Liquiritigenin inhibits hepatic fibrogenesis and TGF-β1/Smad with Hippo/YAP signal

Article abstract of DOI:10.1016/j.phymed.2018.12.003

Background: Recent reports highlighted the possibility that Yes-associated protein (YAP) and transforming growth factor-β1 (TGF-β1) can act as critical regulators of hepatic stellate cells (HSCs) activation; therefore, it is natural for compounds targeting Hippo/YAP and TGF-β1/Smad signaling pathways to be identified as potential anti-fibrotic candidates. Purpose: Liquiritigenin (LQ) is an aglycone of liquiritin and has been reported to protect the liver from injury. However, its effects on the Hippo/YAP and TGF-β1/Smad pathways have not been identified to date. Methods: We conducted a series of experiments using CCl₄-induced fibrotic mice and cultured LX-2 cells. Result: LQ significantly inhibited liver fibrosis, as indicated by decreases in regions of hepatic degeneration, inflammatory cell infiltration, and the intensity of α-smooth muscle actin (α-SMA) staining in mice. Moreover, LQ blocked the TGF-β1-induced phosphorylation of Smad 3, and the transcript levels of plasminogen activator inhibitor-1 (PAI-1) and matrix metalloproteinase-2 (MMP-2) in LX-2 cells, which is similar with resveratrol and oxyresveratrol (positive controls). Furthermore, LQ increased activation of large tumor suppressor kinase 1 (LATS1) with the induction of YAP phosphorylation, thereby preventing YAP transcriptional activity and suppressing the expression of exacerbated TGF-β1/Smad signaling molecules. Conclusion: These results clearly show that LQ ameliorated experimental liver fibrosis by acting on the TGF-β1/Smad and Hippo/YAP pathways, indicating that LQ has the potential for effective treatment of liver fibrosis.

Full text of DOI:10.1016/j.phymed.2018.12.003

Accepted Manuscript
Liquiritigenin inhibits hepatic fibrogenesis and TGF-β1/Smad with
Hippo/Yap signal
Eun Hye Lee , Kwang-Il Park , Kwang-Youn Kim , Ju-Hee Lee ,
Eun Jeong Jang , Sae Kwang Ku , Sang Chan Kim ,
Ho Young Suk , Ji Young Park , Su Youn Baek , Young Woo Kim
PII:
S0944-7113(18)30597-X
DOI:
Reference:
https://doi.org/10.1016/j.phymed.2018.12.003
PHYMED 52780
To appear in:
Phytomedicine
Received date:
Revised date:
Accepted date:
18 April 2018
23 November 2018
9 December 2018
Please cite this article as: Eun Hye Lee , Kwang-Il Park , Kwang-Youn Kim , Ju-Hee Lee ,
Eun Jeong Jang , Sae Kwang Ku , Sang Chan Kim , Ho Young Suk , Ji Young Park ,
Su Youn Baek , Young Woo Kim , Liquiritigenin inhibits hepatic fibrogenesis and TGF-β1/Smad
with Hippo/Yap signal, Phytomedicine (2018), doi: https://doi.org/10.1016/j.phymed.2018.12.003
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ACCEPTED MANUSCRIPT
Liquiritigenin inhibits hepatic fibrogenesis and TGF-β1/Smad with
Hippo/Yap signal
Eun Hye Leeb,#, Kwang-Il Parkc,#, Kwang-Youn Kimc,#, Ju-Hee Leed, Eun Jeong Janga,
Sae Kwang Kua, Sang Chan Kima, Ho Young Suke, Ji Young Parkb,
Su Youn Baeka,*, Young Woo Kima,*
a College of Oriental Medicine, Daegu Haany University, Gyeongsan 38610, Korea
bDepartment of Pathology, School of Medical Science, Kyungpook National University,
Daegu 41566, Korea
c Korea Institute of Oriental Medicine, Daegu 41062, Korea
d College of Korean Medicine, Dongguk University, Gyeongju, 38066 Korea
e Yeungnam University, Gyeongsan 38541, Korea
*Correspondence authors: Dr. YW Kim, E-mail: ywkim@dhu.ac.kr; Dr. SY Baek, E-mail:
rhodeus@dhu.ac.kr, College of Oriental Medicine, Daegu Haany University, Gyeongsan,
38610, Korea. Fax: +82-53-819-1860, Tel: +82-53-819-1867
#These authors equally contributed to this work.
Running title: Liquiritigenin inhibits liver fibrosis
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Abstract
Backgroud: Recent reports highlighted the possibility that Yes-associated protein (Yap) and
transforming growth factor-β1 (TGF-β1) can act as critical regulators of hepatic stellate cells
(HSCs) activation; therefore, it is natural for compounds targeting Hippo/Yap and TGF-
β1/Smad signaling pathways to be identified as potential anti-fibrotic candidates.
Purpose: Liquiritigenin (LQ) is an aglycone of liquiritin and has been reported to protect the
liver from injury. However, its effects on the Hippo/Yap and TGF-β1/Smad pathways have
not been identified to date.
Methods: We conducted a series of experiments using CCl4-induced fibrotic mice and
cultured LX-2 cells.
Result: LQ significantly inhibited liver fibrosis, as indicated by decreases in regions of
hepatic degeneration, inflammatory cell infiltration, and the intensity of α-smooth muscle
actin (α-SMA) staining in mice. Moreover, LQ blocked the TGF-β1-induced phosphorylation
of Smad 3, and the transcript levels of plasminogen activator inhibitor-1 (PAI-1) and matrix
metalloproteinase-2 (MMP-2) in LX-2 cells, which is similar with resveratrol and
oxyresveratrol (positive controls). Furthermore, LQ increased activation of large tumor
suppressor kinase 1 (LATS1) with the induction of YAP phosphorylation, thereby preventing
YAP transcriptional activity and suppressing the expression of exacerbated TGF-β1/Smad
signaling molecules.
