Antifibrotic Effects of a Barbituric Acid Derivative on Liver Fibrosis by Blocking the NF-κB Signaling Pathway in Hepatic Stellate Cells

Article abstract of DOI:10.3389/fphar.2020.00388

Hepatic stellate cells (HSCs) are the major profibrogenic cells that promote the pathogenesis of liver fibrosis. The crosstalk between transforming growth factor-β1 (TGF-β1) signaling and lipopolysaccharide (LPS)-induced NF-κB signaling plays a critical role in accelerating liver fibrogenesis. Until now, there have been no FDA-approved drug treatments for liver fibrosis. Barbituric acid derivatives have been used as antiasthmatic drugs in the clinic; however, the effect of barbituric acid derivatives in treating liver fibrosis remains unknown. In this study, we synthesized a series of six barbituric acid (BA) derivatives, and one of the compounds, BA-5, exhibited the best ability to ameliorate TGF-β1-induced HSC activation without overt cytotoxic effects. Then, we treated HSCs and RAW264.7 macrophages with BA-5 to analyze the cross-talk of anti-fibrotic and anti-inflammatory effects. Carbon tetrachloride (CCl₄)-induced liver fibrosis mouse model was used to evaluate the therapeutic effects of BA-5. Treatment with BA-5 inhibited TGF-β1-induced α-SMA, collagen1a2, and phosphorylated smad2/3 expression in HSCs. Furthermore, BA-5 treatment reversed the LPS-induced reduction in BAMBI protein and decreased IκBα and NF-κB phosphorylation in HSCs. NF-κB nuclear translocation, MCP-1 secretion, and ICAM-1 expression were also inhibited in BA-5-treated HSCs. Conditioned medium collected from BA-5-treated HSCs showed a reduced ability to activate RAW264.7 macrophages by inhibiting the MAPK pathway. In the mouse model, BA-5 administration reduced CCl₄-induced liver damage, liver fibrosis, and F4/80 expression without any adverse effects. In conclusion, our study showed that the barbituric acid derivative BA-5 inhibits HSCs activation and liver fibrosis by blocking both the TGF-β1 and LPS-induced NF-κB signaling pathways and further inhibits macrophages recruitment and activation.

Full text of DOI:10.3389/fphar.2020.00388

ORIGINAL RESEARCH
published: 31 March 2020
doi: 10.3389/fphar.2020.00388
Antifibrotic Effects of a Barbituric
Acid Derivative on Liver Fibrosis by
Blocking the NF-kB Signaling
Pathway in Hepatic Stellate Cells
Yuan-Hsi Wang1,2, Fat-Moon Suk3,4, Chao-Lien Liu2, Tzu-Lang Chen5, Yuh-Ching Twu1
,
*
Ming-Hua Hsu6 and Yi-Jen Liao
2
*
*
1 Department of Biotechnology and Laboratory Science in Medicine, School of Biomedical Science and Engineering, National
Yang-Ming University, Taipei, Taiwan, 2 School of Medical Laboratory Science and Biotechnology, College of Medical
Science and Technology, Taipei Medical University, Taipei, Taiwan, 3 Division of Gastroenterology, Department of Internal
Medicine, Wan Fang Hospital, Taipei Medical University, Taipei, Taiwan, 4 Department of Internal Medicine, School of
Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan, 5 Department of Medical Education, Far Eastern
Memorial Hospital, New Taipei City, Taiwan, 6 Department of Chemistry, National Changhua University of Education,
Changhua, Taiwan
Edited by:
Jose C. Fernandez-Checa,
Spanish National Research Council,
Spain
Reviewed by:
Guoxun Chen,
The University of Tennessee,
Knoxville, United States
Anna Moles,
Hepatic stellate cells (HSCs) are the major profibrogenic cells that promote the
pathogenesis of liver fibrosis. The crosstalk between transforming growth factor-b1 (TGF-
b1) signaling and lipopolysaccharide (LPS)-induced NF-kB signaling plays a critical role in
accelerating liver fibrogenesis. Until now, there have been no FDA-approved drug
treatments for liver fibrosis. Barbituric acid derivatives have been used as antiasthmatic
drugs in the clinic; however, the effect of barbituric acid derivatives in treating liver fibrosis
remains unknown. In this study, we synthesized a series of six barbituric acid (BA)
derivatives, and one of the compounds, BA-5, exhibited the best ability to ameliorate
TGF-b1-induced HSC activation without overt cytotoxic effects. Then, we treated HSCs and
RAW264.7 macrophages with BA-5 to analyze the cross-talk of anti-fibrotic and anti-
inflammatory effects. Carbon tetrachloride (CCl4)-induced liver fibrosis mouse model was
used to evaluate the therapeutic effects of BA-5. Treatment with BA-5 inhibited TGF-b1-
induced a-SMA, collagen1a2, and phosphorylated smad2/3 expression in HSCs.
Furthermore, BA-5 treatment reversed the LPS-induced reduction in BAMBI protein and
decreased IkBa and NF-kB phosphorylation in HSCs. NF-kB nuclear translocation, MCP-1
secretion, and ICAM-1 expression were also inhibited in BA-5-treated HSCs. Conditioned
medium collected from BA-5-treated HSCs showed a reduced ability to activate RAW264.7
macrophages by inhibiting the MAPK pathway. In the mouse model, BA-5 administration
reduced CCl4-induced liver damage, liver fibrosis, and F4/80 expression without any
adverse effects. In conclusion, our study showed that the barbituric acid derivative BA-5
inhibits HSCs activation and liver fibrosis by blocking both the TGF-b1 and LPS-induced
NF-kB signaling pathways and further inhibits macrophages recruitment and activation.
Newcastle University,
United Kingdom
*Correspondence:
Yuh-Ching Twu
yctwu@ym.edu.tw
Ming-Hua Hsu
minghuahsu@cc.ncue.edu.tw
Yi-Jen Liao
yjliao@tmu.edu.tw
Specialty section:
This article was submitted to
Gastrointestinal and
Hepatic Pharmacology,
a section of the journal
Frontiers in Pharmacology
Received: 06 December 2019
Accepted: 13 March 2020
Published: 31 March 2020
Citation:
Wang Y-H, Suk F-M, Liu C-L,
Chen T-L, Twu Y-C, Hsu M-H and
Liao Y-J (2020) Antifibrotic Effects
of a Barbituric Acid Derivative on
Liver Fibrosis by Blocking the
NF-kB Signaling Pathway in
Hepatic Stellate Cells.
Front. Pharmacol. 11:388.
Keywords: barbituric acid derivative, fibrosis, hepatic stellate cells, liver, NF-kB signaling, TGF-b1/Smad, in vitro
doi: 10.3389/fphar.2020.00388
study, animal model
Frontiers in Pharmacology | www.frontiersin.org
1
March 2020 | Volume 11 | Article 388

