplementary Fig. 10F). In hepatocytes with TGF-β1
exposure, Mul-suppressed NOX2 and NOX4 gene expression levels were
almost abolished upon Nrf2 deletion (Supplementary Fig. 10G). Western
blotting conrmed that after TGF-β1 stimulation, Nrf2 was almost un-
detectable both in nucleus and cytoplasm either with or without Mul
exposure (Supplementary Fig. 10H). Taken together, all these data
depicted that Mul exerted antioxidant function mainly through
improving Nrf2 signaling.
3.9. Effects of mulberrin on brosis markers of LX-2 cells cultured in
conditional medium from TGF-β1-treated hepatocytes
Given that the inammatory response and oxidative stress in hepa-
tocytes are crucial for HSCs activation and brosis, conditional medium
(CM) derived from L02 cells was collected and subjected to LX-2 culture
to further explore the underlying mechanisms (Fig. 10A). CM from TGF-
β1-treated L02 cells markedly increased the expression of
α
-SMA, Col1a1
and Col3a1 in LX-2 cells, which was signicantly ameliorated by Mul
(Fig. 10B). IF staining by
α
-SMA indicated that CM derived from TGF-β1-
exposed L02 cells led to HSCs activation, while being greatly amelio-
rated upon Mul treatment (Fig. 10C and D). CM obtained from TGF-β1-
stimulated L02 cells signicantly promoted the TGF-β1/SMADs
signaling in LX-2 cells, which was almost diminished after Mul treat-
ment, as indicated by the decreased expression of TGF-β1, p-SMAD2/3
and
α
-SMA (Fig. 10E). These ndings elucidated that under brotic
stresses, hepatocyte damage contributed to HSCs activation, and such
effect could be abolished by Mul.
3.10. Mulberrin inhibits brosis via the improvements of TRIM31 and
Nrf2 signaling pathways
Persistent inammation can promote HSCs activation and liver
brosis [7]. We therefore explored the effect of Mul-mediated hepato-
cyte inammation on HSCs activation. To study the cell- to- cell cross-
talk between hepatocytes and HSCs, we established an in vitro CM
system derived from L02 cells, which was then collected for LX-2 cell
culture [40]. We found that CM derived from TGF-β1-incubated L02
cells indeed promoted
α
-SMA, Col1a1, Col3a1 and Fibronectin expres-
sion levels in LX-2 cells, which were, however, accelerated upon
TRIM31 and Nrf2 deletion. Additionally, in LX-2 cells exposed to CM
from TGF-β1-stimulated L02 cells, Mul-inhibited expression of these
brotic genes was considerably abolished by siTRIM31 and siNrf2
(Fig. 11A). Activation of LX-2 cells provoked by CM-TGF-β1 was further
aggravated when TRIM31 and Nrf2 was ablated in L02 cells, as proved
by the increased expression of
α
-SMA in LX-2 cells. Meanwhile,
siTRIM31 and siNrf2 signicantly eliminated the inhibitory effect of Mul
on
α
-SMA expression in LX-2 cells cultured in CM derived from
TGF-β1-treated L02 cells (Fig. 11B and C). Under brotic pressure, CM
derived from siTRIM31 and siNrf2 L02 cells markedly exacerbated
TGF-β1, p-SMAD2/3 and
α
-SMA expression in LX-2 cells. What’s more,
CM obtained from TGF-β1-incubated L02 cells with TRIM31 or Nrf2
deletion signicantly abolished the capacity of Mul to reduce the
Fig. 10. Effects of mulberrin on brosis markers of LX-2 cells cultured in conditional medium from TGF-β1-treated hepatocytes. L02 cells were incubated
with or without TGF-β1 (10 ng/ml) for 24 h. Then, the culture media was collected and mixed with fresh media at 1:1 ratio, which was served as the conditional
medium (CM). Next, LX-2 cells were incubated in the CM with or without Mul-H (100
μ
M) treatment for another 24 h. Finally, all LX-2 cells were collected for studies
as follows. (A) Scheme for the in vitro experimental design. (B) RT-qPCR results for brosis markers including
α
-SMA, Col1a1 and Col3a1. (C,D) IF staining for
α
-SMA
expression in LX-2 cells. Positive
α
-SMA uorescent intensity was quantied. (E) Western blotting analysis for TGF-β1,
α
-SMA, p-SMAD2/3 protein expression levels.
Representative data were expressed as mean ±SEM (n =3 per group). Scale bar, 10
μ
m. **P <0.01 and ***P <0.001 vs the CM-Ctrl/Veh group;
++
P <0.01 vs the
CM-TGF-β1/Veh group.
