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Citation: Inoue, T.; Fu, B.; Nishio, M.;
Tanaka, M.; Kato, H.; Tanaka, M.;
Itoh, M.; Yamakage, H.; Ochi, K.; Ito,
A.; et al. Novel Therapeutic
Potentials of Taxifolin for
Obesity-Induced Hepatic Steatosis,
Fibrogenesis, and Tumorigenesis.
Nutrients 2023,15, 350. https://
doi.org/10.3390/nu15020350
Academic Editor: Jean-Louis
Guéant
Received: 28 November 2022
Revised: 5 January 2023
Accepted: 6 January 2023
Published: 10 January 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
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Attribution (CC BY) license (https://
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4.0/).
nutrients
Article
Novel Therapeutic Potentials of Taxifolin for Obesity-Induced
Hepatic Steatosis, Fibrogenesis, and Tumorigenesis
Takayuki Inoue 1, †, Bin Fu 2 ,3,†, Miwako Nishio 4, Miyako Tanaka 2,3,5,*, Hisashi Kato 1, Masashi Tanaka 1,6 ,
Michiko Itoh 2, Hajime Yamakage 1, Kozue Ochi 2, Ayaka Ito 2,3,7, Yukihiro Shiraki 8, Satoshi Saito 9,
Masafumi Ihara 9, Hideo Nishimura 10, Atsuhiko Kawamoto 10 , Shian Inoue 4, Kumiko Saeki 4,
Atsushi Enomoto 8, Takayoshi Suganami 2,3,5,11 and Noriko Satoh-Asahara 1,12 ,*
1
Department of Endocrinology, Metabolism and Hypertension Research, Clinical Research Institute, National
Hospital Organization Kyoto Medical Center, Kyoto 612-8555, Japan
2
Department of Molecular Medicine and Metabolism, Research Institute of Environmental Medicine, Nagoya
University, Nagoya 464-8601, Japan
3Department of Immunometabolism, Nagoya University Graduate School of Medicine,
Nagoya 464-8601, Japan
4Department of Laboratory Molecular Genetics of Hematology, Graduate School of Medical and Dental
University, Tokyo Medical and Dental University, Tokyo 113-8510, Japan
5Institute of Nano-Life-Systems, Institutes of Innovation for Future Society, Nagoya University,
Nagoya 464-8601, Japan
6Department of Physical Therapy, Health Science University, Yamanashi 401-0380, Japan
7Institute for Advanced Research, Nagoya University, Nagoya 464-8601, Japan
8Department of Pathology, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan
9Department of Neurology, National Cerebral and Cardiovascular Center, Osaka 564-8565, Japan
10
Translational Research Center for Medical Innovation, Foundation for Biomedical Research and Innovation at
Kobe, Kobe 650-0047, Japan
11 Center for One Medicine Innovative Translational Research, Gifu University Institute for Advanced Study,
Gifu 501-11193, Japan
12 Department of Metabolic Syndrome and Nutritional Science, Research Institute of Environmental Medicine,
Nagoya University, Nagoya 466-8550, Japan
*Correspondence: tanaka@riem.nagoya-u.ac.jp (M.T.); nsatoh@kuhp.kyoto-u.ac.jp (N.S.-A.);
Tel.: +81-75-641-9161 (M.T.); +81-52-789-3883 (N.S.-A.)
† These authors contributed equally to this work.
Abstract:
The molecular pathogenesis of nonalcoholic steatohepatitis (NASH) includes a complex
interaction of metabolic stress and inflammatory stimuli. Considering the therapeutic goals of NASH,
it is important to determine whether the treatment can prevent the progression from NASH to
hepatocellular carcinoma. Taxifolin, also known as dihydroquercetin, is a natural bioactive flavonoid
with antioxidant and anti-inflammatory properties commonly found in various foods and health
supplement products. In this study, we demonstrated that Taxifolin treatment markedly prevented
the development of hepatic steatosis, chronic inflammation, and liver fibrosis in a murine model of
NASH. Its mechanisms include a direct action on hepatocytes to inhibit lipid accumulation. Taxifolin
also increased brown adipose tissue activity and suppressed body weight gain through at least two
distinct pathways: direct action on brown adipocytes and indirect action via fibroblast growth factor
21 production in the liver. Notably, the Taxifolin treatment after NASH development could effectively
prevent the development of liver tumors. Collectively, this study provides evidence that Taxifolin
shows pleiotropic effects for the treatment of the NASH continuum. Our data also provide insight
into the novel mechanisms of action of Taxifolin, which has been widely used as a health supplement
with high safety.