Wang et al.
Barbituric-Acid Derivative Inhibits Liver Fibrosis
(Marecki et al., 2019), anti-inflammatory (Xu et al., 2016),
anticancer, and antitumor properties (Singh et al., 2009;
Penthala et al., 2015; Laxmi et al., 2016). In 2011, a study
showed that a novel barbituric and thiobarbituric acid
derivative inhibited high-fat/high-calorie diet-induced non-
alcoholic fatty liver disease in male Wistar rats (Ma et al.,
2011). These results provide the impetus for us to develop
novel and potent therapeutic agents containing barbituric acid
and to investigate their pharmacological functions in treating
liver fibrosis. Therefore, the purpose of this study was to identify
the effect of novel barbituric acid derivative on the TGF-b1 and
LPS-induced NF-kB signaling pathways in activated hepatic
stellate cells and further influences macrophages recruitment
by using both in vitro and in vivo experiments.
INTRODUCTION
Liver fibrosis is a pathological response of the liver to a variety of
chronic diseases, such as alcohol consumption, non-alcoholic
steatohepatitis, non-alcoholic fatty liver disease, viral hepatitis,
autoimmune hepatitis, and cholestatic liver diseases (Campana
and Iredale, 2017; Aydin and Akcali, 2018). Liver fibrosis is
characterized by the surplus accumulation of extracellular
matrix, which follows chronic liver injury (Trautwein et al.,
2015). If the injury persists, liver fibrosis can progress to
cirrhosis and ultimately result in the development of liver
cancer (Affo et al., 2017). Hepatic stellate cells (HSCs) are the
major cell type responsible for liver fibrogenesis (Barcena-Varela
et al., 2019). Upon liver injury, HSCs become activated and
transdifferentiate into myofibroblast‐like cells that proliferate
and migrate to regions of hepatocyte injury and produce
collagen and a‐smooth muscle actin (a‐SMA) (Barcena-Varela
et al., 2019). HSC activation is driven by multiple mediators, such
as transforming growth factor-b1 (TGF-b1), platelet-derived
growth factor, and tumor necrosis factor-a (Dewidar et al.,
2019). Among these, TGF-b1 is the most potent mediator in
accelerating liver fibrosis by activating smad2/3 signaling (Hu
et al., 2018). Therefore, TGF-b1 signaling represents a potential
therapeutic target for treating liver fibrosis.
Previous studies indicate that the lipopolysaccharide (LPS)/
Toll-like receptor 4 (TLR4) signaling pathway plays an
important role in liver fibrogenesis (Liu et al., 2014; Kiziltas,
2016). In hepatic fibrosis mouse models and cirrhosis patients,
bacterial translocation and LPS levels in the liver were increased
(Pradere et al., 2010). LPS activates TLR4 signaling through the
adaptor protein MyD88 to activate the downstream NF-kB
signaling pathway and further downregulate expression of the
TGF-b1 pseudoreceptor BAMBI (Kiziltas, 2016). Therefore, LPS
sensitizes HSCs to enhance TGF-b1-induced signaling and
further enhances HSC activation (Petrasek et al., 2013; Kiziltas,
2016). In addition, TLR4 activation also leads to MCP-1
secretion and ICAM-1 upregulation in HSCs, thereby guiding
monocyte recruitment to the liver (Tacke and Zimmermann,
2014). Many studies have demonstrated that hepatic macrophage
infiltration promotes liver fibrosis through the recruitment of
immune cells and the secretion of cytokines and chemokines
(Wynn and Barron, 2010; Tacke and Zimmermann, 2014; Li
et al., 2016). Although several new compounds have been
developed in recent years (Levada et al., 2019), no antifibrotic
therapy has yet been approved by the FDA. Therefore,
modulation of TLR4-MyD88-NF-kB signaling might represent
a feasible strategy for the treatment of liver fibrosis.
MATERIALS AND METHODS
Barbituric Acid Derivative Synthesis
Chemistry
All reactions were carried out in oven-dried glassware (120°C)
under an atmosphere of nitrogen, unless as indicated otherwise.
Acetone, dichloromethane, ethyl acetate, hexane, and
tetrahydrofuran were purchased Mallinckrodt Chemical Co.
Acetone was dried with 4Å molecular sieves and distilled.
Dichloromethane, ethyl acetate, and hexane were dried and
distilled from CaH2. Tetrahydrofuran were dried by distillation
from sodium and benzophenone under an atmosphere of
nitrogen. Melting point was obtained with a Buchi 535 melting
point apparatus. Analytical thin layer chromatography (TLC)
was performed on precoated plates (silica gel 60 F–254),
purchased from Merck Inc. Ultraviolet (UV) spectra were
measured on Hitachi U3300 UV/VIS spectrophotometer.
Infrared (IR) spectra were measured on Jasco FT–IR-5300
Fourier transform infrared spectrometer. The wave numbers
reported are referenced to the polystyrene 1601 cm–1
absorption. Absorption intensities are recorded by the
following abbreviations: s, strong; m, medium; w, weak. 1H
NMR spectra were obtained on a Varian Mercury-400 (400
MHz) spectrometer by use of DMSO-d6 as the solvent and
sodium 3-(trimethylsilyl) propionate as internal standard. 13C
NMR spectra were obtained on a Varian Mercury-400 (100
MHz) spectrometer by use of DMSO-d6 as the solvent.
Multiplicities are recorded by the following abbreviations: s,
singlet; d, doublet; t, triplet; q, quartet; m, multiplet; J, coupling
constant (hertz).
Barbituric acid is an organic compound based on a
pyrimidine heterocyclic skeleton and was first discovered by
the German chemist Adolf von Baeyer in 1864 (Baeyer, 1864).
Although barbituric acid is not pharmacologically active, it can
form a large variety of derivatives called barbiturates that have
been used in many ways (Shahzad et al., 2016). Barbituric acid
derivatives possess different biological activities, such as hypnotic
(Shonle and Moment, 1923; Wisner, 1925), sedative
(Kliethermes et al., 2004), anticonvulsant (Srivastava and
Kumar, 2004), antimicrobial (Dhorajiya et al., 2014), antiviral
Standard Procedure for the Syntheses of
Barbiturate-Conjugated Derivatives
To a mixture of barbituric acid (0.5g, 3.9 mmol) and
arylaldehyde (3.9 mmol) in ethanol (5.0 ml) were set into a
microwave reaction vial tube. The mixture was heated in
microwave reactor at 140°C for 10 min. After the reaction cool
down to room temperature, the mixture was poured into 15 ml
water causing product precipitated and stir for 10 min. The
precipitate was collected and washed by water twice to give
Frontiers in Pharmacology | www.frontiersin.org
2
March 2020 | Volume 11 | Article 388

Wang et al.
Barbituric-Acid Derivative Inhibits Liver Fibrosis
corresponding compounds. All the compounds were confirmed
by 1H NMR and the purity was checked by HPLC (Schemes 1
and 2).
7.017~7.101 (m, 2H, Ar-H), 7.180 (m, 2H, Ar-H), 7.216 (s, 1H,
Ar-H), 11.009 (s, 1H, N-H), 11.586 (s, 1H, N-H).
5,5′-(1,4-phenylenebis(methanylylidene))bis(pyrimidine-
2,4,6(1H,3H,5H)-trione) (8)
1H NMR (DMSO- d6; 400MHz), d: 8.034 (s, 4H, Ar-H), 8.273 (s,
2H, CH=), 11.288 (s, 2H, N-H), 11.436 (s, 2H, N-H). MS (ESI):
m/z, 355.067 (ES+).
5-benzylidenepyrimidine-2,4,6(1H,3H,5H)-trione (4a)
Its spectroscopic characteristics are consistent with those of the
same compound reported (Khalafi-Nezhad and Hashemi, 2001).
5-(4-methoxybenzylidene)pyrimidine-2,4,6(1H,3H,5H)-
trione (4b)
1H NMR (DMSO- d6; 400MHz), d: 3.88 (s, 3H, OCH3), 7.06 (d,
J = 8.8Hz, 2H, Ar-H), 8.25 (s, 1H, CH=), 8.37 (d, J = 8.8Hz, 2H,
Ar-H), 11.18 (s, 1H, N-H), 11.30 (s, 1H, N-H). MS (ES), m/z:
231.2. Its spectroscopic characteristics are consistent with those
of the same compound reported (Luo et al., 2010).
5,5′-(1,3-phenylenebis(methanylylidene))bis(pyrimidine-
2,4,6(1H,3H,5H)-trione) (9)
1H NMR (DMSO- d6; 400MHz), d: 7.541 (t, J = 7.2Hz, 1H, Ar-
H), 8.187 (d, J = 8.8Hz, 2H, Ar-H), 8.264 (s, 1H, Ar-H), 8.472 (s,
2H, CH=), 11.275 (s, 2H, N-H), 11.430 (s, 2H, N-H). MS (ESI):
m/z, 355.068 (ES+).
5-(benzo[d][1,3]dioxol-5-ylmethylene)pyrimidine-2,4,6
(1H,3H,5H)-trione (4c)
1H NMR (DMSO-d6; 400MHz), d: 6.170 (s, 2H, O2CH2), 7.069
(d, J = 8.4 Hz, 1H, Ar-H), 7.729 (d, J = 8.4 Hz, 1H, Ar-H), 8.194
(s, 1H, Ar-H), 8.246 (s, 1H, CH=), 11.207 (s, 1H, N-H), 11.323 (s,
1H, N-H). MS (ESI): m/z, 277.050 (ES+).
Cell Culture
LX2 cells and HSC-T6 cells (an immortalized strain of rat HSCs)
(Twu et al., 2016) were cultured in Dulbecco’s modified Eagle’s
medium (DMEM; Gibco BRL, Grand Island, NY, USA) with 1%
heat-inactivated fetal bovine serum (FBS; HyClone, Logan, UT,
USA), penicillin and streptomycin (100 U/ml), nonessential
amino acids (0.1 mM), and L-glutamine (2 mM) at 37°C in a
5% CO2 incubator. Murine RAW264.7 macrophages (Bioresource
Collection and Research Center, BCRC 60001, Taiwan) were
cultured in DMEM supplemented with 10% FBS, penicillin and
(E)-5-(2-methyl-3-phenylallylidene)pyrimidine-2,4,6
(1H,3H,5H)-trione
(5): 1H NMR (DMSO-d6; 400MHz), d: 2.086 (s, 3H, CH3), 3.949
(s, 1H, CH=C), 4.192 (s, 1H, CH=), 6.492 (s, 1H, Ar-H),
SCHEME 1 | Synthesis of compound 4a, 4b, 4c, and 5.
SCHEME 2 | Synthesis of compound 8 and 9.
Frontiers in Pharmacology | www.frontiersin.org
3
March 2020 | Volume 11 | Article 388