C. Ge et al.
Redox Biology 51 (2022) 102274
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(caption on next page)
C. Ge et al.
Redox Biology 51 (2022) 102274
16
expression of TGF-β1, p-SMAD2/3 and
α
-SMA in LX-2 cells (Fig. 11D and
E). Collectively, these ndings demonstrated that the TRIM31-Nrf2 axis
in hepatocytes was crucial for Mul to restrain HSCs activation in vitro.
Moreover, CM derived from TGF-β1-incubated L02 cells led to higher
contents of IL-1β, TNF-
α
, IL-18 and CXCL-10 in supernatants, which
were signicantly accelerated upon TRIM31 and Nrf2 knockdown.
Similarly, Mul-reduced concentrations of these inammatory factors
were markedly restrengthened by siTRIM31 or siNrf2 (Fig. 11F).
Western blotting suggested that CM-TGF-β1 exposure strongly reduced
TRIM31 and nuclear Nrf2 protein expression levels in LX-2 cells, and
such effects were further weakened upon TRIM31 and Nrf2 deletion.
Mul-improved expression of TRIM31 and Nrf2 was also signicantly
abolished when TRIM31 and Nrf2 were ablated. In contrast, NLRP3,
ASC, Caspase-1 and p-NF-κB protein expression levels potentiated by
CM-TGF-β1 were further exacerbated by siTRIM31 and siNrf2. Consis-
tently, Mul-inhibited expression of these signals was strongly restored
Fig. 11. Mulberrin inhibits brosis via the improvements of TRIM31 and Nrf2 signaling pathways. L02 cells were transfected with siTRIM31-1# or siNRf2-2#
for 24 h for the knockdown of TRIM31 and Nrf2, respectively, and were then stimulated by TGF-β1 (10 ng/ml) for another 24 h in the absence or presence of Mul-H
(100
μ
M). The cultured medium was then collected, and mixed with fresh medium at 1:1 ratio, served as CM. The CM was then subjected to the culture of LX-2 cells.
After 24 h, LX-2 cells and the cultured supernatants were harvested for the following studies. (A) RT-qPCR results for brosis markers including
α
-SMA, Col1a1,
Col3a1 and Fibronectin. (B,C) IF staining for
α
-SMA expression in LX-2 cells. Quantication for positive
α
-SMA uorescent intensity was shown. (D,E) Western
blotting results for TGF-β1,
α
-SMA, p-SMAD2/3 protein expression levels in LX-2 cells. (F) ELISA analysis for IL-1β, TNF-
α
, IL-18 and CXCL-10 in the collected
supernatants. (G) Western blotting analysis for protein expression levels of cellular TRIM31, NLRP3, ASC, Caspase-1 and p-NF-κB/P65, and nuclear Nrf2. Repre-
sentative data were expressed as mean ±SEM (n =3 per group). Scale bar, 10
μ
m. **P <0.01 and ***P <0.001 vs the CM-Ctrl group;
+
P <0.05 and
++
P <0.01 vs
the CM-TGF-β1 group;
#
P <0.05 and
##
P <0.01.
Fig. 12. Hepatic protective effects of mulberrin are TRIM31-dependent in vivo. (A) Body weights of TRIM31
Flox
or TRIM31
Hep-cKO
mice were recorded. Serum
(B) TBIL, (C) LDH levels, (D) ALT, AST, ALP contents, (E) hyaluronic acid, (F) PC III, and (G) Collagen IV levels were examined. Representative data were expressed as
mean ±SEM (n =9 per group).
#
P <0.05,
##
P <0.01 and
###
P <0.001; ns, no signicant difference.
C. Ge et al.
Redox Biology 51 (2022) 102274
17
upon TRIM31 and Nrf2 silence (Fig. 11G). The expression changes of
TRIM31/NLRP3 and Nrf2 signaling in LX-2 cells might be attributed to
the severer inammatory conditions.
3.11. Anti-brosis effects of mulberrin are TRIM31-dependent in CCl
4
-
treated mice
To conrm the interaction between hepatocytes and HSCs mediated
by Mul under brotic stresses in vivo, the hepatocyte-specic TRIM31
knockout (TRIM31
Hep-cKO
) mice were generated (Supplementary
Fig. 11A). Western blotting results conrmed that TRIM31 was not
detected in liver of TRIM31
Hep-cKO
mice (Supplementary Figs. 11B and
C). H&E staining demonstrated that there were no evident histological
changes in liver sections between TRIM31
Flox
and TRIM31
Hep-cKO
groups
of mice (Supplementary Fig. 11D). IF staining validated the successful
TRIM31 deletion in liver of TRIM31
Hep-cKO
mice (Supplementary
Fig. 11E). In response to CCl
4
, Mul improved the body weights of mice,
which was, however, abolished in TRIM31
Hep-cKO
mice (Fig. 12A). As
shown in Fig. 12B–D, Mul failed to mitigate the elevation of serum TBIL,
LDH, ALT, AST and ALP in the CCl
4
/TRIM31
Hep-cKO
mice. Likewise, the
effects of Mul to reduce brotic parameters including hyaluronic acid,
PC III and Collagen IV levels were impeded in TRIM31
Hep-cKO
mice after
Fig. 13. Anti-brosis effects of mulberrin are TRIM31-dependent in CCl
4
-treated mice. (A) H&E, Sirius red and Masson’s Trichrome staining of liver sections
from TRIM31
Flox
or TRIM31
Hep-cKO
mice. Quantication results for (B) brosis scores, (C) Sirius red- and (D) Masson’s Trichrome-positive staining areas were
exhibited. Representative data were expressed as mean ±SEM (n =6 or 9 per group). Magnication: ×100.