Conclusion: These results clearly show that LQ ameliorated experimental liver fibrosis by
acting on the TGF-β1/Smad and Hippo/Yap pathways, indicating that LQ has the potential for
effective treatment of liver fibrosis.
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Key words: Liquiritigenin; liver; fibrosis; Hippo/YAP; TGF-β1/Smad
Abbreviations: α-SMA, α-smooth muscle actin; ALT, alanine aminotransferase; APAP,
acetaminophen; CCl4, carbon tetrachloride; CTGF, connective tissue growth factor; ECM,
extracellular matrix; H&E, hematoxylin and eosin; HCC, hepatocellular carcinoma; HNE,
hydroxynonenal; HO, heme oxygenase ; HSC, hepatic stellate cells; LATS1, Large Tumor
Suppressor Kinase 1; LQ, Liquiritigenin; PAI, plasminogen activator inhibitor; PDGF,
platelet-derived growth factor; TGF, transforming growth factor; TNF, tumor necrosis factor;
ROS, reactive oxygen species; PGC, peroxisome proliferator-activated receptor-gamma
coactivator; SMA, smooth muscle actin; SPP, secreted phosphate protein; YAP, Yes-
associated protein
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Introduction
Liquiritigenin (LQ) is an aglycone of liquiritin in licorice, and has been shown to have
various pharmacological activities including antioxidant, anti-inflammatory, anti-tumor, and
anti-diabetic activities (Gaur et al., 2014; Kim et al., 2008; Wang et al., 2014). A number of
studies have demonstrated that LQ plays a role in the liver as a hepatic protectant. LQ exerts
cytoprotective effects against heavy metals-induced toxicity in vitro as well as against acute
liver injuries induced by acetaminophen (APAP) in vivo (Kim et al., 2004; Kim et al., 2006).
LQ has also been shown to induce the secretion of bile by induction of antioxidant phase-II
enzymes (Kim et al., 2009), as well as to exert protective effects against liver injuries via the
PGC-1α pathway (Zhang et al., 2015). However, no detailed molecular evidence of the
effects of LQ on the liver fibrogenesis and TGF-β1/Smad pathways has been provided to date.
Liver fibrosis is a wound-healing process caused by most types of chronic damage to the
liver, including alcohol abuse, hepatitis virus infection, biliary obstruction, and nonalcoholic
fatty liver disease (Bataller and Brenner, 2005; Friedman, 2008; Ramachandran and Iredale,
2012). Fibrosis is normally characterized by the accumulation of extracellular matrix (ECM)
proteins, which form fibrous scars and develop nodules, distorting the hepatic architecture. If
fibrogenesis persists, cirrhosis may develop and the risk of developing hepatocellular
carcinoma (HCC) increases (Friedman, 2008; Sakurai and Kudo, 2013).
Activation of hepatic stellate cells (HSCs) has been recognized as an important
component during the initiation and development of liver fibrosis (Puche et al., 2014). In
normal livers, the quiescent HSCs reside in the space known as the Disse and store retinoids.
However, upon liver injury, HSCs are activated to undergo a transition to contractile, matrix-
producing myofibroblasts. The transition to myofibroblasts, in turn, induces gene
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transcription of fibrillar collagen and α-smooth muscle actin (α-SMA), leading to formation
of stress fiber, deposition of extracellular matrix and enlarged cell-extracellular matrix
contact (Higashi et al., 2017; Puche et al., 2014). Thus, elucidating the molecular
mechanisms that trigger HSC activation is the most important part of regulating fibrotic
responses.
The TGF-β1/Smad signaling is known to play a critical role in activation of HSCs and
regulation of the production and accumulation of ECM proteins (Fabregat et al., 2016). The
onset of TGF-β1/Smad signaling occurs when TGF-β binds to the serine/threonine kinase
receptor, TβRII, phosphorylating its transducer receptor, TβRI. The activated TβRI then
recruits and phosphorylates R-Smads (Smad2/3), which are transcription factors that typically
bind co-Smad (Smad4), after which these R-Smad complexes accumulate in the cell nucleus,
where they increase the transcriptional level of specific target genes (e.g., collagen-I) (Inagaki
and Okazaki, 2007).
Recent studies suggested that the Hippo pathway and its downstream effector, Yes-
associated protein (YAP), act as critical regulators of HSC activation (Mannaerts et al., 2015).
In recent studies, YAP was found to be elevated in the nucleus through HSC activation and
the inhibition of YAP prevented early HSC activation in vitro as well as the overall processes
of fibrogenesis in vivo (Mannaerts et al., 2015). Therefore, the present study investigated the
mechanistic action of LQ on the Hippo/Yap and TGF-β1/Smad pathways in liver fibrogenesis
associated with HSCs activation using an in vivo mouse model of liver fibrosis induced by
carbon tetrachloride (CCl4) and an in vitro model based on LX-2 cells treated with or without
TGF-β1.
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Materials and Methods
Materials
LQ was isolated from Glycyrrhizae radix (Sejong, Daegu, Korea) warranted as a standard
herb for medicine by Korea Food and Drug Administration (Ref. No. GLUR2008). Isolation
of the compound was verified by a series of spectroscopic analyses by Dr. Jong-Rok Lee
(Daegu Haany University) as previously described (Kim et al., 2006)(figure 1). Briefly, G.
radix (3 kg) was extracted with 15 L methanol for 72 h, and this extracts were concentrated to
make a dark gum. The fraction of CHCl3–MeOH was concentrated to produce an additional
residue, and these were evaporated to have a liquiritin-rich fraction, which were moved to
silica gel CC to obtain liquiritin. And then, LQ was prepared by acid hydrolysis of this
liquiritin. Briefly, liquiritin was reconstituted in DMSO and with 0.1N HCl, all of which were
heated at 100 ◦C. Conversion of liquiritin to LQ was verified by HPLC and NMR analyses.