Wang et al.
Barbituric-Acid Derivative Inhibits Liver Fibrosis
streptomycin (100 U/ml), nonessential amino acids (0.1 mM), and
L-glutamine (2 mM) at 37°C in a 5% CO2 incubator.
Nuclear/Cytosolic Fractionation
LX2 cells were seeded in 6-well plates (1 × 105 per well),
pretreated with or without 5 mM BA-5 for 3 h and further
treated with 100 ng/ml LPS for 1 h. Nuclear/cytosolic
fractionation was measured by using a commercial
colorimetric kit (BioVision, Mountain View, CA, USA).
Nuclear/cytosolic fractionation was carried out according to
the manufacturer’s instructions.
AlamarBlue Cell Viability Assay
Cell viability was determined by the commercial alamarBlue cell
viability reagent (Life Technologies, Carlsbad, CA, USA) which
functions as a cell health indicator by using the reducing power
of living cells to quantitatively measure the proliferation of
cultured cells (O’Brien et al., 2000). LX2 cells were seeded (2 ×
104) in a 96-well plate. After 24 and 48 h, 10 ml of alamarBlue
reagent was added to 100 ml culture medium and incubated for
2.5 h at 37°C in a 5% CO2 incubator. The nontoxic alamarBlue
(resazurin) enters living cells and is reduced by mitochondrial
FMNH2, FADH2, NADH, NADPH, and cytochromes. The
reagent transforms from blue nonfluorescent resazurin to pink
fluorescent resorufin. The absorbance was measured at 570 nm,
and we used 600 nm as the reference wavelength (normalized to
the 600 nm value) to quantitatively measure the metabolically
active cells.
ELISA
LX2 cells were seeded in 6-well plates (1 × 105 per well), pretreated
with or without 5 mM BA-5 for 24 h and further treated with 100
ng/ml LPS for 9 h. R&D ELISA kits (MCP-1, IL-6, and TNF-a)
were used to measure the cytokine content of cell supernatants.
ELISAs were carried out according to the manufacturer’s
instructions (R&D Systems, Minneapolis, MN, USA).
Production of HSC‐Derived Conditioned
Medium
LX2 cells were pretreated with 5 mM BA-5 overnight and further
treated with 100 ng/ml LPS for 9 h. The conditioned media were
collected and centrifuged at 1500 rpm for 5 min, and the
supernatant fractions were transferred to new tubes. The
generated conditioned media were kept at -80°C until use.
TGF-b1 Treatment
To study the effect of BA-5 on TGF-b1-induced fibrogenesis,
LX2 cells were (1) seeded in 6-well plates (1 × 105 per well) and
treated with 0, 1.25, 2.5, and 5 mM BA-5 and 10 ng/ml TGF-b1
(R&D Systems, Minneapolis, MN, USA) for 24 h; and (2) seeded
in 6-well plates (1 × 105 per well), pretreated with or without 5
mM BA-5 for 24 h and further treated with 10 ng/ml TGF-b1 for
0, 15, 30, and 60 min. HSC-T6 cells were (1) seeded in 6-well
plates (2.5 × 105 per well) and treated with 5 mM BA-5 and 10 ng/
ml TGF-b1 for 24 h; and (2) seeded in 6-well plates (2 × 105 per
well), pretreated with or without 5 mM BA-5 for 24 h and further
treated with 10 ng/ml TGF-b1 for 30 min.
Animal Model
C57BL/6 male mice, aged 7–8 weeks, were purchased from the
Taiwan National Laboratory Animal Center. All mice were
maintained on a standard chow diet (No. 5001, LabDiet, St
Louis, MO, USA) and housed in conditions with a 12-h light-
dark cycle. The fibrosis model was generated using carbon
tetrachloride (CCl4, Sigma-Aldrich, St Louis, MO, USA). The
mice were assigned randomly to four groups: (1) vehicle control
(0.9% Normal Saline; daily intraperitoneal injection for 7 weeks),
n=5, (2) CCl4 (2 ml/kg; twice-weekly intraperitoneal injection for
6 weeks), n=6, (3) BA-5 (4 mg/kg; daily intraperitoneal injection
for 7 weeks), n=5, and (4) CCl4 plus BA-5 (administered as
previously described; BA-5 pretreatment for 1 week and co-
treatment with CCl4 for 6 weeks), n=6. All animal experiments
were carried out in accordance with the guidelines of the National
Institute of Heath’s Guide for the Care and Use of Laboratory
Animals, and were approved by the Institutional Animal Care and
Use Committee of Taipei Medical University (LAC-2017-0476).
Mouse body weight was monitor once weekly by using an
electronic balance. Liver tissues and serum were collected at the
end of the experiments. The samples used in protein and RNA
analyses were frozen in liquid nitrogen and stored at -80°C, while
those used in IHC staining were fixed in 10% formalin.
LPS Treatment
To study the effect of BA-5 on LPS-induced inflammation, LX2
cells were seeded in 6-well plates (1 × 105 per well), pretreated
with or without 5 mM BA-5 for 24 h and further treated with 100
ng/ml LPS (Sigma-Aldrich, St Louis, MO, USA) for 30 min.
Immunofluorescence Staining
LX2 cells were seeded in 24-well plates (2 × 104 per well),
pretreated with or without 5 mM BA-5 for 3 h and further
treated with 100 ng/ml LPS for 1 h. The culture medium was
removed and then the cells were fixed in 4% paraformaldehyde
for 30 min. After permeabilization with 0.5% Triton X-100 for
5 min at room temperature, the cells were blocked with 0.01 g/ml
albumin for 1 h. Subsequently, the cells were incubated with
antibodies against total-p65 (1:100; Cell Signaling, Beverly, MA,
USA) overnight at 4 °C. Then, the LX2 cells were incubated with
anti-IgG (1:200) for 1 h at room temperature. Finally, the cells
were counterstained with 4′,6-diamidino-2-phenylindole
(DAPI) (1:600; Molecular Probes, Eugene, OR, USA) before
capturing images by using a fluorescence microscope (Nikon
Eclipse TE 2000-U, Tokyo, Japan).
Immunohistochemistry (IHC) Staining and
Blood Biochemical Parameters
The liver from each mouse was removed and fixed in freshly
prepared 10% formalin. The sections were stained with
hematoxylin and eosin (H&E) for histopathological
examination. Sirius red staining (Abcam, Cambridge, MA,
USA) of paraffin-embedded liver sections was used to
Frontiers in Pharmacology | www.frontiersin.org
4
March 2020 | Volume 11 | Article 388

Wang et al.
Barbituric-Acid Derivative Inhibits Liver Fibrosis
qualitatively assess the collagen architecture and the extent of
fibrosis in accordance with the manufacturer’s instructions.
Masson’s Trichrome staining (Abcam, Cambridge, MA, USA)
was carried out in accordance with the manufacturer’s
instructions to investigate the collagen architecture. Paraffin-
embedded liver sections were incubated with the antibodies
against F4/80 (1:200; Cell Signaling, Beverly, MA, USA) and
detected using the Universal LSABTM2 kit (DakoCytomation,
Carpinteria, CA, USA) according to the manufacturer’s
instructions. All sections were investigated by a light
microscope (Olympus CKX41, Olympus Corp., Tokyo, Japan).
Histopathological diagnosis of the mouse liver tissue was
analyzed by gastroenterological physician. Serum alanine
aminotransferase (ALT), albumin (ALB), and blood urea
nitrogen (BUN) values were measured with a biochemical
analyzer (VetTest™, IDEXX, USA).
reverse, 5′-ACATGTTCAGCTTTGTGGACC-3′. a-SMA,
forward, 5′-GTTCAGTGGTGCCTCTGTCA-3′, reverse, 5′-AC
TGGGACGACATGGAAAAG-3′. TGF-b1, forward, 5′-CGAAG
CGGACTACTATGC-3′, reverse, 5′-GTTGCTCCACACTTG
ATTT-3′. MMP-2, forward, 5′-CTCAGATCCGTGGTGAG
AT-3′, reverse, 5′-AGGCTGGTCAGTGGCTTGG-3′. MCP-1,
forward, 5′-ACCGAGAGGCTGAGACTAAC-3′, reverse, 5′-
CAGGTGACTGGGGCATTGAT-3′. IL-6, forward, 5′-TGGCT
GAAAAAGATGGATGCT-3′, reverse, 5′-TCTGCACAGCTC
TGGCTTGT-3′. TNF-a, forward, 5′-TGTAGCCCATGTTGT
AGCAAACC-3′, reverse, 5′-GAGGACCTGGGAGTAGATG
AGGTA-3′. IL-1, forward, 5′-CTGAGCACCTTCTTTCCCTT
CA-3′, reverse, 5′-TGGACCAGACATCACCAAGCT-3′. IL-8,
forward, 5′-CAGCCTTCCTGATTTCTGCAG-3′, reverse, 5′-
AGACAGAGCTCTCTTCCATCAG-3′. IL-1b, forward, 5′-
GCTGATGGCCCTAAACAGATG-3′, reverse, 5′-TTCTCCTG
GAAGGTCTGTGG-3′. F4/80, forward, 5′-CAAGACTGACA
ACCAGACG-3′, reverse, 5′-ACAGAAGCAGAGATTAT
GACC-3′. GAPDH, forward, 5′-TCACCACCATGGAGAA
GGC-3′, reverse, 5′-GCTAAGCAGTTGGTGGTGCA-3′.
Western Blotting
Isolation of total protein from cultured cells was performed by
using lysis buffer supplemented with protease and phosphatase
inhibitors. The protein concentration was measured by protein
assay (BioRad, Hercules, CA, USA), and all samples were
normalized to 30 mg. Cellular proteins were separated by SDS-
PAGE and transferred onto polyvinylidene difluoride (PVDF)
membranes. Primary and secondary antibodies were used to
label the target on the membrane. The a-SMA (ab5694) and
BAMBI (ab57043) antibody used in this study were purchased
from Abcam (Cambridge, MA, USA). The following antibodies
were purchased from Cell Signaling (Beverly, MA, USA): T/p
(Ser465/467; Ser423/425)-smad2/3 (#8685, #8828), T/p
(Ser536)-p65 (#8242, #3033), T/p (Ser176/180)-IKKa/b
(#11930, #8943, #2697), T/p (Ser32)-IkBa (#4814, #2859), T/p
(Thr202/Tyr204)-ERK (#4695, #9101), T/p (Thr183/
Tyr185467)-JNK (#9252, #4668), and T/p (Thr180/Tyr182)-
p38 (#8690, #4511). The immunoblotting signals were
normalized to a-tubulin (T9026) (Sigma-Aldrich, St Louis,
MO, USA). The bands were visualized using an ECL detection
reagent (Millipore Corporation, Billerica, MA, USA).
Statistical Analysis
Data are shown as the mean SD. Data from cell studies were
evaluated by nonparametric tests. For this purpose, the Mann-
Whitney U test was used to compare two independent groups.
Data from animal studies were evaluated by parametric tests. For
this purpose, a two-way analysis of variance was used to make
multiple comparisons. Statistical analyses were performed using
the SPSS program (SPSS Inc., Chicago, IL, USA), and p < 0.05
was considered to indicate statistical significance.
RESULTS
The Compound BA-5 Has the Best Ability
to Ameliorate TGF-b1-Induced HSC
Activation Without Overt Cytotoxic Effects
Regarding the antifibrotic effects of the six different barbituric
acid derivatives on TGF-b1-induced HSC activation, the protein
expression level of a-SMA was analyzed. The results showed that
TGF-b1-induced upregulation of a-SMA was markedly reduced
after treatment with BA-5 (Figure 1A). While in the absence of
TGF-b1 treatment, cells treated with the six different barbituric
acid derivatives did not alter the protein expression of a-SMA
(Figure 1B). To evaluate the cytotoxic effect of BA-5 on HSCs, an
alamarBlue assay was performed. The results showed that after
treatment with the maximum concentration of BA-5 (50 mM) for
48 h, more than 80% of the LX2 cells had survived (Figure 1C).
Therefore, BA-5 was selected for further experiments.
RNA Extraction and Real-Time PCR
Total RNA was isolated from cultured cells by using TRIzol
reagent (Ambion, Carlsbad, CA, USA) according to the
manufacturer’s protocol. High-capacity cDNA reverse
transcription kits (Applied Biosystems, Carlsbad, CA, USA)
were used to produce complementary DNA from cellular RNA
(2 mg). A total of 4 µl template cDNA (20 ng), 5 µl KAPA SYBR®
FAST qPCR master mix (2X), and 1 µl forward/reverse primer
mix (6 µM each) (KAPA Biosystems, Boston, MA, USA) were
added into 48-well PCR plates for each reaction (10 ml). Thermal
cycling consisted of 15 min at 95°C, followed by 40 cycles at 95°C
for 15 s and 60°C for 60 s, using the StepOne System (Applied
Biosystems, Foster City, CA, USA). The predicted cycle
threshold (Ct) values were exported into Excel worksheets for
analysis. Comparative Ct methods, normalized to GAPDH, were
used to determine the gene expression levels. Sequences of
oligonucleotides used as primers were as follows: Collagen1a2
(Col1a2), forward, 5′-TAGGCCATTGTGTATGCAGC-3′,
Treatment With BA-5 Inhibits TGF-b1-
Induced Fibrogenesis in HSCs Cell Lines
To delineate the effect of BA-5 on the inhibition of HSC
activation, the expression levels of a-SMA and Col1a2 were
analyzed. BA-5 significantly inhibited TGF-b1-induced a-SMA
and Col1a2 protein and mRNA expression in HSCs in a dose-
Frontiers in Pharmacology | www.frontiersin.org
5
March 2020 | Volume 11 | Article 388