#
P <0.05,
##
P <0.01 and
###
P <0.001; ns, no
signicant difference. (For interpretation of the references to color in this gure legend, the reader is referred to the Web version of this article.)
C. Ge et al.
Redox Biology 51 (2022) 102274
18
CCl
4
challenge (Fig. 12E–G). Furthermore, histological staining sug-
gested that Mul treatment failed to restrain the collagen deposition in
liver of TRIM31
Hep-cKO
mice induced by CCl
4
, along with restored
brosis scores (Fig. 13A–D). IF staining conrmed that after CCl
4
injection, the function of Mul to reduce
α
-SMA and Col-I accumulation in
liver sections was almost diminished upon hepatocyte-specic TRIM31
knockout (Fig. 14A–D). Consistently, Mul failed to repress
α
-SMA,
Col1a1, Col3a1 and TGF-β1 gene expression levels in liver of CCl
4
-
Fig. 14. Mulberrin suppresses collagen deposition by improving TRIM31 in CCl
4
-challenged mice. IF staining for (A)
α
-SMA and (B) Col-I expression in hepatic
sections from TRIM31
Flox
or TRIM31
Hep-cKO
mice. Quantication for positive uorescent intensity of (C)
α
-SMA and (D) Col-I following IF assays. (E) RT-qPCR results
for
α
-SMA, Col1a1, Col3a1 and TGF-β1 in liver of all groups of mice. (F) Western blotting analysis for TGF-β1, p-SMAD2/3 and
α
-SMA protein expression in liver
tissues of all groups of mice. Representative data were expressed as mean ±SEM (n =4 per group). Magnication: ×200.
#
P <0.05,
##
P <0.01 and
###
P <0.001;
ns, no signicant difference.
C. Ge et al.
Redox Biology 51 (2022) 102274
19
treated TRIM31
Hep-cKO
mice (Fig. 14E). Similarly, the effects of Mul to
decrease TGF-β1, p-SMAD2/3 and
α
-SMA protein expression levels were
also completely diminished in CCl
4
/TRIM31
Hep-cKO
mice (Fig. 14F).
Together, these ndings elucidated that Mul might exert its anti-brotic
effects through improvement of hepatocyte TRIM31 signaling.
3.12. Anti-inammatory effects of mulberrin are TRIM31-dependent in
CCl
4
-treated mice
In this regard, IHC staining suggested that in response to CCl
4
stimuli, Mul failed to mitigate F4/80 and CD68 positive expression in
liver sections of TRIM31
Hep-cKO
mice (Fig. 15A and B). TRIM31
Hep-cKO
also abolished the capacity of Mul to reduce serum IL-1β, TNF-
α
and IL-6
contents in CCl
4
-challenged mice (Fig. 15C). Consistently, after CCl
4
Fig. 15. Anti-inammatory actions of mulberrin in liver brosis mice regulated by TRIM31 in vivo. (A) IHC staining for F4/80 and CD68 expression in hepatic
sections from TRIM31
Flox
or TRIM31
Hep-cKO
mice. (B) F4/80- and CD68-positive areas were quantied after IHC assays. (C) ELISA analysis for serum contents of IL-1β,
TNF-
α
and IL-6 from the shown groups of mice. (E) RT-qPCR analysis for IL-1β, TNF-
α
, IL-6, IL-18, MCP-1 and CXCL-10 in liver tissues from the shown groups of mice.
(F) Western blotting analysis for hepatic NLRP3, ASC, Caspase-1, p-IKK
α
, p-IκB
α
, IκB
α
, p-NF-κB/p65, mIL-1β and mIL-18 protein expression levels. Representative
data were expressed as mean ±SEM (n =4 for IHC, RT-qPCR and western blotting assays, or 9 for biological analysis per group). Magnication: ×200.
#
P <0.05,
##
P <0.01 and
###
P <0.001; ns, no signicant difference.