Keywords:
Taxifolin; obesity; antioxidant; nonalcoholic steatohepatitis (NASH); inflammation; brown
adipocytes; fibroblast growth factor-21
Nutrients 2023,15, 350. https://doi.org/10.3390/nu15020350 https://www.mdpi.com/journal/nutrients
Nutrients 2023,15, 350 2 of 19
1. Introduction
Increasing attention has been paid to nonalcoholic steatohepatitis (NASH), a hepatic
phenotype of the metabolic syndrome, because NASH progressively develops into cirrhosis
and hepatocellular carcinoma in the long term. To date, numerous clinical trials for NASH
have been conducted globally; however, there are no approved therapeutic strategies for
NASH [
1
]. As the “multiple parallel hits” hypothesis suggests, the molecular pathogenesis
of NASH includes the complex interaction of metabolic abnormalities, such as insulin
resistance and lipid accumulation and inflammatory stimuli, including endotoxins and
proinflammatory cytokines [
2
]. Therefore, chemical compounds possessing pleiotropic
effects may be applicable for treating NASH. Indeed, combinations of chemical compounds
for distinct molecular targets have been under clinical trials.
Considering the therapeutic goals of NASH, it is important to determine whether the
treatment can prevent its progression to hepatocellular carcinoma, in addition to ameliorat-
ing hepatic steatosis, metabolic derangements, and liver fibrosis. A bottleneck of NASH
research is the limited experimental NASH models that exhibit human NASH-like liver
phenotypes. In this respect, we have shown that genetically obese melanocortin 4 receptor
(Mc4r)-deficient mice on a high-fat diet progressively develop hepatic steatosis, NASH, and
multiple liver tumors [
3
]. Using this unique experimental model, we assessed the effects
of several chemical compounds such as sodium-glucose cotransporter-2 inhibitors on the
development of NASH and subsequent liver tumors [4–6].
Taxifolin, also known as dihydroquercetin, is a natural bioactive flavonoid commonly
contained in various foods, such as green tea, fruits, and several herbs, such as milk
thistle [
7
]. It is also included in health supplements including silymarin [
7
]. Based on
its antioxidant and anti-inflammatory properties, accumulating evidence has indicated
that Taxifolin potently ameliorates various disease models including cardiovascular dis-
eases
[8–10]
. Moreover, we have demonstrated the therapeutic potential of Taxifolin for
amyloid-
β
oligomer formation and cognitive dysfunction in a murine model of Alzheimer’s
disease
[11,12]
. Taxifolin also mitigates the development of obesity and glucose intolerance
in certain experimental models [
13
–
15
], although the molecular mechanisms of action are
currently unknown. These observations indicate the pleiotropic effects of Taxifolin. In
addition, several studies have pointed to the protective effects of Taxifolin with the dose of
20–200 mg/kg/day by oral gavage daily for 7 to 28 days on chemically induced liver injury
in mice [
16
–
18
]. In addition, suppressive effects of Taxifolin have been reported in studies
related to the acute alcohol–induced liver injury in mice [
19
]. However, the therapeutic
efficacy of Taxifolin on NASH and subsequent liver tumors remains to be elucidated.
In this study, we demonstrated that Taxifolin treatment markedly prevented the
development of lipid accumulation, chronic inflammation, and fibrosis of the liver in a
murine NASH model. Its mechanisms include suppressing body weight gain, at least
partly, through increasing brown adipose tissue activity. Taxifolin may also directly act
on hepatocytes to inhibit lipid accumulation. Moreover, Taxifolin treatment after NASH
development could effectively prevent its progression to liver tumors. Collectively, this
study provides evidence that Taxifolin shows pleiotropic effects for the treatment of obesity-
induced hepatic steatosis, fibrogenesis, and tumorigenesis.