Wang et al.
Barbituric-Acid Derivative Inhibits Liver Fibrosis
A
C
B
FIGURE 1 | Effects of BA-5 on TGF-b1-induced LX2 cells activation. (A) LX2 cells were treated with 10 ng/ml TGF-b1 and 5 mM of six different barbituric acid
derivatives for 24 h. The protein expression of a-SMA was assessed by the Western blot. (B) LX2 cells were treated with 5 mM of six different barbituric acid
derivatives for 24 h. The protein expression of a-SMA was assessed by the Western blot. (C) LX2 cells were exposed to 0, 3.125, 6.25, 12.5, 25, and 50 mM of BA-
5 for the indicated time periods, and the cell viability was assessed by using an alamarBlue assay kit. The data are expressed as the mean SD. Each experiment
was performed in three independent replicates with similar results. **, p < 0.01; ***, p < 0.001.
dependent manner (Figures 2A, C). While BA-5 alone
administration showed no effect on changing the protein
expression levels of a-SMA (Figure 2B). However, the mRNA
expression of a-SMA and Col1a2 were reduced after treated with
5 mM of BA-5 (Figure 2D). The TGF-b1-induced
phosphorylation of smad2/3 was diminished in BA-5-treated
LX2 cells compared with that of the control (Figure 2E). While
in the absence of TGF-b1 treatment, cells treated with BA-5
showed no change on the protein expression of phosphorylated-
smad2/3 (Figure 2F). To confirm the effect of BA-5 on HSCs
cells, we further used an immortalized strain of rat HSCs cell line,
HSC-T6, in the experiment. The result showed that BA-5
significantly inhibited TGF-b1-induced a-SMA and
phosphorylated-smad2/3 protein expression in HSC-T6 cells,
while BA-5 alone treatment also reduced the protein expression
compared with that of the control (Figure 2G). Besides, BA-5
treatment significantly inhibited TGF-b1-induced a-SMA and
Col1a2 mRNA expression in HSC-T6 cells while BA-5 alone
administration also reduced the mRNA expression of a-SMA
and Col1a2 (Figure 2H). These results demonstrate that BA-5
exhibits an inhibitory effect on TGF-b1-induced HSC activation
in both LX2 and HSC-T6 cells.
treatment with TGF-b1 and LPS significantly elevated the
expression level of a-SMA in HSCs, whereas treatment with
BA-5 reversed LPS-induced BAMBI downregulation and
significantly inhibited a-SMA expression (Figure 3A). In
contrast, BA-5 alone treatment did not affect the protein
expression of BAMBI compared with that of the control
(Figure 3B). To investigate the anti-inflammatory effect of BA-
5 on HSCs, LPS-induced TLR4-MyD88-NF-kB signaling
pathway activation was analyzed. The protein expression of
TLR4 and MyD88 showed no change after treatment with LPS
and BA-5. On the other hand, BA-5 administration ameliorated
LPS-induced IkBa and NF-kB phosphorylation, while
phosphorylated-IKKa/b remained unchanged (Figure 3C). In
the absence of TGF-b1 treatment, cells treated with BA-5 showed
no effect on the regulation of TLR4-MyD88-NF-kB signaling
pathway activation (Figure 3D). Moreover, treatment with BA-5
significantly inhibited LPS-induced total-p65 nuclear
translocation, and these results were confirmed by
immunofluorescence staining (Figures 3E, F). These results
show that BA-5 inhibits LPS-induced NF-kB pathway activation.
BA-5 Inhibits LPS-Induced Chemokine,
Inflammatory Cytokine, and Adhesion
Molecule Expression in HSCs
To assess whether BA-5 could inhibit the production of LPS-
induced chemokines and inflammatory cytokines, HSCs were
pretreated with BA-5 and stimulated with LPS. The results
showed that treatment with BA-5 significantly inhibited LPS-
induced mRNA expression of MCP-1, IL-6, and TNF-a in a
dose-dependent manner, while the expression of IL-1, IL-8, and
IL-1b was not changed (Figure 4A). The secretion levels of
BA-5 Reverses LPS-Induced BAMBI
Downregulation and Inhibits NF-kB
Pathway Activation and p65 Nuclear
Translocation in HSCs
Since LPS is another important factor that influences HSC
activation by downregulating the TGF-b1 pseudoreceptor-
BAMBI (Seki et al., 2007), the effect of BA-5 on BAMBI
expression was also analyzed. The results showed that co-
Frontiers in Pharmacology | www.frontiersin.org
6
March 2020 | Volume 11 | Article 388

Wang et al.
Barbituric-Acid Derivative Inhibits Liver Fibrosis
C
A
B
D
E
F
G
H
FIGURE 2 | BA-5 inhibits TGF-b1 induced HSCs activation in both LX2 and HSC-T6 cells. (A–D) LX2 cells were treated with or without 10 ng/ml TGF-b1 and 0,
1.25, 2.5, and 5 mM of BA-5 for 24 h. Western Blot and real-time PCR were used to evaluate the protein and mRNA expression of a-SMA and Col1a2. (E) LX2 cells
were treated with 5 mM of BA-5 for 24 h and then subjected to 10 ng/ml TGF-b1 for the indicated time periods, and the lysates were analyzed by Western blot and
quantification to detect smad2/3 phosphorylation (►, smad2, ►►, smad3). (F) LX2 cells were treated with 5 mM of BA-5 for 24 h and the lysates were analyzed by
Western blot and quantification to detect smad2/3 phosphorylation. (G) HSC-T6 cells were treated with or without 5 mM of BA-5 and 10 ng/ml TGF-b1 for 24 h.
Western Blot was used to evaluate the protein expression of a-SMA. For detection of smad2/3 phosphorylation, HSC-T6 cells were treated with 5 mM of BA-5 for 24
h and then subjected to 10 ng/ml TGF-b1 for 30 min, and the lysates were analyzed by Western blot. (H) HSC-T6 cells were treated with or without 5 mM of BA-5
and 10 ng/ml TGF-b1 for 24 h. Real-time PCR were used to evaluate the mRNA expression of a-SMA and Col1a2. *, p < 0.05; **, p < 0.01; ***, p < 0.001 vs. white
bar. ##p < 0.01; ###p < 0.001 vs. black bar. Data are shown as mean SD. Each experiment was performed in three independent replicates with similar results.
MCP-1, IL-6, and TNF-a in the culture supernatant were
detected by ELISA. The results showed that treatment with
BA-5 significantly inhibited the production of LPS-induced
MCP-1 in HSCs, while IL-6 was not changed (Figure 4B), and
TNF-a was not detected (data not shown). Since LPS-induced
TLR4 activation also leads to ICAM-1 upregulation in HSCs
(Paik et al., 2003), the protein expression of ICAM-1 was
analyzed. The results showed that treatment with BA-5
significantly inhibited LPS-induced ICAM-1 upregulation in
HSCs (Figure 4C) while BA-5 alone administration did not
change the protein expression level of ICAM-1 (Figure 4D).
These results suggest that BA-5 treatment inhibits LPS-induced
MCP-1 and ICAM-1 expression in HSCs.
The Conditioned Medium From BA-5-
Treated LX2 Cells Showed Reduced Ability
to Activate RAW264.7 Macrophages
Since liver fibrosis is driven by the crosstalk between
macrophages and activated HSCs (Wynn and Barron, 2010;
Tacke and Zimmermann, 2014), we designed experiments to
determine the effect of BA-5 on the interplay between
macrophages and HSCs. LX2 cells were treated with or without
Frontiers in Pharmacology | www.frontiersin.org
7
March 2020 | Volume 11 | Article 388