C. Ge et al.
Redox Biology 51 (2022) 102274
20
injection, hepatic IL-1β, TNF-
α
, IL-6, IL-18, MCP-1 and CXCL-10 gene
expression levels were also failed to be diminished by Mul upon
hepatocyte-specic TRIM31 deletion (Fig. 15D). As expected, Mul lost
its inhibitory effects on NLRP3, ASC, Caspase-1, p-IKK
α
, p-IκB
α
, p-NF-
κB, mIL-1β and mIL-18 protein expression levels in liver of CCl
4
/
TRIM31
Hep-cKO
mice (Fig. 15E). These ndings suggested that hepato-
cyte TRIM31 expression was required for Mul to perform its anti-
inammatory role.
3.13. Anti-brotic effect of mulberrin is Nrf2-dependent in the primary
HSCs
To further explore whether Nrf2 in hepatocytes was necessary for
Mul to control HSCs activation, primary hepatocytes were isolated from
the wild type (WT) and Nrf2-knockout (KO) mice and were employed in
vitro. Western blotting and IF staining assays conrmed the successful
knockout of Nrf2 in the extracted primary mouse hepatocytes (Supple-
mentary Figs. 12A and B). We then established an in vitro CM system
derived from Nrf2
WT
or Nrf2
KO
primary hepatocytes with or without
Fig. 16. Anti-brotic effect of mulberrin is Nrf2-dependent in the primary HSCs. Primary hepatocytes isolated from Nrf2
WT
or Nrf2
KO
mice were stimulated by
TGF-β1 (10 ng/ml) for 24 h in the absence or presence of Mul-H (100
μ
M). The cultured medium was then collected, and mixed with fresh medium at 1:1 ratio, served
as CM. The CM was then subjected to the culture of primary HSCs. After 24 h, these primary HSCs were collected for the following studies. (A) Scheme for the in vitro
experimental design. (B) Western blotting analysis for nuclear and cytoplastic Nrf2 protein expression levels in the treated primary hepatocytes. (C) IF staining for
α
-SMA expression in the treated primary HSCs. (D) Positive uorescent intensity of
α
-SMA expression was quantied. (E) RT-qPCR results for
α
-SMA, Col1a1, Col3a1
and Fibronectin in primary HSCs. (F) Western blotting analysis for TGF-β1, p-SMAD2/3 and
α
-SMA protein expression levels in primary HSCs. Representative data
were expressed as mean ±SEM (n =4 per group). Scale bar, 50
μ
m.
#
P <0.05,
##
P <0.01 and
###
P <0.001; ns, no signicant difference.
C. Ge et al.
Redox Biology 51 (2022) 102274
21
TGF-β1 stimuli, which was then collected for mouse primary HSCs cul-
ture in the presence or absence of Mul treatment (Fig. 16A). Firstly, we
showed that Mul remarkably improved nucleus Nrf2 expression, but
decreased cytoplastic Nrf2 in primary Nrf2
WT
hepatocytes after TGF-β1
stimulation. As expected, Nrf2 was undetectable in Nrf2
KO
hepatocytes
(Fig. 16B). We then found that Mul failed to reduce
α
-SMA, Col1a1,
Col3a1 and Fibronectin expression levels in primary HSCs cultured in
CM derived from TGF-β1-incubated Nrf2
KO
hepatocytes (Fig. 16C–E).
Consistently, the effects of Mul to down-regulate TGF-β1, p-SMAD2/3
and
α
-SMA protein expression levels were also signicantly diminished
Fig. 17. Exploration of the interaction between TRIM31 and Nrf2. (A,B) The interaction of endogenous TRIM31 with Nrf2 in L02 cells was examined by
immunoprecipitation assay using the indicated antibodies. (C) After TGF-β1 (10 ng/ml) incubation for 24 h, interaction between TRIM31 and Nrf2 in L02 cells was
assessed by immunoprecipitation analysis using anti-TRIM31 antibody and by immunoblotting assay with anti-Nrf2 antibody. (D,E) The interaction of endogenous
TRIM31 with Nrf2 in LX2 cells was explored with immunoprecipitation assay using the shown antibodies. (F) LX2 cells were exposed to TGF-β1 (10 ng/ml) culture for
24 h and were then harvested to explore the interaction between TRIM31 and Nrf2 by immunoprecipitation analysis using anti-TRIM31 antibody and by immu-
noblotting assay with anti-Nrf2 antibody. (G) After siTRIM31 transfection for 24 h, L02 and LX2 cells were stimulated by TGF-β1 (10 ng/ml) for another 24 h. Then,
all cells were collected for western blotting analysis of TRIM31 protein expression levels in whole cells, and Nrf2 expression in nucleus and cytoplasm, respectively;
siCtrl was used as a control. (H) Representative western blot analysis of cellular TRIM31, nuclear and cytoplastic Nrf2 protein expression levels in L02 and LX2 cells
infected with Ad-Flag-TRIM31 for 24 h, followed by an additional 24 h of TGF-β1 (10 ng/ml) exposure. (I) IHC staining for Nrf2 positive expression in hepatic
sections from TRIM31
Flox
or TRIM31
Hep-cKO
mice. (J) Quantication for Nrf2-positive nucleus was shown. (K) Western blotting analysis for nuclear and cytoplastic
Nrf2 protein expression levels in liver of the indicated groups of mice. Representative data were expressed as mean ±SEM (n =4 per group). Magnication: ×200.