The hydrolysate was solved with 40% methanol and analyzed by HPLC using an H-80
column (250 mm×10 mm). The amount of LQ was quantitated by monitoring absorbance at
254 nm, and the efficiency was calculated from LQ produced (>95% purity). NMR analysis
also determined the authenticity of LQ. TGF β1 was purchased from R&D Systems
(Minneapolis, MN, USA). Anti-p-Smad 3, anti-p-YAP, anti-p-LATS1, anti-PAI-1 and anti-β-
actin antibody were supplied from Cell Signaling Technologies (Danvers, MA, USA). Anti-
YAP and anti-mouse IgG kappa binding protein (m-IgGκ BP) conjugated to fluorescein
(FITC) antibody were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Anti-α-SMA, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) and
other reagents were purchased from Sigma–Aldrich (St. Louis, MO, USA).
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Animals and experimental protocol
Animal studies were conducted in accordance with the institutional guidelines for animal
experinents, and were approved by the Institutional Animal Care and Use Committee of the
Daegu Haany University. To induce liver fibrosis, CCl4 (0.5 ml/kg) dissolved in olive oil
(5 %) was intraperitoneally injected into the C57BL/6 mice (male, 20 – 22 g) twice a week
for 4 weeks (Lee et al., 2014). LQ, dissolved in 40% PEG 400, was orally administered (four
times per week for 4 weeks) at the dose of 10 or 30 mg/kg/day (Fig. 2A), which were the
effective doses to treat the hepatic oxidative damage in the previous study (Park et al., 2015).
The mice were assigned into four groups, control, CCl4, CCl4 + LQ (lower dose) and CCl4
+LQ (higher dose).
Blood chemistry
Serum level of alanine aminotransferase was measured based on the standard enzymatic
techniques using ALT Colorimetric Activity Assay Kit (Cayman, Ann Arbor, MI, USA)
according to manufacturer’s instructions (Lee et al., 2014).
Histological process and immunohistochemistry
Histological analysis was performed and determined by Dr. Sae-Kwang Ku (Department
Histology, College of Oriental Medicine, Daegu Haany University) as previously described
(Lee et al., 2007; Lee et al., 2014). Liver were embedded in paraffin and sectioned (3 ~ 4 μm)
(Lee et al., 2014). Sections were stained with hematoxylin and eosin (H&E) for general
histopathology or with Masson’s trichrome for collagen fibers. The expression levels of α-
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SMA, nitrotyrosine, and 4-hydroxynonenal (4-HNE) were determined using the
immunohistochemistry tool as previously described (Cho et al., 2010). Tissue sections were
stained using avidin – biotin methods (Vector Labs., Inc.).
Histomorphometry
The percentages of collagen fiber occupied area in hepatic parenchyma, numbers of
degenerative hepatocytes and numbers of inflammatory cells infiltrated were calculated using
a digital image analyzer (iSolution FLver 9.1, IMT i-solution Inc., Quebec, Canada),
according to our previously established methods (Lee et al., 2007; Lee et al., 2014). In
addition, the effects on the α-SMA, 4-HNE and nitrotyrosine immunoreactivities were also
measured using digital image analyzer in the restricted view fields of liver tissue.
Cell culture and Western blot analysis
LX-2 cells, an immortalized human HSCs line, were kindly provided by Dr. SL Friedman
(Mount Sinai School of Medicine, NY, USA) (Lee et al., 2014). Cells were treated with LQ
(30 and/or 100 μM). Preparation of protein lysates from cell line and liver tissue were
performed as previously described protocol (Lee et al., 2014).
Real-time RT-PCR analysis
Total RNA was extracted using a Trizol (Invitrogen, Carlsbad, CA, USA)(Cho et al., 2010).
Real-time PCR was performed using MG 2X qPCR mastermix (MGmed, Seoul, Korea) with
a SYBR green premix, and primer sets were shown in Table 1.
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Immunocytochemistry
Untreated or LQ (100 μM for 1h)-treated LX-2 cells was fixed for 10 min in 4 %
paraformaldehyde at room temperature. The cells were incubated with anti-YAP antibody
(1:200) for overnight, and FITC-conjugated secondary antibodies (1:200). The slides were
mounted with 80 % glycerol in PBS with DAPI (0.3 µg/ml).
Data analysis
A Multiple comparison tests for different dose groups were conducted as previously
described (Lee et al., 2014). Variance homogeneity was determined by using the Levene test
(Levene, 1981). The statistical significance of the differences among the treatment groups
was verified by one-way analysis of variance (ANOVA). An unpaired t-test was also used to
analyze the differences, and a non-parametric test was used (Mann-Whitney U test) if the
distribution of data was abnormal. All the statistical analyses were performed using Stat View
5.0 (Abacus Concepts, Berkeley, CA). P values were displayed with asterisks (*** ; P <
0.001, **; P < 0.01, *; P < 0.05, ns; P > 0.05).
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Results
Liquiritigenin protected the liver from CCl4-induced toxicity
CCl4 is known to induce liver toxicity and resultantly produce highly reactive metabolites
(Manibusan et al., 2007). And, a long exposure of this insult results severe hepatocyte injury
and initiated pathological process of fibrosis (Manibusan et al., 2007). Repeated injections of
CCl4 (2 times/week, 4 weeks) significantly elevated the level of ALT (P < 0.01). However,
the level was attenuated by oral administration of two concentrations of LQ (10 mg/kg and 30
mg/kg; both P < 0.05)(Fig. 2A). Histopathological analysis showed that CCl4 treatment
induced the ballooning of hepatocytes and the infiltration of inflammatory cells in the liver
tissue (Fig. 2B). The number of degenerative hepatocytes as well as the number of
inflammatory cells was markedly increased in the CCl4 treated mice (Table 2). However, LQ
protected these kinds of hepatic damage (Fig. 1B and Table 2).