2. Materials and Methods
2.1. Materials
All reagents and materials were obtained from Sigma-Aldrich (St. Louis, MO, USA),
Cell Signaling Technology (CST, Beverly, MA, USA), or Nacalai Tesque (Kyoto, Japan),
unless otherwise noted.
2.2. Animals
The C57BL/6J mice were obtained from CLEA Japan. Fibroblast growth factor 21
(Fgf21)-deficient mice and Mc4r-deficient mice on the C57BL/6J background were kindly
gifted by Nobuyuki Itoh (Kyoto University, Kyoto, Japan) and Joel K. Elmquist (Univer-
Nutrients 2023,15, 350 3 of 19
sity of Texas Southwestern Medical Center), respectively. The animals were housed in
a temperature-, humidity-, and light-controlled animal room (12 h light and 12 h dark
cycle) and allowed free access to food and water. All animal experiments were carried out
according to the ARRIVE guidelines.
2.3. Diet-Induced Obesity Model
The eight-week-old male C57BL/6J mice were fed a standard diet (SD) or a high-fat
diet (HD) (HFD-60; 506 kcal/100 g, 60% energy as fat; Oriental Yeast, Tokyo, Japan) with or
without Taxifolin (0.05% for the low-dose group (TX-L) and 3% for the high-dose group (TX-
H); Ametis JSC, Blagoveshchensk, Russia). Twelve weeks after the start of the experiment,
an intraperitoneal glucose tolerance test (IPGTT; 1.0 g/kg body weight) was performed
under overnight fasting conditions. For the experiments using Fgf21-deficient mice, the
mice were fed an SD or a TX-H for 6 weeks. The rectal temperature was evaluated with a
thermometer (Physitemp BAT7001H, Fisher scientific, Clifton, NJ) 8 weeks after the start
of the experiment. The mice were sacrificed after overnight fasting under intraperitoneal
pentobarbital anesthesia (30 mg/kg) at the end of each experiment.
2.4. NASH and Liver Tumor Models
To examine the preventive effects of Taxifolin in a NASH model, 8 week old male
Mc4r-deficient mice were fed a Western diet (WD) (D12079B; 468 kcal/100 g, 41% energy
as fat, 34.0% sucrose, 0.21% cholesterol; Research Diets, New Brunswick, NJ, USA) with
or without 3% Taxifolin for up to 20 weeks. As a control, wild-type mice were fed an
SD. To examine the therapeutic effects, Mc4r-deficient mice were fed a WD for 16 weeks,
and then the mice were treated with or without 3% Taxifolin for an additional 8 weeks.
For evaluating the effects on hepatocellular carcinoma development, the Mc4r-deficient
mice were fed a WD for 20 weeks, and then the mice were treated with or without 3%
Taxifolin for an additional 30 weeks. The mice were sacrificed, when fed ad libitum, under
intraperitoneal pentobarbital anesthesia (30 mg/kg) at the end of each experiment.
2.5. Blood Analysis
The concentrations of blood glucose, serum alanine aminotransferase (ALT), aspar-
tate aminotransferase (AST), total cholesterol (TC), triglyceride (TG), and nonesterified
fatty acid (NEFA) were measured as described previously [
11
,
20
]. The serum concentra-
tions of insulin and FGF21 were measured by means of commercially available ELISA
kits (Morinaga Ultra Sensitive Mouse Insulin ELISA kit (Morinaga Institute of Biologi-
cal Science, Kanagawa, Japan) and Mouse FGF21 ELISA kit (R&D Systems, MN, USA),
respectively). The homeostasis model assessment of insulin resistance (HOMA-IR) was
calculated as (fasting serum glucose
×
fasting serum insulin (mg/dL
×
ng/mL)) to assess
the insulin resistance.