#
P <0.05,
##
P <0.01 and
###
P <0.001; ns, no signicant difference.
C. Ge et al.
Redox Biology 51 (2022) 102274
22
in mouse primary HSCs cultured in CM obtained from Nrf2
KO
hepato-
cytes under TGF-β1 stimuli (Fig. 16F). These data elucidated that the
anti-brotic effects of Mul were at least in part attributed to hepatocyte
Nrf2 expression.
Finally, to further explore the underlying molecular mechanisms, we
examined whether there might be a possible interaction between
TRIM31 and Nrf2. Endogenous immunoprecipitation and western blot-
ting assays conrmed the interaction between TRIM31 and Nrf2 in
human hepatocytes (Fig. 17A and B). Additionally, the interaction be-
tween TRIM31 and Nrf2 in L02 cells was weakened upon TGF-β1 stim-
ulation (Fig. 17C). The interaction between TRIM31 and Nrf2 was
veried in human HSCs (Fig. 17D and E), and was receded under TGF-β1
stress (Fig. 17F). Moreover, knockdown of TRIM31 expression by siRNA
further reduced Nrf2 nuclear translocation after TGF-β1 treatment in
L02 and LX2 cells (Fig. 17G). On the contrary, Nrf2 nuclear expression
was up-regulated by TRIM31 in a concentration-dependent manner
(Fig. 17H). As expected, IHC staining showed that Mul-rescued expres-
sion of nuclear Nrf2 in liver of CCl
4
-challenged mice was completely
abrogated upon hepatic-specic TRIM31 knockout (Fig. 17I and J),
which was validated by western blotting assay (Fig. 17K), partially
demonstrating the requirement of TRIM31 for Mul to improve Nrf2
signaling. Together, these ndings suggested that there might be an
interaction between TRIM31 and Nrf2.
4. Discussion
Hepatic inammatory response and oxidative stress contribute to
brosis progression [2,4,7,8,18]. Active TGF-β promotes hepatocyte
injury and the activation of HSCs and broblast, leading to collagen
formation [41]. TGF-β1 binds to its receptor and contributes to the
phosphorylation of mothers against decapentaplegic homolog
SMAD2/3, which is required for their nuclear translocation and tran-
scriptional modulation of their brotic target genes including
α
-SMA,
Col1a1, Col3a1 and Fibronectin, ultimately resulting in collagen depo-
sition and brosis [2,4,7,42]. Unfortunately, liver brosis is still an
intractable disease without effective therapeutic options due to its
intricate pathogenesis [3,5]. Approaches to restrain inammation, ROS
production and collagen deposition are pivotal and promising for the
treatment of liver brosis [10,20,22]. Mulberrin (Mul), as a key
component of Chinese medicine Romulus Mori, has anti-inammatory
and antioxidant properties to meliorate tissue injury [13,14].
Here in our present study, we for the rst time demonstrated that
Mul exerted protective effects against liver brosis both in vivo and in
vitro. Briey, our ndings showed that Mul administration signicantly
reduced hepatic dysfunction and toxicity in CCl
4
-challenged mice,
proved by the reduced serum AST, ALT, AKP, TBIL and LDH levels. We
then revealed that CCl
4
-indcued hepatic collagen deposition was
considerably ameliorated by Mul supplementation through suppressing
TGF-β1/SMAD2/3 signaling, thereby decreasing the expression of
brotic genes, such as
α
-SMA, Co1a1 and Col3a1. More studies indicated
that Mul remarkably suppressed inammatory response in liver of CCl
4
-
injected mice, evidenced by the down-regulated expression and releases
of pro-inammatory cytokines or chemokines through blocking NF-κB
signaling pathway. We surprisingly found that CCl
4
injection signi-
cantly reduced TRIM31, while promoted NLRP3 inammasome in liver
samples; however, these effects were dramatically reversed by Mul
treatments. Moreover, CCl
4
-triggered hepatic oxidative stress was
strongly ameliorated in mice with Mul administration through
improving Nrf2 signaling. Our in vitro studies subsequently conrmed
that Mul could suppress TGF-β1-induced HSCs activation by decreasing
α
-SMA expression. In line with in vivo ndings, TGF-β1- or LPS-induced
inammatory response in L02 cells and primary mouse hepatocytes was
remarkably alleviated by Mul through restraining NF-κB and NLRP3
signaling pathways, and such anti-inammatory property of Mul was
largely TRIM31-dependent. Additionally, ROS production induced by
TGF-β1 or H
2
O
2
was also strongly repressed by Mul exposure both in
human and mouse hepatocytes through improving Nrf2 activation.