Liquiritigenin ameliorates CCl4-induced liver fibrosis
To examine the potential effects of LQ on collagen occupation during fibrosis, the tissues
were stained with α-SMA and Masson's trichrome. α-SMA-immunolabeled cells were
significantly (P < 0.01) increased in the central vein regions in response to CCl4 treatment
(Fig. 3A). These CCl4 treatment-related increases of α-SMA immunoreactivities in hepatic
parenchyma were dose-dependently inhibited by treatment with LQ (Fig. 3B). Moreover,
CCl4 markedly induced collagen fibers, as indicated with fibrotic septa between each nodule,
which were significantly decreased by LQ treatment (Fig. 3B). Moreover, the collagen fiber
occupied percentages in the CCl4-treated group increased significantly when compared with
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the vehicle control, while they were attenuated in both LQ 10 and 30 mg/kg treated mouse
livers (data not shown).
Because oxidative stress was believed to be an important mediator of liver fibrosis
(Sánchez-Valle et al., 2012), additional analyses were performed to examine whether LQ
could inhibit oxidative stress in CCl4-induced liver fibrosis. The numbers of nitrotyrosine or
4-HNE-positive cells in the CCl4 control were increased relative to the intact control, whereas
the level was attenuated in response to treatment with LQ (Fig. 3C).
Liquiritigenin suppressed TGF-β1/Smad signaling and HSC activation in vitro
To study the mechanisms of LQ inhibition of hepatic fibrosis in vivo, we conducted in vitro
studies using LX-2 cells, a human HSC cell line. Before examination, we tested the
cytotoxicity of LQ in LX-2 cells. MTT assay showed no significant differences in LX-2 cells
treated with LQ 100 μM (Supplementary Fig. S1). Therefore, we used concentrations of 30
μM (lower effect) and 100 μM (higher effect).
The TGF-β1-mediated signaling pathway is known to mediated with the phosphorylation
of Smad 2/3 (Inagaki and Okazaki, 2007). To reveal the underlying mechanisms responsible
for the inhibition of HSCs activation, authors determined the effects of the compound on
signal transduction of TGF-β/Smad. As expected, TGF-β1 enhanced α-SMA levels, but this
was inhibited by LQ pretreatment (Fig. 4A). Real-time-PCR analysis showed that the LQ
markedly suppressed the TGF-β1-induced gene expression of PAI-1 and MMP-2 (Fig. 4B).
Moreover, LQ also inhibited autophagy-related protein induction (i.e. LC3-II) by TGF-β1
(Fig. 4C).
Next, we determined signaling pathway of Smad. Smad proteins are of great importance
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in the progression of fibrosis. Smad3 is related with R-Smad, the representative in three
subtype of Smads, which also include Co-Smad (Smad4) and I-Smad (Smad 7). Immunoblot
analysis revealed an increase in the Smad3 phosphorylation by TGF-β1, as well as inhibition
of this increase by LQ treatment (Fig. 5A). Moreover, LQ inhibited dysregulation of Smad4
and 7 induced by TGF-β1 (Fig. 5B). As a positive control experiment, resveratrol and
oxyresveratrol also inhibited the Smad3 activation and α-SMA as similar with LQ (Fig. 5C).
Liquiritigenin activated hippo signaling
Recently, YAP has been shown to play essential roles in liver cell fate and HSC activation.
YAP activity is regulated by phosphorylation, which causes YAP inactivation and nuclear to
cytoplasmic translocation (Mannaerts et al., 2015; Meng et al., 2016). To examine the time
course of the effects of LQ on the hippo signaling pathway, we treated LX-2 cells with 100
μM LQ. The level of phospho LATS1, a negative regulator of YAP, increased after treatment
with LQ for 0.5 –3 h. In addition, the phosphorylation level of YAP was robustly elevated 1 h
after treatment with LQ at 100 μM (Fig. 6A). To verify the effects of LQ on the hippo
signaling pathway, the levels of Yap were measured by immunocytochemistry analysis (Fig.
5B). In the vehicle control (LX-2 cells) the Yap signal was low and mostly located in the
nucleus (Fig. 6B). In comparison, Yap was robustly expressed with high amounts present in
the cytoplasm of LQ treated LX-2 cells (Fig. 6B). Moreover, we tested the effect of
verteporfin, a chemical inhibitor of YAP. As expected, the treatment of verteporfin (0.4 M)
inhibited TGF-b1-induced -SMA, and co-treatment of LQ and verteporfin attenuated the
expression of -SMA more than LQ alone (Fig. 6C).
We next tested whether LQ could affect YAP signaling in fibrogenesis in vivo. Both the
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phosphorylated form of YAP and LATS1 were significantly decreased in the CCl4-induced
fibrotic group compared to the control group (Fig. 7A). However, LQ treatment of the CCl4-
induced group led to increase them relative to CCl4-induced mice (Fig. 7A), indicating that
LQ has profound effects on YAP activity and HSC activation. Collectively, these results
suggest that LQ activates the hippo signaling pathway, which might contribute LQ’s
protection against liver fibrosis (Fig. 7B).
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Discussion
Here, we investigated the efficiency of LQ as a candidate for hepatic fibrosis control and
attempted to examine its functional mechanism across a series of experimental tests. Hepatic
fibrosis was induced using carbon tetrachloride (CCl4), a representative hepatotoxic
substance (Manibusan et al., 2007; Yazdani et al., 2017). When CCl4 enters the hepatocytes, it
is converted to CCl3 by cytochrome P450 (CYP450) located in the smooth endoplasmic
reticulum. Both CCl3 and CCl3O2, which are created by binding with intracellular oxygen, are
highly reactive, resulting in damage to various cellular functions, such as destruction of the
endogenous reticulum or disruption of mitochondrial and intracellular enzyme activities,
which ultimately leads to the development of cirrhosis, usually after 4 to 8 weeks (Lee et al.,
2014; Manibusan et al., 2007). As predicted, we observed significant liver damage in the
experimental mice subjected to intraperitoneal injection of CCl4 for 4 weeks. However,
treatment with CCl4 in the presence of LQ resulted in a marked decrease in local fibrosis and
α-SMA expression, as well as pathological histologic liver damage such as increased CCl4-
induced ALT activity, inflammation and necrosis. Moreover, these effects occurred in a dose
dependent manner, indicating its beneficial effects at protecting the liver from damage and
the gene expression of liver fibrosis. The results of these in vivo experiments support those of
previous studies showing that LQ has protective effects on hepatocyte injury induced by
heavy metals or liver damage induced by acetaminophen (Kim et al., 2004; Kim et al., 2006).