Wang et al.
Barbituric-Acid Derivative Inhibits Liver Fibrosis
A
B
C
D
F
E
FIGURE 3 | BA-5 reversed LPS-induced BAMBI downregulation and inhibits NF-kB pathway activation and nuclear translocation of p65 in HSCs. (A) LX2 cells were co-
treated with 10 ng/ml TGF-b1, 100 ng/ml LPS, and 5 mM of BA-5 for 72 h. The protein expression of a-SMA and BAMBI were analyzed by the Western blot. (B) LX2 cells
were treated with 5mM of BA-5 for 24 h. The protein expression of BAMBI were analyzed by the Western blot. (C) LX2 cells were pre-treated with 0, 2.5, and 5 mM of BA-5
for 16 h and 100 ng/ml LPS was further treated for 30 min or 48 h. Western blot was used to access the protein expression of TLR4, MyD88, and the phosphorylation of
IKKa/b, IkBa, and NF-kB. (D) LX2 cells were treated with 0, 2.5, and 5 mM of BA-5 for 16 h and western blot was used to access the protein expression of TLR4, MyD88,
and the phosphorylation of IKKa/b, IkBa, and NF-kB. (E) LX2 cells were pre-treated with 5 mM of BA-5 for 3 h and 100 ng/ml LPS was further treated for 1 h. Nuclear/
cytosol fractionation kit was used to separate the cytosol and nuclear protein and the expression of NF-kB was analyzed by using the Western blot. (F) LX2 cells were pre-
treated with 5 mM of BA-5 for 3 h and 100 ng/ml LPS was further treated for 1 h. Immunofluorescence assay was performed to evaluate the total p65 translocation. Each
experiment was performed in three independent replicates with similar results. *p < 0.05; **p < 0.01; ***p < 0.001.
LPS and BA-5 for 9 h, and then the conditioned medium, labeled
A, B, and C, were collected and used to treat the RAW264.7 cells
(Figure 5A). Macrophage stimulation by LPS results in the
phosphorylation of the mitogen-activated protein kinase
(MAPK) family (Su and Karin, 1996); thus, activation of the
MAPK pathway was analyzed. The results showed that
conditioned medium B from LPS-activated HSCs significantly
enhanced RAW264.7 cell activation by upregulating ERK, JNK,
and p38 phosphorylation (Figure 5B). Importantly, the
phosphorylation of ERK, JNK and P38 was decreased when
RAW264.7 cells were exposed to conditioned medium C
(collected from BA-5-treated HSCs) (Figure 5B). When the
MAPK pathway is activated, macrophages activate and express
inflammation-related genes (Lloberas et al., 2016). BA-5-treated
HSC-conditioned medium C showed reduced IL-6, IL-1, TNF-a,
and TGF-b1 mRNA expression compared with that of
conditioned medium B, which was collected from LPS-
activated HSCs (Figure 5C). To determine whether BA-5 can
directly inhibit macrophage activation, RAW264.7 cells were
treated with LPS and compounds BA-4a and BA-5. The
protein phosphorylation of ERK, JNK, and p38 showed no
change in LPS- and BA-5-treated RAW264.7 cells (Figure 5D).
These results demonstrate that BA-5-treated LX2 cells possess
limited ability to activate macrophages.
Frontiers in Pharmacology | www.frontiersin.org
8
March 2020 | Volume 11 | Article 388

Wang et al.
Barbituric-Acid Derivative Inhibits Liver Fibrosis
A
B
D
C
FIGURE 4 | BA-5 decreases MCP-1, IL-6, and ICAM-1 expression in HSCs. (A) LX2 cells were pre-treated with 0, 2.5, and 5mM of BA-5 for 16 h and the cells
were further treated with 100 ng/ml LPS for 3 h. Real-time PCR was used to evaluate the mRNA expression of MCP-1, IL-6, TNF-a, IL-1, IL-8, and IL-1b. (B) LX2
cells were pre-treated with 0, 2.5, and 5mM of BA-5 for 16 h and the cells were further treated with 100 ng/ml LPS for 9 h. DuoSet ELISA DEVELOPMENT SYSTEM
(R&D Systems) was used to evaluate the protein concentration of MCP-1 and IL-6 in the cultured medium. (C) LX2 cells were co-treated with 10 ng/ml TGF-b1 (T),
100 ng/ml LPS (L), and 5 mM of BA-5 for 24 h. The protein expression of ICAM-1 was analyzed by the Western blot. (D) LX2 cells were treated with 5 mM of BA-5
for 24 h. The protein expression of ICAM-1 was analyzed by the Western blot. *p < 0.05; **p < 0.01; ***p < 0.001 vs. white bar. #p < 0.05; ##p < 0.01 vs. black bar.
Data are shown as mean SD. Each experiment was performed in three independent replicates with similar results.
kB and MAPK pathways. The results showed that BA-5
BA-5 Pretreatment Attenuates CCl4-
administration significantly inhibited CCl4-induced NF-kB and
Induced Liver Damage, Liver Fibrosis, and
ERK protein phosphorylation (Figure 6F). The liver function
(ALT and ALB) and kidney function (BUN) values were also
analyzed, and the results showed that CCl4 administration
Macrophage Infiltration in Mice
To further correlate the in vivo antifibrotic effects of BA-5 with the
mechanisms identified in vitro, a CCl4-induced liver fibrosis mouse
significantly increased ALT level while BA-5 administration did
model was used (Figure 6A). The results showed that the protein
not influence the CCl4-induced serum values of ALT (Figure 6G).
expression of a-SMA was increased after the development of CCl4-
Besides, both CCl4 and BA-5 administration did not influence the
induced liver fibrosis, and BA-5 administration significantly
serum values of ALB and BUN (Figure 6G). These data
reduced the protein expression of a-SMA (Figure 6B). The
demonstrate that BA-5 pretreatment ameliorates CCl4-induced
liver damage, liver fibrosis, and macrophage infiltration.
mRNA expression levels of a-SMA, Col1a2, TGF-b1, and MMP2
were significantly increased in the CCl4 group, while BA-5
administration resulted in significant inhibition of the expression
levels of these genes (Figure 6C). H&E staining showed that CCl4
treatment induced more severe fatty change, irregular perinuclear
BA-5 Administration Does Not Induce
Adverse Effects in Mice
Next, we further investigate whether BA-5 alone administration will
affect liver function or cause any adverse effects in vivo. The result
showed that the protein expression of a-SMA was similar between
control and BA-5 treated group (Figure 7A) while the mRNA
expression levels of a-SMA, Col1a2, TGF-b1, and MMP2 were
decreased in BA-5 administration group (Figure 7B). Histological
liver section staining with H&E, Sirius red, and Masson’s trichrome
showed no hepatic damage and fibrillary collagen deposition in BA-
5-treated mice (Figure 7C). The mRNA expression levels of F4/80
was reduced in BA-5 administration group (Figure 7D) while the
IHC staining of F4/80 showed that the protein expression of F4/80
remained unchanged compared with that of vehicle-treated mice
(Figure 7C). In addition, the protein expression of phosphorylated-
vacuoles, and regional inflammation in mice liver (Figure 6D). In
contrast, BA-5 treatment ameliorated CCl4-induced liver damage
(Figure 6D). Collagen deposition, a marker for liver fibrosis, was
assessed by Sirius red and Masson’s trichrome staining. As shown in
Figure 6D, fibrillary collagen deposition was observed in CCl4-
treated mice, notably, this phenomenon was attenuated in BA-5
treated mice. Macrophage infiltration in the liver was assessed by
the IHC staining of F4/80, and the results showed that BA-5
administration significantly inhibited CCl4-induced F4/80
expression (Figure 6D). The mRNA expression levels of F4/80
were also analyzed, and the results showed that BA-5 administration
significantly inhibited CCl4-induced F4/80 mRNA expression
(Figure 6E). Furthermore, we analyzed the regulation of the NF-
Frontiers in Pharmacology | www.frontiersin.org
9
March 2020 | Volume 11 | Article 388