Importantly, we found that CM derived from TGF-β1-stimulated L02
cells led to LX-2 cell activation, whereas being restrained by Mul. Of
note, these anti-brotic effects mediated by Mul were signicantly
abolished upon TRIM31 and Nrf2 knockdown in L02 cells. More in vivo
and in vitro studies by using TRIM31
Hep-cKO
mice and Nrf2
KO
hepatocytes
supported that the anti-brotic and liver protective effects of Mul were
mainly dependent on the expression of TRIM31 and Nrf2 in hepatocytes
with considerably ameliorated inammatory response and oxidative
stress. Immunoprecipitation analysis surprisingly showed that there
might be an interaction between TRIM31 and Nrf2. Mul-improved Nrf2
nuclear translocation in liver was almost eliminated upon TRIM31
Hep-
cKO
under brotic stress. Taken together, all our ndings demonstrated
that inammation and oxidative stress in hepatocytes contributed to
HSCs activation and collagen deposition, which could be mitigated by
Mul through improving TRIM31-regulated Nrf2 signaling pathways,
consequently ameliorating hepatic brosis (Fig. 18). Therefore, Mul
may be a promising therapeutic strategy for the treatment of liver
brosis.
Inammasome activation plays a key role in the progression of liver
disease. NLRP3 is the most well-studied inammasome and is a multi-
protein cytoplasmic complex that consists of ASC and the effector
molecule pro-Caspase-1 [15–17]. Under stimulation by
pathogen-associated molecular patterns and damage-related molecular
patterns, NLRP3 inammasome activation mediates the cleavage of
Capase-1, leading to the maturation and extracellular releases of IL-1β
and IL-18 [43,44]. IL-1β and IL-18 are synthesized as inactive precursor
forms (pro-IL-1β and pro-IL-18), which is dependent on the activation of
NF-κB under various stimuli conditions [10,15]. The activation of
NLRP3 inammasome can result in severe liver inammation, hepato-
cyte pyroptotic cell death, and hepatic brosis in mice [45], whereas
depressing NLRP3 inammasome ameliorates liver brosis. For
instance, in an animal model of non-alcoholic steatohepatitis (NASH),
NLRP3 inammasome activation was indispensable in brotic response,
Fig. 18. Proposed mechanisms indicate the effects of mulberrin on liver
brosis. In brief, under brotic stimuli, TRIM31-mediated activation of Nrf2
signaling pathway was restrained, resulting in inammatory response and
oxidative stress in hepatocytes, which contributed to the HSC activation and
hepatic brosis. Notably, mulberrin treatment could improve TRIM31/Nrf2
signaling to ameliorate hepatic inammation and ROS production, conse-
quently attenuating liver brosis.
C. Ge et al.
Redox Biology 51 (2022) 102274
23
and blockage of NLRP3 by an NLRP3 selective inhibitor attenuated
nonalcoholic steatohepatitis (NASH) proles and brosis [16,46].
Additionally, NLRP3 suppression by MCC950, a selective NLRP3 in-
hibitor, could markedly ameliorate liver injury and retard the progres-
sion of bile-duct-ligation (BDL)-induced hepatic brosis in mice [47].
Herein, suppressing NLRP3 inammasome activation may be a prom-
ising therapeutic option to effectively depress liver injury and brosis.
TRIM31, a member of the TRIM protein family, has been recognized to
play important roles in regulating tumor growth and
immuno-inammatory diseases [12,48]. TRIM31 restrained the activa-
tion NLRP3 inammasome through promoting proteasomal degradation
of NLRP3, consequently maintaining immune homeostasis [13]. A
recent study revealed that TRIM31 could promote NLRP3 ubiquitina-
tion, thereafter, suppressing NLRP3 inammasome and pyroptosis in
human retinal pigment epithelial (RPE) cells, accompanied by decreased
expression of mature IL-1β [14]. In pancreatic cancer, TRIM31 promoted
NF-κB/p65 nuclear translocation through catalyzing the K63-linked
polyubiquitination of TRAF2 and thus facilitated NF-κB activation to
accelerate tumor growth consequently [49]. TRIM31 also promoted
colorectal cancer cell proliferation and invasion by activating NF-κB
signaling pathway [50]. Here in our present study, we found that CCl
4
treatment signicantly reduced TRIM31 expression levels in liver of
mice, and promoted NLRP3 inammasome activation, as indicated by
the increased expression of NLRP3, ASC and Caspase-1, which was along
with up-regulated expression of mIL-1β and mIL-18. Consistent with
previous studies [9,51], NF-κB signaling pathway in liver was highly
stimulated by CCl
4
, proved by the elevated expression of p-IKK
α
, p-IκB
α
and p-NF-κB/p65, resulting in the high expression of pro-inammatory
factors including IL-1β, TNF-
α
, IL-6, IL-18, MCP-1 and CXCL-10.