Moreover, LQ has been known to be metabolized to glucuronide conjugates (Kim et al.,
2009). In our previous finding, the time profiles of LQ and its metabolites showed that the
contents of metabolites rapidly increased after treatment and remained for a long time (Kim
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et al., 2009). However, it needs to clarify the exact pharmacokinetic profile to arrange the
time schedule and oral dose in the chronic disease model.
The increased number of nitrotyrosine- or 4-HNE-positive cells by CCl4 injections was
attenuated by LQ treatments, indicating that oxidative stress can also be alleviated in the
fibrous liver by LQ. Oxidative stress is known to induce the synthesis of inflammatory
cytokines and modulates HSC activation (Sánchez-Valle et al., 2012). Thus, ROS is an
important initiator and amplifier during pathological progress of fibrosis in the liver
(Sánchez-Valle et al., 2012; Seki and Schwabe, 2015). Our results clearly support those of a
previous report showing that LQ plays an important role in mitigating CCl4-induced liver
damage by regulating PGC-1α and its downstream genes (Zhang et al., 2015). Taken together,
the results of our study showed that LQ can inhibit hepatic damage and HSC activation
caused by oxidative stress and eventually liver fibrosis.
Inflammatory reactions caused by hepatocyte injury stimulate liver parenchymal cells,
endothelial cells, or Kupffer cells to release various cytokines and chemokines. Among these,
TGF-β1 is known to act as a key factor in liver fibrosis (Fabregat et al., 2016; Friedman,
2008). Upon activation of TGF-β1, HSCs are transformed into myofibroblast-like form and
rapidly proliferate to over-synthesize ECM proteins such as collagen, proteoglycan, and
fibronectin for the establishment of connective tissues, thereby promoting liver fibrosis (Cho
et al., 2010). In addition, TGF-β1 inhibits the degradation of ECM by regulating matrix
metalloproteinase (MMP), tissue inhibitors of metalloproteinases (TIMP), which inhibits
MMP, and plasminogen activator inhibitor (PAI) (Dudás et al., 2001). TGF-β expression is
also known to be associated with progressive liver disease (Friedman, 2008; Lee and
Friedman, 2011).
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The standard TGF-β signaling pathway is the TGF-β1/Smad signaling pathway, in which
TGF-β1 binds to receptors present on the plasma membrane to activate the Smad signaling
pathway, thereby increasing the level of procollagen, the collagen precursor, and transcription
of the downstream genes (Moustakas and Heldin, 2009). Our results showed that LQ
inhibited Smad3 phosphorylation and activation by TGF-β1, subsequently blocking fibrotic
gene expression in HSCs (Fig. 4), suggesting that the effects of LQ on liver fibrosis are at
least partly related to regulation of the TGF-β1/Smad signaling pathway.
Recent studies have demonstrated that the YAP protein in the Hippo signaling pathway
plays a key role in the activation of HSCs. One study showed that Yap was involved in the
progression of fibrosis with HSC activation in mice treated with CCl4. This was further
confirmed by the additional experimental results that showed expression of α-SMA and
collagen-1 was drastically reduced and not activated when Yap was completely knocked
down (Mannaerts et al., 2015). Activation of HSCs leads to overproduction of ECM, which
can be used to protect damaged tissues and appears to involve YAP. The function of YAP as a
sensor of stress and a driver of regeneration processes was observed not only in HSCs, but
also other types of cells and organs. For example, a study showed that YAP expression in
epithelial cells of the intestinal crypt was increased in response to toxin-induced tissue
damage, and that lack of YAP would make it impossible to regenerate damaged intestinal
tissue (Barry et al., 2012, Zhubanchaliyev et al., 2016). YAP overexpression in the heart with
ischemic injury promotes cardiac muscle regeneration, indicating that YAP is associated with
wound healing (Del Re et al., 2013; Xin et al., 2013). In addition, fibroblasts expressing
activated YAP have been found to promote fibrosis when implanted into the lungs of rats (Liu
et al., 2015). Overall, YAP activation appears to play a decisive role in causing fibroblast
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responses in vivo.
The YAP/TAZ of the Hippo signaling pathway interacts with TGF-β-induced Smad2/3 in
the nucleus, resulting in a YAP/TAZ•TEAD•Smad2/3 complex, which regulates several
transcription processes induced by TGF-β (Grannas et al., 2015; Nishio et al., 2016; Varelas
et al., 2010). Activation of the Hippo signaling pathway results in phosphorylation of YAP,
which causes it to remain in the cytoplasm. However, when the Hippo signaling pathway is
inactivated, it migrates to the nucleus and interacts with the TEAD1-4 transcription factor,
promoting transcription of genes such as CTGF, SPP1 and TGF-β (Perumal et al., 2017; Zhao
et al., 2008). Therefore, we investigated whether LQ induces phosphorylation of YAP protein.
Liquiritigenin treatment was found to induce phosphorylation of YAP protein in LX-2 cells
and in mice bearing CCl4-induced liver fibrosis. Immunocytochemistry analysis revealed that
phosphorylation of YAP was significantly increased in LX-2 cells treated with LQ, and it was
clearly observed in the cytoplasm, indicating that YAP was inactivated. In summary, LQ
induced phosphorylation of YAP, which inhibited activation of YAP/TAZ, thereby ultimately
blocking HSC activation and TGF-β1/Smad signaling.
Conclusion
The results of our study clearly indicate that LQ significantly inhibited liver fibrosis in mouse
models in vivo and HSC activation in vitro. This protective mechanism might be related to its
ability to hippo signaling and the TGF-β1/Smad signaling pathway. Based on these findings,
LQ is expected to play a beneficial role in the treatment of liver fibrosis and chronic liver
disease.