Wang et al.
Barbituric-Acid Derivative Inhibits Liver Fibrosis
A
B
C
D
FIGURE 5 | The conditioned medium from BA-5-treated LX2 possessed limited ability in activation of RAW264.7 macrophages. (A) Schematic diagram of the effects of BA-5
between HSCs and macrophage. (B) RAW264.7 cells were treated with three different conditioned mediums respectively for 24 h, and the protein phosphorylation of ERK,
JNK, and p-38 were analyzed by the Western blot. (C) RAW264.7 cells were incubated with three different conditioned mediums respectively for 24 h. Real-time PCR was
used to evaluate the mRNA expression of IL-6, IL-1, TNF-a, and TGF-b1. (D) RAW264.7 cells were treated with 100 ng/ml LPS and 5 mM of BA-4a and BA-5 for 24 h, and
the protein phosphorylation of ERK, JNK, and p-38 were analyzed by the Western blot. *p < 0.05; **p < 0.01; ***p < 0.001 vs. white bar. #p < 0.05; ##p < 0.01 vs. black bar.
Data are shown as mean SD. Each experiment was performed in three independent replicates with similar results.
NF-kB and ERK were no significantly differences between BA-5-
treated and vehicle-treated mice (Figure 7E). The serum values of
ALT, ALB, and BUN were not significantly different between BA-5-
treated and vehicle-treated mice (Figure 7F). To assess the in vivo
toxicities mediated by BA-5, mouse body weights were measured
before sacrificed, and the results showed that the mouse body
weights were similar among the various groups (Figure 7G).
urgently needed (Levada et al., 2019). In this study, we generated six
different barbituric acid derivatives and selected compound BA-5 as
the candidate with the most potential. From the in vitro
experiments, we found that BA-5 treatment significantly inhibited
TGF-b1-induced a-SMA, Col1a2, and phosphorylated-smad2/3
expression and effectively increased expression of the TGF-b1
pseudoreceptor BAMBI. Moreover, treatment with BA-5
decreased LPS-induced IkBa and NF-kB phosphorylation, NF-kB
nuclear translocation, MCP-1 protein secretion, and ICAM-1
protein expression in HSCs. In addition, the conditioned medium
from BA-5-treated HSCs showed reduced ability to promote
macrophages activation. In the in vivo experiment, BA-5
administration reduced CCl4-induced liver damage, liver fibrosis,
and F4/80 expression without any adverse effects in mice (Figure 8).
Barbituric acid was first prepared by Adolf Von Baeyer in 1864
from the fusion of urea and malonic acid (Baeyer, 1864). The
DISCUSSION
Liver fibrosis is the first step toward the progression of cirrhosis and
hepatocellular carcinoma (Friedman, 2008). Despite our increasing
understanding of the disease, there are no effective and clinically
approved antifibrotic therapies available (Trautwein et al., 2015).
Thus, the identification of a new drug for treating liver fibrosis is
Frontiers in Pharmacology | www.frontiersin.org
10
March 2020 | Volume 11 | Article 388

Wang et al.
Barbituric-Acid Derivative Inhibits Liver Fibrosis
A
B
C
E
D
F
G
FIGURE 6 | BA-5 pretreatment decreased CCl4-induced liver damage, liver fibrosis, and macrophage infiltration. (A) Study design of the in vivo experiment. (B)
Representative results from Western blot analyses of a-SMA expression in three groups. (C) Results from QPCR analyses of a-SMA, Col1a2, TGF-b1, and MMP2
gene expression in three groups. (D) Representative H&E, Sirius red, Masson’s trichrome, and F4/80 IHC staining images of liver tissues from control, CCl4, and BA-
5+CCl4 mice. Original magnification, ×100. D, fatty change; #, perinuclear vacuoles; ↑, inflammatory infiltration. (E) Results from QPCR analyses of F4/80 gene
expression in three groups. (F) Representative results from Western blot analyses of T/p-p65, and T/p-ERK expression in three groups. (G) Serum samples were
collected at the end of the experiment, and the serum ALT, ALB, and BUN values were assessed.*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 vs. white
bar. #p < 0.05; ##p < 0.01; ###p < 0.001 vs. black bar. Data are shown as mean SD.
Frontiers in Pharmacology | www.frontiersin.org
11
March 2020 | Volume 11 | Article 388

Wang et al.
Barbituric-Acid Derivative Inhibits Liver Fibrosis
A
B
C
D
F
E
G
FIGURE 7 | Adverse effects of BA-5 administration in mice. (A) Representative results from Western blot analyses of a-SMA expression and (B) QPCR analyses of
a-SMA, Col1a2, TGF-b1, and MMP2 gene expression. (C) H&E, Sirius red, Masson’s trichrome, and F4/80 IHC staining of liver tissues from each treatment group.
(D) Results from QPCR analyses of F4/80 gene expression and (E) Western blot analyses of T/p-p65, and T/p-ERK expression. (F) The serum ALT, ALB, and BUN
values in each group. (G) The value of mouse body weight before sacrificed. *p < 0.05; **p < 0.01; ***p < 0.001 vs. black bar.
derivatives of barbituric acid, known as barbiturates, have a special
place in pharmaceutical chemistry because of the susceptibility to
rapid metabolic attack and subsequent degradation of the
compounds by acidic hydrogen within the body (Adams, 1958).
Substances of the barbituric acid group have been used
therapeutically for many years. Barbiturates are known as central
nervous system depressants and are mainly used in the treatment of
anxiety and epilepsy and to induce sleep and anesthesia (Shonle and
Moment, 1923; Sandberg, 1951; Andrews et al., 1979; Srivastava and
Kumar, 2004). Recently, it was found that barbituric acid derivatives
possess anticancer effects (Singh et al., 2009; Dhorajiya et al., 2014).
In liver diseases, barbituric acid derivatives alleviate the
development of non-alcoholic fatty liver disease by regulating
adipocytokine expression and preventing insulin resistance (Ma
et al., 2011; Zheng et al., 2011). However, the effect of barbituric acid
derivatives on liver fibrosis has not been reported until now. In this
study, we synthesized a series of barbituric acid derivatives and
investigated the detailed antifibrotic mechanisms. Our results
demonstrated that the compound BA-5 not only exerts an anti-
inflammatory effect on the HSCs and attenuates subsequent
macrophages activation, but also inhibited CCl4-induced liver
fibrogenesis. Notably, BA-5 alone treatment demonstrated the
Frontiers in Pharmacology | www.frontiersin.org
12
March 2020 | Volume 11 | Article 388

Wang et al.
Barbituric-Acid Derivative Inhibits Liver Fibrosis
FIGURE 8 | Compound BA-5 inhibits HSCs activation through blocking both TGF-b1 and LPS-induced NF-kB signaling pathways and further inhibits macrophage
recruitment and activation. BA-5 inhibited TGF-b1-induced a-SMA, col1a2, and phosphorylated-smad2/3 expression in HSCs. Furthermore, BA-5 treatment
enhanced BAMBI expression and inhibited LPS-induced IkBa and NF-kB phosphorylation, NF-kB nucleus translocation, MCP-1 secretion, and ICAM-1 expression.
And then, reduced the activation of macrophage (RAW264.7) by inhibiting the protein phosphorylation of ERK, JNK, and p38.
ability in inhibiting the mRNA expression of fibrogenesis makers in
vitro and in vivo (Figures 2C, D, H, and Figure 7B). In contrast, the
protein levels of a-SMA and phosphorylated-smad2/3 were not
significantly changed except in the HSC-T6 cell lines (Figure 2G).
This study provides new insight into the potential of barbituric acid
derivatives in treating liver fibrosis. And further study is required to
clarify the inhibitory effects of BA-5 at transcription level.
suppressed the activation of TLR4 downstream signal transduction,
including phosphorylation of IkBa and NF-kB nuclear
translocation (Figure 3). These results indicate that the anti-
inflammatory and antifibrogenic effects of BA-5 may be
associated with the inhibition of both the TGF-b1 and NF-kB
signaling pathways. NF-kB contributes to the survival and
development of the cells that carry out immune responses in
mammals (Zhang et al., 2017). Although there are some FDA-
approved drugs used in treating multiple myeloma with the strategy
to suppress NF-kB, theses agents have been thwarted by side effects
including nephrotoxicity, neuropathy, and the malignancy (Mina
et al., 2016). In previous study, a barbituric acid derivative was
found to posse the ability to block IL-2 production in activated
Jurkat T cells (Xu et al., 2016). In our animal study, we did not find
any body weight lost, liver and kidney damage in BA-5
administrated mice group (Figure 7). However, we cannot
exclude the possibility of disrupting the innate immune response
or affecting immunomodulatory activity while using BA-5.
Therefore, further studies are required in the future to confirm
the safety of using this agent.
A previous study found that fibrogenesis is a dynamic process that
is tightly regulated by macrophage-stellate cell interactions (Chang
et al., 2013). Crosstalk between macrophages and stellate cells drives
fibrogenic processes via juxtacrine or paracrine signaling pathways
(Holt et al., 2010). In the liver, HSCs regulate leukocyte trafficking and
activation through secretion of MCP-1 and overexpression of ICAM-
1 (Tacke and Zimmermann, 2014). It has been found that the
expression of chemokine (MCP-1 and IL-8) and adhesion molecule
(ICAM-1 and VCAM-1) genes are regulated by NF-kB (Baeuerle and
Henkel, 1994; Tacke and Zimmermann, 2014). Moreover, patients
Liver fibrosis is tightly associated with chronic liver
inflammation. After liver injury, intestinal mucosal permeability
and bacterial translocation are increased and lead to elevated LPS
levels in the portal and systemic circulation (Ponziani et al., 2018). A
previous study found that LPS was increased in hepatic fibrosis
mouse models and cirrhosis patients (Wiest and Garcia-Tsao, 2005;
Pradere et al., 2010). The molecular link between liver fibrosis and
inflammation was shown to be TLR4 signaling, which promotes
HSC activation and modulates TGF-b1 signaling (Seki et al., 2007).
Therefore, the investigation of potential inhibitors of both TLR4
signaling and TGF-b1 signaling represents an attractive strategy for
the treatment of liver fibrosis. Our results demonstrated that the
compound BA-5 exerts a suppressive effect on the TGF-b1 signaling
pathway by inhibiting a-SMA, Col1a2, and phosphorylated-smad2/
3 expression (Figure 2). In addition, BA-5 not only directly
suppressed TGF-b1-induced HSC activation but also inhibited
LPS-enhanced sensitivity of HSCs to TGF-b1. Similar to Seki el
al. (Seki et al., 2007), we found that LPS-induced TLR4 pathway
activation could sensitize HSCs to TGF-b1 stimulation through
downregulation of the TGF-b1 pseudoreceptor BAMBI (Figure
3A). BA-5 was able to inhibit the LPS-mediated sensitization of LX2
cells to TGF-b1. The mechanism by which BA-5 inhibits LPS-
mediated profibrogenic effects might relate to the findings that BA-5
Frontiers in Pharmacology | www.frontiersin.org
13
March 2020 | Volume 11 | Article 388