2.6. Lipid Contents and Hydroxyproline Levels of the Liver
The hepatic total lipids were extracted with ice-cold 2:1 (vol/vol) chloroform/methanol,
and the triglyceride and cholesterol contents were measured by commercially available
kits (FUJIFILM Wako Pure Chemical, Osaka, Japan). The hepatic hydroxyproline levels
were determined as described previously [5].
2.7. Serum and Hepatic Malondialdehyde Contents
The serum and hepatic malondialdehyde (MDA) contents were measured using a
Colorimetric TBARS Microplate Assay kit (Oxford Biomedical Research, Upper Heyford,
UK) according to the manufacturer’s instructions.
2.8. Quantitative Real-Time PCR
Quantitative real-time PCR was conducted as previously described [
21
]. In brief, the
total RNA was extracted from cultured cells or tissues using RNeasy Mini kit (QIAGEN,
Nutrients 2023,15, 350 4 of 19
Germantown, MD, USA), and real-time PCR amplification was performed with the SYBR
GREEN detection protocol in a thermal cycler (StepOne Plus; Thermo Fisher Scientific,
Waltham, MA, USA). The primers used in this study are listed in Supplementary Materials
Tables S1 and S2. As internal controls, 18s, 36B4, or GAPDH was used, and the data were
normalized by the comparative cycle threshold method.
2.9. Western Blotting
Western blotting analysis was performed as described with minor modifications [
22
].
Homogenate from the liver was prepared with a RIPA lysis buffer (150 mM NaCl, 1%
NP-40, 0.5% sodium deoxycholate, 50 mM Tri-HCl (pH 7.4) supplemented with HaltTM
Protease and Phosphatase Inhibitor Cocktail (Thermo Scientific, Tokyo, Japan). The same
concentration of protein (20–40
µ
g per each sample) was resolved by SDS-polyacrylamide
gel electrophoresis and then transferred to PVDF membranes. The membrane was blocked
with a blocking solution (Nacalai Tesque), followed by incubation with the following
primary antibodies: anti-FAS (diluted 1:1000; #3180; Cell Signaling Technology, CST), anti-
ACC (diluted 1:1000; #3676; CST), anti-SCD-1 (diluted 1:1000; #2794; CST), anti-TNF
α
(diluted 1:800; #11948; CST), and anti-
β
-actin (diluted 1:3000; #4980; CST). After washing,
each band was incubated with an HRP-conjugated anti-rabbit IgG secondary antibody
(#7074; CST) and detected with the ECL Prime Western Blotting Detection System (GE
Healthcare, Uppsala, Sweden). We captured each band images using the ChemiDoc XRS
Plus imaging system (Bio-Rad, Hercules, CA, USA) and quantified the protein levels by
analyzing the band intensities using ImageJ (NIH, Bethesda, MD, USA).
2.10. Histological Analysis
The histological analysis was performed as described [
4
,
5
,
23
]. Four-micromillimeter-
thick paraffin-embedded liver sections were stained with hematoxylin and eosin and Sirius
red. Type III collagen and F4/80-positive macrophages were immunohistochemically
detected using anti-type III collagen (1330-01, SouthernBiotech, Birmingham, AL, USA)
and anti-F4/80 (MCA497GA, Bio-Rad Laboratories, Hercules, CA, USA) antibodies, re-
spectively [
23
]. Liver fibrosis was measured as positive areas for Sirius red or type III
collagen using BZ-X710 (KEYENCE, Osaka, Japan). F4/80 immunostaining was used to
detect crown-like structures (CLS), and the number of CLS was counted in the whole area
of each section. Following the NASH clinical research network scoring system, the scores
for steatosis, inflammation, and hepatocyte ballooning were assessed. The stages of fibrosis
were determined with Sirius red staining. For the assessment of tumor development, lumps
were analyzed in the liver, in which the lumps less than 1 mm and larger than
1 mm
were
considered as foci and tumors, respectively (Supplementary Materials
Figure S1
). The his-
tological evaluation for the presence of histologically malignant areas (i.e., carcinoma-like
lesions) and microscopic dysplastic nodules were performed independently by two board-
certified pathologists (Y. S. and A. E.), according to the guidelines of the 5th edition of the
WHO Classification of Tumors of the digestive system. The areas of individual macroscopic
tumors, HCC-like lesions, and dysplastic nodules were measured using ImageJ software
(version 1.51j8), followed by the quantification of the percentage of HCC-like lesions in the
entire macroscopic tumors with diameters more than 2 mm. The microscopic dysplastic
nodules were defined as areas with diameters between 0.5 and 2 mm that are composed of
atypical hepatocytes with a clonal appearance.