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Conflict of interest
The authors declare that there are no conflicts of interest
Acknowledgements
This work was supported by the National Research Foundation of Korea (NRF) grant funded
by the Korea Government [MSIP] (No. 2017R1D1A3B03027847), and also by the grant
K18102 awarded to Korea Institute of Oriental Medicine (KIOM) from Korea Ministry of
Education, Science and Technology (MEST). Lee EH would like to thank to the Ph.D.’s
program of Kyungpook National University for completing the Ph.D. thesis through this
work (Academic advisor: Dr. Ji Young Park). Authors appreciate Dr. Jong-Rok Lee (Daegu
Haany University) for the isolation of liquiritigenin from Glycyrrhizae radix, and the
laboratory of Dr. Sae Kwang Ku (Daegu Haany University) for histological analysis.
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References
Barry, E.R., Morikawa, T., Butler, B.L., Shrestha, K., de la Rosa, R., Yan, K.S., Fuchs, C.S.,
Magness, S.T., Smits, R., Ogino, S., Kuo, C.J., Camargo, F.D., 2012. Restriction of
intestinal stem cell expansion and the regenerative response by YAP. Nature 493(7430),
106-10. http://dx.doi.org/10.1038/nature11693
Bataller, R., Brenner, D.A., 2005. Liver fibrosis. J. Clin. Invest. 15(2), 209-18.
http://dx.doi.org/10.1172/JCI24282
Cho, I.J., Kim, Y.W., Han, C.Y., Kim, E.H., Anderson, R.A., Lee, Y.S., Lee, C.H., Hwang,
S.J., Kim, S.G., 2010. E-cadherin antagonizes transforming growth factor β1 gene
induction in hepatic stellate cells by inhibiting RhoA-dependent Smad3 phosphorylation.
Hepatology 52(6), 2053-64. http://dx.doi.org/10.1002/hep.23931
Del Re, D.P., Yang, Y., Nakano, N., Cho, J., Zhai, P., Yamamoto, T., Zhang, N., Yabuta, N.,
Nojima, H., Pan, D., Sadoshima, J., 2013. Yes-associated protein isoform 1 (Yap1)
promotes cardiomyocyte survival and growth to protect against myocardial ischemic
injury. J. Biol. Chem. 288(6), 3977-88. http://dx.doi.org/10.1074/jbc.M112.436311
Dudás, J., Kovalszky, I., Gallai, M., Nagy, J.O., Schaff, Z., Knittel, T., Mehde, M., Neubauer,
K., Szalay, F., Ramadori, G., 2001. Expression of decorin, transforming growth factor-
beta 1, tissue inhibitor metalloproteinase 1 and 2, and type IV collagenases in chronic
hepatitis. Am. J. Clin. Pathol. 115(5), 725-35. http://dx.doi.org/10.1309/J8CD-E9C8-
X4NG-GTVG
Fabregat, I., Moreno-Càceres, J., Sánchez, A., Dooley, S., Dewidar, B., Giannelli, G., Ten
Dijke, P.; IT-LIVER Consortium, 2016. TGF-β signalling and liver disease. FEBS J.
283(12), 2219-32. http://dx.doi.org/10.1111/febs.13665
19

ACCEPTED MANUSCRIPT
Friedman, S.L., 2008. Mechanisms of hepatic fibrogenesis. Gastroenterology 134(6), 1655-69.
http://dx.doi.org/10.1053/j.gastro.2008.03.003
Gaur, R., Yadav, K.S., Verma, R.K., Yadav, N.P., Bhakuni, R.S., 2014. In vivo anti-diabetic
activity of derivatives of isoliquiritigenin and liquiritigenin. Phytomedicine 21(4), 415-
22. http://dx.doi.org/10.1016/j.phymed.2013.10.015
Grannas, K., Arngården, L., Lönn, P., Mazurkiewicz, M., Blokzijl, A., Zieba, A., Söderberg,
O., 2015. Crosstalk between Hippo and TGFβ: Subcellular Localization of
YAP/TAZ/Smad
Complexes.
J.
Mol.
Biol.
427(21),
3407-15.
http://dx.doi.org/10.1016/j.jmb.2015.04.015
Higashi, T., Friedman, S.L., Hoshida, Y., 2017. Hepatic stellate cells as key target in liver
fibrosis. Adv. Drug Deliv. Rev. 121, 27-42. http://dx.doi.org/10.1016/j.addr.2017.05.007
Inagaki, Y., Okazaki, I., 2007. Emerging insights into transforming growth factor beta Smad
signal
in
hepatic
fibrogenesis.
Gut
56(2),
284-92.
http://dx.doi.org/10.1136/gut.2005.088690
Kim, S.C., Byun, S.H., Yang, C.H., Kim, C.Y., Kim, J.W., Kim, S.G., 2004. Cytoprotective
effects of Glycyrrhizae radix extract and its active component liquiritigenin against
cadmium-induced toxicity (effects on bad translocation and cytochrome c-mediated
PARP cleavage). Toxicology 197(3), 239-51. http://dx.doi.org/10.1016/j.tox.2004.01.010
Kim, Y.W., Kang, H.E., Lee, M.G., Hwang, S.J., Kim, S.C., Lee, C.H., Kim, S.G., 2009.
Liquiritigenin, a flavonoid aglycone from licorice, has a choleretic effect and the ability
to induce hepatic transporters and phase-II enzymes. Am. J. Physiol. Gastrointest. Liver
Physiol. 296(2), G372-81. http://dx.doi.org/10.1152/ajpgi.90524.2008
Kim, Y.W., Ki, S.H., Lee, J.R., Lee, S.J., Kim, C.W., Kim, S.C., Kim, S.G., 2006.
20

ACCEPTED MANUSCRIPT
Liquiritigenin, an aglycone of liquiritin in Glycyrrhizae radix, prevents acute liver
injuries in rats induced by acetaminophen with or without buthionine sulfoximine. Chem.