Notably, Mul treatments considerably improved TRIM31 expression,
and reduced the activation of NLRP3 inammasome and NF-κB
signaling, thereby mitigating hepatic inammatory response. These
regulatory effects of Mul on TRIM31/NLRP3 and NF-κB signaling were
validated both in L02 cells and mouse primary hepatocytes in vitro under
TGF-β1 or LPS irritating status. Our more in vitro studies showed that in
response to TGF-β1 stimulus, TRIM31 knockdown accelerated NLRP3
inammasome and NF-κB activation in L02 cells, thereby aggravating
inammatory response. In line with previous research [13,14], TRIM31
exerted inhibitory effect on NLRP3 inammasome activation. However,
the negative regulatory effect of TRIM31 on NF-κB/p65 here we
observed was different with previous studies [49,50]. We hypothesized
that it might be associated with the different types of cells. As for this,
more studies are still required to explore the correlation between
TRIM31 and NF-κB under different physiological status. More impor-
tantly, we found that siTRIM31 signicantly abolished the function of
Mul to retard inammation via restrengthening NLRP3 inammasome
and NF-κB activation in TGF-β1-stimulated L02 cells. In vivo studies
conrmed that Mul failed to suppress NLRP3 inammasome activation
and inammatory response in liver of CCl
4
-challened mice with
hepatocyte-specic TRIM31 knockout, disclosing the necessity of
TRIM31 for Mul to perform its anti-inammatory capacities. Collec-
tively, both our in vivo and in vitro ndings demonstrated that Mul could
restrain hepatic inammation by suppressing NLRP3 and NF-κB
signaling pathways through the improvement of TRIM31 signaling
(Fig. 18).
Nrf2 pathway plays an essential role in defending against oxidative
stress. As a transcription factor, Nrf2 induces the expression of numerous
cytoprotective and detoxifying genes, including HO-1 and NQO1 [18,19,
21]. As reported, Nrf2 activation could increase the expression of its
down-streaming antioxidant factors HO-1 and NQO1 to meliorate he-
patic brosis [20–23]. GCLC and GCLM are two crucial enzymes during
the synthesis of GSH. As the major components of the Nrf2 pathway,
GCLC and GCLM increases are involved in the suppression of liver
brosis [52]. NOXs are a major source of ROS in liver and modulate
brogenic responses induced by angiotensin II, platelet derived growth
factor (PDGF), and TGF-β in cells [34,36,53,54]. The function of Mul to
improve Nrf2 signaling to subsequently ameliorate spinal cord injury
has been recognized [25]. In the current study, we consistently found
that Mul treatments restored the expression of HO-1, NQO1, GCLC, and
GCLM in liver of CCl
4
-challenged mice, and such effect was concomitant
with the up-regulated expression of nuclear Nrf2. Therefore, Mul exer-
ted its antioxidant effect through modifying Nrf2 signaling. Meanwhile,
NOX1, NOX2, NOX4 and p22
phox
stimulated by CCl
4
were strongly
restrained by Mul supplements in liver of mice. These regulatory effects
of Mul on Nrf2 were validated in TGF-β1- or H
2
O
2
-stimulated L02 cells
and mouse primary hepatocytes. Intriguingly, we found that siNrf2
markedly diminished the effects of Mul against ROS production and
oxidative stress in TGF-β1-treated L02 cells, and accelerated
TGF-β1-induced oxidative damage in vitro. Herein, we concluded that
Mul-suppressed oxidative stress in liver was Nrf2-dependent, contrib-
uting to the amelioration of hepatic injury (Fig. 18).