2.11. Experiments Using HepG2
The human hepatocellular carcinoma cell line, HepG2, was purchased from the Amer-
ican Type Culture Collection (Manassas, VA). HepG2 was cultured in high-glucose Dul-
becco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum
(BSA), 100 U/mL penicillin, and 100
µ
g/mL streptomycin and incubated in 5% CO
2
at
37 ◦C
.
The effects of Taxifolin on cell viability were evaluated using the 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay according to the manufacturer’s in-
Nutrients 2023,15, 350 5 of 19
structions (Nacalai Tesque). The administration of palmitic acid (PA) was conducted as
previously described with minor modifications. [
24
]. The PA (Sigma-Aldrich) was solu-
bilized in ethanol until the PA particles were completely dissolved. Then, the PA was
combined with fatty-acid-free BSA solution at a volume ratio of 10:1 (BSA sol.: PA sol.)
immediately and with sufficient mixing at 37
◦
C. A control solution containing ethanol and
BSA was prepared similarly. The HepG2 cells were treated with PA at 400
µ
M for 24 h. The
lipid accumulation of the HepG2 cells were also assessed by Oil Red O staining. Briefly, the
HepG2 cells were with PBS, fixed in formalin (10%) for 1 h, stained with Oil Red O solution
for 1 h, and washed with distilled water. To quantitate the lipid contents, Oil Red O was
extracted from each well with isopropanol and read spectophotometrically at 540 nm.
2.12. Experiments Using Human iPS Cell-Derived Brown Adipocytes
The human iPS cell line (hiPSCs) was established from human umbilical vein en-
dothelial cells by introducing Yamanaka factors (Oct3/4, Sox2, Klf4, and c-Myc) using
CytoTune-iPS ver.1.0 (ID Pharma, Ibaraki, Japan). The hiPSCs were maintained by the
feeder-free system (StemFit AK02N, Ajinomoto Healthy Supply, Tokyo, Japan) on 60 mm
diameter plates precoated with iMatrix (10
µ
L/60 mm plate) (Nippi, Tokyo, Japan). The
media were changed every other day. Once the cell density reached a 70–80% confluency,
the hiPSCs were treated by a treatment with 1 mL TrypLE Express (Thermo Fisher Sci-
entific) for 5–10 min at 37
◦
C and collected by gentle pipetting with a 100
µ
L tip. After
washing with PBS, the hiPSCs were seeded onto new 60 mm diameter precoated plates
(
0.5–1 ×105 cells/plate
) using 4 mL StemFit AK02N supplemented with 40
µ
L RevitaCell
supplement (Thermo Fisher Scientific), ROCK inhibitor. The cell proliferation rate was
2.0 logs in 5–7 days. To differentiate the hiPSCs into brown adipocytes, the hiPSCs were
harvested, dissociated into single cells by TrypLE Express treatment, and suspended in
differentiation medium with RevitaCell supplement. The cells were cultured at 37
◦
C in
a CO
2
incubator (5% CO
2
) for 6–8 days. Half of the differentiation media were refreshed
every other day. Then, hiPSC-derived brown adipocytes were treated with 100
µ
M Taxifolin
for 48 h.
2.13. Statistical Analysis
The data are expressed as the mean
±
SEM. The statistical analysis was conducted
using one-way ANOVA followed by the Tukey–Kramer test. The comparisons of the body
weight and serum glucose concentrations during the IPGTT were performed using a two-
way factorial ANOVA with repeated measurement followed by the Tukey–Kramer test. A
p-value < 0.05 was considered statistically significant. The analyses were performed with
GraphPad Prism version 9 (GraphPad Software, San Diego, CA, USA).