Wang et al.
Barbituric-Acid Derivative Inhibits Liver Fibrosis
with hepatitis (Leifeld et al., 2003) and experimental animal liver
injury models (Paik et al., 2003) have elevated expression levels of
MCP-1. Our data showed that BA-5 treatment inhibited LPS-induced
mRNA expression and secretion of MCP-1 and protein expression of
ICAM-1 (Figure 4). To further explore the interplay between HSCs
and macrophages, we used a conditioned medium study, and the
results demonstrated that LPS-activated HSCs drove the activation of
macrophages, and BA-5 treatment reversed the effects (Figure 5).
Furthermore, in CCl4-induced liver fibrogenesis mouse models, we
showed that BA-5 not only inhibited liver fibrosis but also reduced
macrophage infiltration in vivo (Figure 6).
In conclusion, our research showed that the barbituric acid
derivative BA-5 efficiently reduces hepatic stellate cells activation
through inhibition of the NF-kB and TGF-b1 signalling
pathways and further alleviates liver fibrosis formation in
CCl4-induced mouse models. Altogether, these results suggest
the dual therapeutic potential of BA-5 as an inhibitor of both
liver fibrosis and inflammation pathways.
ETHICS STATEMENT
The animal study was reviewed and approved by Institutional
Animal Care and Use Committee of Taipei Medical University
(LAC-2017-0476).
AUTHOR CONTRIBUTIONS
Conceptualization, Y-JL and Y-CT. Methodology, C-LL, M-HH,
and T-LC. Validation, M-HH. Formal analysis, F-MS and Y-HW.
Data curation, Y-CT and M-HH. Writing—original draft
preparation, Y-HW and M-HH. Writing—review and editing,
F-MS and C-LL. Supervision, Y-JL.
FUNDING
DATA AVAILABILITY STATEMENT
This study was partially supported by a grant from the Ministry
of Science and Technology of the Republic of China (MOST 107-
2320-B-038-021 and 108-2628-B-038-004).
All datasets generated for this study are included in the article/
supplementary material.
Holt, D. J., Chamberlain, L. M., and Grainger, D. W. (2010). Cell-cell signaling in
co-cultures of macrophages and fibroblasts. Biomaterials 31 (36), 9382–9394.
doi: 10.1016/j.biomaterials.2010.07.101
Hu, H. H., Chen, D. Q., Wang, Y. N., Feng, Y. L., Cao, G., Vaziri, N. D., et al.
(2018). New insights into TGF-beta/Smad signaling in tissue fibrosis. Chem.
Biol. Interact. 292, 76–83. doi: 10.1016/j.cbi.2018.07.008
Khalafi-Nezhad, A., and Hashemi, A. (2001). Microwave enhanced knoevenagel
condensation of barbituric acid with aromatic aldehydes on basic alumina. Iran
J. Chem. Chem. Eng. 20 (1), 9–11.
Kiziltas, S. (2016). Toll-like receptors in pathophysiology of liver diseases. World J.
Hepatol. 8 (32), 1354–1369. doi: 10.4254/wjh.v8.i32.1354
REFERENCES
Adams, E. (1958). Barbiturates. Sci. Am. 198 (1), 60–67. doi: 10.1038/
scientificamerican0158-60
Affo, S., Yu, L. X., and Schwabe, R. F. (2017). The Role of Cancer-Associated
Fibroblasts and Fibrosis in Liver Cancer. Annu. Rev. Pathol. 12, 153–186.
doi: 10.1146/annurev-pathol-052016-100322
Andrews, P. R., Jones, G. P., and Lodge, D. (1979). Convulsant, anticonvulsant and
anaesthetic barbiturates. 5-Ethyl-5-(3′-methyl-but-2′-enyl)-barbituric acid and
related compounds. Eur. J. Pharmacol. 55 (2), 115–120. doi: 10.1016/0014-
2999(79)90382-0
Aydin, M. M., and Akcali, K. C. (2018). Liver fibrosis. Turk J. Gastroenterol. 29 (1),
14–21. doi: 10.5152/tjg.2018.17330
Baeuerle, P. A., and Henkel, T. (1994). Function and activation of NF-kappa B in
the immune system. Annu. Rev. Immunol. 12, 141–179. doi: 10.1146/
annurev.iy.12.040194.001041
Kliethermes, C. L., Metten, P., Belknap, J. K., Buck, K. J., and Crabbe, J. C. (2004).
Selection for pentobarbital withdrawal severity: correlated differences in
withdrawal from other sedative drugs. Brain Res. 1009 (1-2), 17–25.
doi: 10.1016/j.brainres.2004.02.040
Laxmi, S. V., Rajitha, G., Rajitha, B., and Rao, A. J. (2016). Photochemical
synthesis and anticancer activity of barbituric acid, thiobarbituric acid,
thiosemicarbazide, and isoniazid linked to 2-phenyl indole derivatives. J.
Chem. Biol. 9 (2), 57–63. doi: 10.1007/s12154-015-0148-y
Leifeld, L., Dumoulin, F. L., Purr, I., Janberg, K., Trautwein, C., Wolff, M., et al.
(2003). Early up-regulation of chemokine expression in fulminant hepatic
failure. J. Pathol. 199 (3), 335–344. doi: 10.1002/path.1298
Levada, K., Omelyanchik, A., Rodionova, V., Weiskirchen, R., and Bartneck, M.
(2019). Magnetic-Assisted Treatment of Liver Fibrosis. Cells 8 (10), 1279.
doi: 10.3390/cells8101279
Li, H., You, H., Fan, X., and Jia, J. (2016). Hepatic macrophages in liver fibrosis:
pathogenesis and potential therapeutic targets. BMJ Open Gastroenterol. 3 (1),
e000079. doi: 10.1136/bmjgast-2016-000079
Liu, C., Chen, X., Yang, L., Kisseleva, T., Brenner, D. A., and Seki, E. (2014).
Transcriptional repression of the transforming growth factor beta (TGF-beta)
Pseudoreceptor BMP and activin membrane-bound inhibitor (BAMBI) by
Nuclear Factor kappaB (NF-kappaB) p50 enhances TGF-beta signaling in
hepatic stellate cells. J. Biol. Chem. 289 (10), 7082–7091. doi: 10.1074/
jbc.M113.543769
Baeyer, A. (1864). Mittheilungen aus dem organischen Laboratorium des
Gewerbeinstitutes in Berlin: Untersuchungen über die Harnsäuregruppe.
Justus Liebigs Ann. der Chem. 130 (2), 129–175. doi: 10.1002/jlac.18641300202
Barcena-Varela, M., Colyn, L., and Fernandez-Barrena, M. G. (2019). Epigenetic
Mechanisms in Hepatic Stellate Cell Activation During Liver Fibrosis and
Carcinogenesis. Int. J. Mol. Sci. 20 (10), 2507–2523. doi: 10.3390/ijms20102507
Campana, L., and Iredale, J. P. (2017). Regression of Liver Fibrosis. Semin. Liver
Dis. 37 (1), 1–10. doi: 10.1055/s-0036-1597816
Chang, J., Hisamatsu, T., Shimamura, K., Yoneno, K., Adachi, M., Naruse, H., et al.
(2013). Activated hepatic stellate cells mediate the differentiation of macrophages.
Hepatol. Res. 43 (6), 658–669. doi: 10.1111/j.1872-034X.2012.01111.x
Dewidar, B., Meyer, C., Dooley, S., and Meindl-Beinker, A. N. (2019). TGF-beta in
Hepatic Stellate Cell Activation and Liver Fibrogenesis-Updated 2019. Cells 8
(11), 1419–1454. doi: 10.3390/cells8111419
Dhorajiya, B. D., Dholakiya, B. Z., and Mohareb, R. M. (2014). Hybrid probes of
aromatic amine and barbituric acid: highly promising leads for anti-bacterial,
anti-fungal and anti-cancer activities. Med. Chem. Res. 23 (9), 3941–3952. doi:
10.1007/s00044-014-0973-5
Friedman, S. L. (2008). Mechanisms of hepatic fibrogenesis. Gastroenterology 134
(6), 1655–1669. doi: 10.1053/j.gastro.2008.03.003
Lloberas, J., Valverde-Estrella, L., Tur, J., Vico, T., and Celada, A. (2016). Mitogen-
Activated Protein Kinases and Mitogen Kinase Phosphatase 1: A Critical
Frontiers in Pharmacology | www.frontiersin.org
14
March 2020 | Volume 11 | Article 388