Hepatic inammation is a pan-etiology driver of hepatic damage and
liver brosis. Inammatory factors such as IL-1β and IL-6 released by
hepatocytes are important mediators of the inammatory response
which initiate and perpetuate an abnormal wound-healing response and
facilitate the progression of hepatic brosis through promoting HSCs
activation [55]. Therefore, blockade of inammation in hepatocytes
emerges as a novel therapeutic target to reduce liver inammation and
brosis in different types of hepatic diseases such as NASH [56]. NLRP3
inammasome and inammatory response in hepatocytes led to the HSC
activation, which is then responsible for collagen deposition and brosis
[16,17,46,47]. The probrogenic effects of ROS are compounded by the
fact that NOX4 up-regulation in hepatocytes results in the cell damage,
further inducing the cascade of cellular events that lead to HSCs acti-
vation and cirrhosis [52,57]. Similarly, here in our present study, we
found that after exposure to CM derived from TGF-β1-stimulated L02
cells containing higher levels of inammatory factors and oxidative
markers, HSCs activation of LX-2 cells was promoted, which might be
mainly attributed to the elevated inammatory and oxidative micro-
environment; however, these phenomena were strongly abolished by
Mul. Notably, the effects of Mul to suppress LX-2 cell activation induced
by CM collected from TGF-β1-stimulated L02 cells were almost abro-
gated upon TRIM31 or Nrf2 deletion. More animal studies using
TRIM31
Hep-cKO
mice demonstrated that Mul failed to ameliorate liver
dysfunction and collagen deposition, revealing that the anti-brotic and
liver protective effects of Mul were mainly TRIM31-dependent in he-
patocytes with reduced inammatory factors. Similarly, Mul lost its
function to reduce the activation of primary mouse HSCs when cultured
in CM derived from Nrf2
KO
hepatocytes under stimuli. Therefore, he-
patocytes played the main role in restraining HSCs activation and liver
brosis through regulating Nrf2 signaling by Mul treatment. All these in
vitro and in vivo studies elucidated that TRIM31 and Nrf2 expression in
hepatocytes was necessary for Mul to perform its suppressive effects on
HSCs activation and hepatic brosis. Growing studies have reported that
TRIMs family members such as TRIM16 and TRIM25 regulate Nrf2
signaling and its activation to control numerous cellular events,
including oxidative stress, ECM process and inammatory response by
governing the protein quality control [58,59]. Here in our study, we
found that Mul-improved expression of nuclear Nrf2 was considerably
abrogated by hepatocyte-specic knockout of TRIM31 in liver of
CCl
4
-treated mice, partially revealing the benecial effect of TRIM31 on
Nrf2 activation. Notably, we initially showed that there might be an
interaction between TRIM31 and Nrf2 by immunoprecipitation analysis.
Nevertheless, how TRIM31 regulates Nrf2 signaling deserves further
attention. As reported, there are numerous types of cells in liver tissues,
such as Kupffer cells, Dendritic cells (DCs) and NK cells, which play
crucial roles in mediating inammation and brosis [60,61]. Given that
the inammation and macrophage activation at different stages of liver
injury is a pivotal character to regulate brosis progression [62], more
studies are still warranted to explore whether and how Mul performs its
biological functions in macrophage to subsequently control hepatic
damage. In addition, if TRIM31 expression changes in HSCs could
C. Ge et al.
Redox Biology 51 (2022) 102274
24
govern hepatic brosis, further studies are required to address this issue.
In conclusion, we established a link between hepatocyte TRIM31 and
liver brosis in mouse models and revealed the therapeutic potential of
Mul on the disease. Specically, we found that Mul treatments signi-
cantly ameliorated HSCs activation and collagen deposition both in
CCl
4
-induced mouse models and TGF-β1-stimulated HSCs. Mul mainly
mitigated inammatory response and oxidative stress in hepatocytes
through improving TRIM31/Nrf2 signaling pathway, contributing to the
blockage of HSCs activation and ameliorating liver brosis consequently
(Fig. 18). Therefore, Mul can be considered as a promising therapeutic
strategy for liver brosis management through improving TRIM31/Nrf2
axis.
Declaration of competing interest
The authors see no conict of interest.
Acknowledgments
This work was supported by (1) National Natural Science Foundation
of China (NSFC Grant No.: 81703527); (2) Chongqing Research Program
of Basic Research and Frontier Technology (Grant No. cstc2018jcy-
jAX0393, cstc2018jcyjAX0811, cstc2018jcyjA3533); (3) Science and
Technology Research Program of Chongqing Education Commission of
China (Grant No.: KJQN201901608, KJQN201901615, KJZD-
M201801601, KJZD-K202001603); (4) Chongqing Professional Talents
Plan for Innovation and Entrepreneurship Demonstration Team
(CQCY201903258, cstc2021ycjh-bgzxm0202); (5) School-level
Research Program of Chongqing University of Education (Grant No.:
2019BSRC001); (6) Advanced Programs of Post-doctor of Chongqing
(Grant No.: 2017LY39); (7) Supported by Youth Project of Science and
Technology Research Program of Chongqing Education Commission of
China (Grant No.: KJQN201901606).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.redox.2022.102274.
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