3. Results
3.1. Preventive Effects of Taxifolin on Body Weight Gain, Metabolic Derangements, and Hepatic
Steatosis in Diet-Induced Obese Mice
To test the therapeutic potentials of Taxifolin in obesity, different doses of Taxifolin
were orally administered to male C57BL6/J mice fed an HD for 12 weeks (Figure 1A).
Taxifolin treatment dose-dependently suppressed the increase in body weight and liver
and epididymal fat weights (Figure 1B,C). The mice fed an HD containing high-dose
Taxifolin (TX-H) had a significantly increased rectal temperature relative to those fed a
control HD (Figure 1D), although there was no significant difference in the food intake
between the treatments. For the metabolic parameters, the blood glucose concentrations
under fasted conditions were significantly lower in the TX-H group than in the HD group
(Figure 1E). The serum insulin concentrations and HOMA-IR were also suppressed in
the TX-H group (Figure 1F,G). The intraperitoneal glucose tolerance test confirmed the
ameliorated glucose metabolism by Taxifolin treatment (Figure 1H,I). As it is known as an
antioxidant, the Taxifolin treatment significantly reduced the serum MDA concentrations
in the diet-induced obese mice (Figure 1J). In addition, the serum levels of triglyceride,
Nutrients 2023,15, 350 6 of 19
total cholesterol, and NEFA were significantly lower in the TX-H group than in the HD
group (Figure 1K–M).
Nutrients 2023, 15, x FOR PEER REVIEW 6 of 20
3. Results
3.1. Preventive Effects of Taxifolin on Body Weight Gain, Metabolic Derangements, and Hepatic
Steatosis in Diet-Induced Obese Mice
To test the therapeutic potentials of Taxifolin in obesity, different doses of Taxifolin
were orally administered to male C57BL6/J mice fed an HD for 12 weeks (Figure 1A).
Taxifolin treatment dose-dependently suppressed the increase in body weight and liver
and epididymal fat weights (Figure 1B,C). The mice fed an HD containing high-dose Tax-
ifolin (TX-H) had a significantly increased rectal temperature relative to those fed a control
HD (Figure 1D), although there was no significant difference in the food intake between
the treatments (data not shown). For the metabolic parameters, the blood glucose concen-
trations under fasted conditions were significantly lower in the TX-H group than in the
HD group (Figure 1E). The serum insulin concentrations and HOMA-IR were also sup-
pressed in the TX-H group (Figure 1F,G). The intraperitoneal glucose tolerance test con-
firmed the ameliorated glucose metabolism by Taxifolin treatment (Figure 1H,I). As it is
known as an antioxidant, the Taxifolin treatment significantly reduced the serum MDA
concentrations in the diet-induced obese mice (Figure 1J). In addition, the serum levels of
triglyceride, total cholesterol, and NEFA were significantly lower in the TX-H group than
in the HD group (Figure 1K–M).
Figure 1. The preventive effects of Taxifolin on obesity and metabolic derangements in diet-induced
obese mice. (A) Experimental protocol: male C57BL/6J mice were divided into the following 4
groups—SD group with a standard diet, HD group with a high-fat diet, TX-L group with a high-fat
diet containing 0.05% (wt/wt) of Taxifolin, and TX-H group with a high-fat diet containing 3%
(wt/wt) of Taxifolin. n = 6 in each group. (B) Growth curve; (C) tissue weights; (D) rectal tempera-
ture; (E–G) blood glucose levels (E), serum insulin concentrations (F), and homeostasis model as-
sessment of insulin resistance (HOMA-IR) under fasting conditions (G); (H,I) intraperitoneal glu-
cose tolerance test (injection of 1.0 g/kg of glucose) after 12 weeks of high-fat diet feeding: (H) blood
glucose levels; (I) area under the curve (AUC) values for the blood glucose concentrations during
the glucose tolerance test; (J–M) serum concentrations of malondialdehyde (MDA), triglyceride,
Figure 1.