Biol. Interact. 161(2), 125-38. http://dx.doi.org/10.1016/j.cbi.2006.03.008
Kim, Y.W., Zhao, R.J., Park, S.J., Lee, J.R., Cho, I.J., Yang, C.H., Kim, S.G., Kim, S.C., 2008.
Anti-inflammatory effects of liquiritigenin as a consequence of the inhibition of NF-
kappaB-dependent iNOS and proinflammatory cytokines production. Br. J. Pharmacol.
154(1), 165-73. http://dx.doi.org/10.1038/bjp.2008.79
Lee, H.S., Keum, K.Y., Ku, S.K.., 2007. Effects of Picrorrhiza rhizoma water extracts on the
subacute liver damages induced by carbon tetrachloride. J. Med. Food. 10(1), 110-7.
http://dx.doi.org/10.1089/jmf.2006.0114
Lee, J.H., Jang, E.J., Seo, H.L., Ku, S.K., Lee, J.R., Shin, S.S., Park, S.D., Kim, S.C., Kim,
Y.W., 2014. Sauchinone attenuates liver fibrosis and hepatic stellate cell activation
through TGF-β/Smad signaling pathway. Chem. Biol. Interact. 224, 58-67.
http://dx.doi.org/10.1016/j.cbi.2014.10.005
Lee, U.E., Friedman, S.L., 2011. Mechanisms of hepatic fibrogenesis. Best Pract. Res. Clin.
Gastroenterol. 25(2), 195-206. http://dx.doi.org/10.1016/j.bpg.2011.02.005
Levene, A., 1981. Pathological factors influencing excision of tumours in the head and neck.
Part I. Clin. Otolaryngol. Allied Sci. 6(2), 145-51
Liu, F., Lagares, D., Choi, K.M., Stopfer, L., Marinković, A., Vrbanac, V., Probst, C.K.,
Hiemer, S.E., Sisson, T.H., Horowitz, J.C., Rosas, I.O., Fredenburgh, L.E., Feghali-
Bostwick, C., Varelas, X., Tager, A.M., Tschumperlin, D.J., 2015. Mechanosignaling
through YAP and TAZ drives fibroblast activation and fibrosis. Am. J. Physiol. Lung
Cell Mol. Physiol. 308(4), L344-57. http://dx.doi.org/10.1152/ajplung.00300.2014
21

ACCEPTED MANUSCRIPT
Manibusan, M.K., Odin, M., Eastmond, D.A., 2007. Postulated carbon tetrachloride mode of
action: a review. J. Environ. Sci. Health C. Environ. Carcinog. Ecotoxicol. Rev. 25(3),
185-209. http://dx.doi.org/10.1080/10590500701569398
Mannaerts, I., Leite, S.B., Verhulst, S., Claerhout, S., Eysackers, N., Thoen, L.F., Hoorens, A.,
Reynaert, H., Halder, G., van Grunsven, L.A., 2015. The Hippo pathway effector YAP
controls mouse hepatic stellate cell activation. J. Hepatol. 63(3), 679-88.
http://dx.doi.org/10.1016/j.jhep.2015.04.011
Meng, Z., Moroishi, T., Guan, K.L., 2016. Mechanisms of Hippo pathway regulation. Genes
Dev. 30(1), 1-17. http://dx.doi.org/10.1101/gad.274027.115
Moustakas, A., Heldin, C.H., 2009. The regulation of TGFbeta signal transduction.
Development 136(22), 3699-714. http://dx.doi.org/10.1242/dev.030338
Nishio, M., Sugimachi, K., Goto, H., Wang, J., Morikawa, T., Miyachi, Y., Takano, Y., Hikasa,
H., Itoh, T., Suzuki, S.O., Kurihara, H., Aishima, S., Leask, A., Sasaki, T., Nakano, T.,
Nishina, H., Nishikawa, Y., Sekido, Y., Nakao, K., Shin-Ya, K., Mimori, K., Suzuki, A.1.,
2016. Dysregulated YAP1/TAZ and TGF-β signaling mediate hepatocarcinogenesis in
Mob1a/1b-deficient mice. Proc. Natl. Acad. Sci. U.S.A. 113(1), E71-80.
http://dx.doi.org/10.1073/pnas.1517188113
Park, S.M., Ki, S.H., Han, N.R., Cho, I.J., Ku, S.K., Kim, S.C., Zhao, R.J., Kim, Y.W. 2015.
Tacrine, an oral acetylcholinesterase inhibitor, induced hepatic oxidative damage, which
was blocked by liquiritigenin through GSK3-beta inhibition. Biol. Pharm. Bull. 38(2),
184-192. http://dx.doi.org/10.1248/bpb.b14-0043
Perumal, N., Perumal, M., Halagowder, D., Sivasithamparam, N., 2017. Morin attenuates
diethylnitrosamine-induced rat liver fibrosis and hepatic stellate cell activation by co-
22

ACCEPTED MANUSCRIPT
ordinated regulation of Hippo/Yap and TGF-β1/Smad signaling. Biochimie. 140, 10-19.
http://dx.doi.org/10.1016/j.biochi.2017.05.017
Puche, J.E., Saiman, Y., Friedman, S.L., 2013. Hepatic stellate cells and liver fibrosis. Compr.
Physiol. 3(4), 1473-92. http://dx.doi.org/10.1002/cphy.c120035
Ramachandran, P., Iredale, J.P., 2012. Liver fibrosis: a bidirectional model of fibrogenesis
and resolution. Q. J. M. 105(9), 813-7. http://dx.doi.org/10.1093/qjmed/hcs069
Sakurai, T., Kudo, M., 2013. Molecular link between liver fibrosis and hepatocellular
carcinoma. Liver Cancer 2(3-4), 365-6. http://dx.doi.org/10.1159/000343851
Sánchez-Valle, V., Chávez-Tapia, N.C., Uribe, M., Méndez-Sánchez, N., 2012. Role of
oxidative stress and molecular changes in liver fibrosis: a review. Curr. Med. Chem.
19(28), 4850-60.