Wang et al.
Barbituric-Acid Derivative Inhibits Liver Fibrosis
Interplay in Macrophage Biology. Front. Mol. Biosci. 3, 28. doi: 10.3389/
fmolb.2016.00028
Singh, P., Kaur, M., and Verma, P. (2009). Design, synthesis and anticancer
activities of hybrids of indole and barbituric acids–identification of highly
promising leads. Bioorg. Med. Chem. Lett. 19 (11), 3054–3058. doi: 10.1016/
j.bmcl.2009.04.014
Srivastava, V., and Kumar, A. (2004). Synthesis of some newer derivatives of
substituted quinazolinonyl-2-oxo/thiobarbituric acid as potent anticonvulsant
agents. Bioorg. Med. Chem. 12 (5), 1257–1264. doi: 10.1016/j.bmc.2003.08.035
Su, B., and Karin, M. (1996). Mitogen-activated protein kinase cascades and
regulation of gene expression. Curr. Opin. Immunol. 8 (3), 402–411. doi:
10.1016/S0952-7915(96)80131-2
Luo, Y., Ma, L., Zheng, H., Chen, L., Li, R., He, C., et al. (2010). Discovery of (Z)-5-
(4-methoxybenzylidene)thiazolidine-2,4-dione, a readily available and orally
active glitazone for the treatment of concanavalin A-induced acute liver injury
of BALB/c mice. J. Med. Chem. 53 (1), 273–281. doi: 10.1021/jm901183d
Ma, L., Li, S., Zheng, H., Chen, J., Lin, L., Ye, X., et al. (2011). Synthesis and
biological activity of novel barbituric and thiobarbituric acid derivatives against
non-alcoholic fatty liver disease. Eur. J. Med. Chem. 46 (6), 2003–2010.
doi: 10.1016/j.ejmech.2011.02.033
Marecki, J. C., Aarattuthodiyil, S., Byrd, A. K., Penthala, N. R., and Crooks, P. A.
(2019). Raney KD. N-Naphthoyl-substituted indole thio-barbituric acid
analogs inhibit the helicase activity of the hepatitis C virus NS3. Bioorg.
Med. Chem. Lett. 29 (3), 430–434. doi: 10.1016/j.bmcl.2018.12.026
Mina, R., Cerrato, C., Bernardini, A., Aghemo, E., and Palumbo, A. (2016). New
pharmacotherapy options for multiple myeloma. Expert Opin. Pharmacother.
17 (2), 181–192. doi: 10.1517/14656566.2016.1115016
O’Brien, J., Wilson, I., Orton, T., and Pognan, F. (2000). Investigation of the Alamar
Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity.
Eur. J. Biochem. 267 (17), 5421–5426. doi: 10.1046/j.1432-1327.2000.01606.x
Paik, Y. H., Schwabe, R. F., Bataller, R., Russo, M. P., Jobin, C., and Brenner, D. A.
(2003). Toll-like receptor 4 mediates inflammatory signaling by bacterial
lipopolysaccharide in human hepatic stellate cells. Hepatology 37 (5), 1043–
1055. doi: 10.1053/jhep.2003.50182
Penthala, N. R., Ketkar, A., Sekhar, K. R., Freeman, M. L., Eoff, R. L., Balusu, R.,
et al. (2015). 1-Benzyl-2-methyl-3-indolylmethylene barbituric acid
derivatives: Anti-cancer agents that target nucleophosmin 1 (NPM1). Bioorg.
Med. Chem. 23 (22), 7226–7233. doi: 10.1016/j.bmc.2015.10.019
Petrasek, J., Csak, T., and Szabo, G. (2013). Toll-like receptors in liver disease. Adv.
Clin. Chem. 59, 155–201. doi: 10.1016/b978-0-12-405211-6.00006-1
Ponziani, F. R., Zocco, M. A., Cerrito, L., Gasbarrini, A., and Pompili, M. (2018).
Bacterial translocation in patients with liver cirrhosis: physiology, clinical
consequences, and practical implications. Expert Rev. Gastroenterol. Hepatol.
12 (7), 641–656. doi: 10.1080/17474124.2018.1481747
Tacke, F., and Zimmermann, H. W. (2014). Macrophage heterogeneity in liver
injury and fibrosis. J. Hepatol. 60 (5), 1090–1096. doi: 10.1016/
j.jhep.2013.12.025
Trautwein, C., Friedman, S. L., Schuppan, D., and Pinzani, M. (2015). Hepatic
fibrosis: Concept to treatment. J. Hepatol. 62 (1 Suppl), S15–S24. doi: 10.1016/
j.jhep.2015.02.039
Twu, Y. C., Lee, T. S., Lin, Y. L., Hsu, S. M., Wang, Y. H., Liao, C. Y., et al. (2016).
Niemann-Pick Type C2 Protein Mediates Hepatic Stellate Cells Activation by
Regulating Free Cholesterol Accumulation. Int. J. Mol. Sci. 17 (7), 1122–1134.
doi: 10.3390/ijms17071122
Wiest, R., and Garcia-Tsao, G. (2005). Bacterial translocation (BT) in cirrhosis.
Hepatology 41 (3), 422–433. doi: 10.1002/hep.20632
Wisner, F. P. (1925). A Comparative Study of the General Systemic and Dental
Conditions of Fourteen Dental Students. Cal West Med. 23 (8), 980–981.
Wynn, T. A., and Barron, L. (2010). Macrophages: master regulators of
inflammation and fibrosis. Semin. Liver Dis. 30 (3), 245–257. doi: 10.1055/s-
0030-1255354
Xu, C., Wyman, A. R., Alaamery, M. A., Argueta, S. A., Ivey, F. D., Meyers, J. A.,
et al. (2016). Anti-inflammatory effects of novel barbituric acid derivatives in T
lymphocytes. Int. Immunopharmacol. 38, 223–232. doi: 10.1016/
j.intimp.2016.06.004
Zhang, Q., Lenardo, M. J., and Baltimore, D. (2017). 30 Years of NF-kappaB: A
Blossoming of Relevance to Human Pathobiology. Cell 168 (1-2), 37–57.
doi: 10.1016/j.cell.2016.12.012
Pradere, J. P., Troeger, J. S., Dapito, D. H., Mencin, A. A., and Schwabe, R. F.
(2010). Toll-like receptor 4 and hepatic fibrogenesis. Semin. Liver Dis. 30 (3),
232–244. doi: 10.1055/s-0030-1255353
Sandberg, F. (1951). Anaesthetic Properties of Some New N-substituted and N, N
′-disubstituted Derivatives of 5, 5-Diallyl-Barbituric Acid. Acta Physiol. Scand.
24 (1), 7–26. doi: 10.1111/j.1748-1716.1951.tb00823.x
Zheng, H., Li, S., Ma, L., Cheng, L., Deng, C., Chen, Z., et al. (2011). A novel
agonist of PPAR-gamma based on barbituric acid alleviates the development of
non-alcoholic fatty liver disease by regulating adipocytokine expression and
preventing insulin resistance. Eur. J. Pharmacol. 659 (2-3), 244–251.
doi: 10.1016/j.ejphar.2011.03.033
Seki, E., De Minicis, S., Osterreicher, C. H., Kluwe, J., Osawa, Y., Brenner, D. A.,
et al. (2007). TLR4 enhances TGF-beta signaling and hepatic fibrosis. Nat.
Med. 13 (11), 1324–1332. doi: 10.1038/nm1663
Conflict of Interest: The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be construed as a
potential conflict of interest.
Shahzad, S., Shahzadi, L., Mahmood, N., Siddiqi, S. A., Rauf, A., Manzoor, F., et al.
(2016). A new synthetic methodology for the preparation of biocompatible and
organo-soluble barbituric- and thiobarbituric acid based chitosan derivatives
for biomedical applications. Mater. Sci. Eng. C. Mater. Biol. Appl. 66, 156–163.
doi: 10.1016/j.msec.2016.04.056
Copyright © 2020 Wang, Suk, Liu, Chen, Twu, Hsu and Liao. This is an open-access
article distributed under the terms of the Creative Commons Attribution License (CC
BY). The use, distribution or reproduction in other forums is permitted, provided the
original author(s) and the copyright owner(s) are credited and that the original
publication in this journal is cited, in accordance with accepted academic practice. No
use, distribution or reproduction is permitted which does not comply with these terms.
Shonle, H., and Moment, A. (1923). Some new hypnotics of the barbituric acid
series. J. Am. Chem. Soc. 45 (1), 243–249. doi: 10.1021/ja01654a033
Frontiers in Pharmacology | www.frontiersin.org
15
March 2020 | Volume 11 | Article 388