Seki, E., Schwabe, R.F., 2015. Hepatic inflammation and fibrosis: functional links and key
pathways. Hepatology 61(3), 1066-79. http://dx.doi.org/10.1002/hep.27332
Varelas, X., Samavarchi-Tehrani, P., Narimatsu, M., Weiss, A., Cockburn, K., Larsen, B.G.,
Rossant, J., Wrana, J.L., 2010. The Crumbs complex couples cell density sensing to
Hippo-dependent control of the TGF-β-SMAD pathway. Dev. Cell 19(6), 831-44.
http://dx.doi.org/10.1016/j.devcel.2010.11.012
Wang, D., Wong, H.K., Feng, Y.B., Zhang, Z., 2014. Liquiritigenin exhibits antitumour action
in pituitary adenoma cells via Ras/ERKs and ROS-dependent mitochondrial signalling
pathways. J. Pharm. Pharmacol. 66(3), 408-17. http://dx.doi.org/10.1111/jphp.12170
Xin, M., Kim, Y., Sutherland, L.B., Murakami, M., Qi, X., McAnally, J., Porrello, E.R.,
Mahmoud, A.I., Tan, W., Shelton, J.M., Richardson, J.A., Sadek, H.A., Bassel-Duby, R.,
Olson, E.N., 2013. Hippo pathway effector Yap promotes cardiac regeneration. Proc.
23

ACCEPTED MANUSCRIPT
Natl. Acad. Sci. U.S.A. 110(34), 13839-44. http://dx.doi.org/10.1073/pnas.1313192110
Yazdani, S., Bansal, R., Prakash, J., 2017. Drug targeting to myofibroblasts: Implications for
fibrosis
and
cancer.
Adv.
Drug
Deliv.
Rev.
121,
101-116.
http://dx.doi.org/10.1016/j.addr.2017.07.010
Zhang, Y., He, Y., Yu, H., Ma, F., Wu, J., Zhang, X., 2015. Liquiritigenin Protects Rats from
Carbon Tetrachloride Induced Hepatic Injury through PGC-1α Pathway. Evid. Based
Complement. Alternat. Med. 2015, 649568. http://dx.doi.org/10.1155/2015/649568
Zhao, B., Ye, X., Yu, J., Li, L., Li, W., Li, S., Yu, J., Lin, J. D., Wang, C.Y., Chinnaiyan, A.M.,
Lai, Z.C., Guan, K.L., 2008. TEAD mediates YAP-dependent gene induction and growth
control. Genes Dev. 22(14), 1962-71. http://dx.doi.org/10.1101/gad.1664408
Zhubanchaliyev, A., Temirbekuly, A., Kongrtay, K., Wanshura, L.C., Kunz, J., 2016.
Targeting mechanotransduction at the transcriptional Level: YAP and BRD4 are novel
therapeutic targets for the reversal of liver fibrosis. Front. Pharmacol. 7, 462.
http://dx.doi.org/10.3389/fphar.2016.00462
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Figure Legends
Fig. 1. The chemical structure of liquiritigenin (LQ).
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Fig. 2. The effect of LQ on CCl4-induced liver toxicity in mice. Mice were injected with CCl4
(0.5 ml/kg) intraperitoneally twice a week and administered LQ (10 or 30 mg/kg) orally four
times per week for 4 weeks. After treatment, (A) the activities ALT were assayed by using
semi-automated blood chemistry analyzer. Data represents the mean ± SD. * P < 0.05; ** P
< 0.01. (B) Representative histological sections of the liver. The liver sections from left
lateral lobes were stained with haematoxylin and eosin (H&E) in mice treated with vehicle,
CCl4, CCl4 + LQ 10 mg/kg, and CCl4 + LQ 30 mg/kg. Scale bars = 80 μm.
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Fig. 3. Effects of LQ on liver fibrosis induced by CCl4. (A) Liver fibrosis was observed by
immunohistochemical staining of α-SMA-positive cells. Scale bars = 80 μm. (B) Quantitation
of α-SMA-positive cells (left) and collagen fiber occupied regions (right). Data represents the
mean ± SD from seven separate experiments. * P < 0.05; *** P < 0.001. (C) Quantitation of
nitrotyrosine (left) and 4-HNE positive cells (right). Data represents the mean ± SD from
seven separate experiments. * P < 0.05; *** P < 0.001.
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Fig. 4. Effects of LQ on TGF-β1-induced HSCs activation. (A) LQ suppresses TGF-β1-
induced expression of the myofibroblast marker protein α-SMA. Western blot analyses were
performed on the lysates of serum starved LX-2 cells that were treated with LQ in the
presence or absence of TGF-β1 (5 ng/ml) for an additional 24 h. β-actin served as a loading
control. Protein levels were presented as relative band intensities to control (vehicle treated)
group. (B) Real-time PCR was assessed to investigate mRNA level of PAI-1 and MMP-2,
TGF-β1 target genes. (C) LQ suppresses TGF-β1-induced expression of the autophagy
related proteins, LC3II. Data represents the mean ± SD from three separate experiments. * P
< 0.05; **P < 0.01.
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Fig. 5. Effects of LQ on TGF-β1/Smad pathway in HSCs. LQ suppresses the activation of
TGF-β1/Smad pathway. (A) Phosphorylation levels of Smad 3 were analyzed by
immunoblotting with specific antibodies. Western blot performed on the lysates of serum
starved LX-2 cells that were treated with LQ in the presence or absence of TGF-β1 (5 ng/ml)
for an additional 24 h. β-actin served as a loading control. Protein levels were presented as
relative band intensities to control (vehicle treated) group. (B) Protein expression levels of
different Smads subtypes (pSmad3, Smad4, and Smad7) were analyzed using western blot. (3)
Effects of resveratrol (Res) and oxyresveratrol (OXY) on TGF-β1-stimulated phosphorylation
of Smad 3. The cells were treated with 30 μM of the compounds for 1 h and then with TGF-
β1 (5 ng/ml, 24 h).
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