The preventive effects of Taxifolin on obesity and metabolic derangements in diet-induced
obese mice. (
A
) Experimental protocol: male C57BL/6J mice were divided into the following
4 groups
—SD group with a standard diet, HD group with a high-fat diet, TX-L group with a high-
fat diet containing 0.05% (wt/wt) of Taxifolin, and TX-H group with a high-fat diet containing 3%
(wt/wt) of Taxifolin. n= 6 in each group. (
B
) Growth curve; (
C
) tissue weights; (
D
) rectal temperature;
(
E
–
G
) blood glucose levels (
E
), serum insulin concentrations (
F
), and homeostasis model assessment
of insulin resistance (HOMA-IR) under fasting conditions (
G
); (
H
,
I
) intraperitoneal glucose tolerance
test (injection of 1.0 g/kg of glucose) after 12 weeks of high-fat diet feeding: (
H
) blood glucose
levels; (
I
) area under the curve (AUC) values for the blood glucose concentrations during the glucose
tolerance test; (
J
–
M
) serum concentrations of malondialdehyde (MDA), triglyceride, total cholesterol,
and nonesterified fatty acid (NEFA). Values are presented as the means
±
SEM; significant differences:
*p< 0.05 and ** p< 0.01 vs. HD.
We further examined the effects of Taxifolin on hepatic steatosis and found that serum
concentrations of AST and ALT, along with the hepatic contents of triglyceride and MDA,
were dose-dependently reduced by Taxifolin treatment (Figure 2A–C). Hematoxylin and
eosin staining of the liver confirmed these data (Figure 2D). In addition, the upregulation
of lipogenic (Srebp1c,Fas,Scd1, and Acc1) and inflammatory (Tnf
α
,Il1b, and Emr1 (F4/80))
genes in the liver was significantly suppressed in the TX-H group relative to the HD
group (Figure 2E,F). The lipogenic (FAS, SCD1, and ACC) and inflammatory (TNF
α
)
protein expression levels were also upregulated in the HD group, and it was significantly
decreased in the TX-H group relative to the HD group (Figure 2G,H). Collectively, these
Nutrients 2023,15, 350 7 of 19
findings suggest that Taxifolin is capable of preventing the development of obesity and
hepatic steatosis.
Nutrients 2023, 15, x FOR PEER REVIEW 7 of 20
total cholesterol, and nonesterified fatty acid (NEFA). Values are presented as the means ± SEM;
significant differences: * p < 0.05 and ** p < 0.01 vs. HD.
We further examined the effects of Taxifolin on hepatic steatosis and found that se-
rum concentrations of AST and ALT, along with the hepatic contents of triglyceride and
MDA, were dose-dependently reduced by Taxifolin treatment (Figure 2A–C). Hematoxy-
lin and eosin staining of the liver confirmed these data (Figure 2D). In addition, the up-
regulation of lipogenic (Srebp1c, Fas, Scd1, and Acc1) and inflammatory (Tnfα, Il1b, and
Emr1 (F4/80)) genes in the liver was significantly suppressed in the TX-H group relative
to the HD group (Figure 2E and F). The lipogenic (FAS, SCD1, and ACC) and inflamma-
tory (TNFα) protein expression levels were also upregulated in the HD group, and it was
significantly decreased in the TX-H group relative to the HD group (Figure 2G,H). Collec-
tively, these findings suggest that Taxifolin is capable of preventing the development of
obesity and hepatic steatosis.
Figure 2. Preventive effects of Taxifolin on hepatic steatosis in diet-induced obese mice. White
square: SD; black square: HD; dark-green square: TX-L; light-green square: TX-H. n = 6 in each
group. (A) Serum concentrations of AST and ALT after 12 weeks of HD feeding; (B,C) hepatic tri-
glyceride and MDA contents; (D) hematoxylin and eosin (HE) staining of the liver. Insets: gross
appearance of the livers. Scale bars: 100 µ m. (E,F) Expression levels of genes related to lipogenesis
(Srebp1c, Fas, Scd1, and Acc1) and inflammation (Tnfα, Il1b, and Emr1 (F4/80)) in the liver; (G,H)
immunoblot analysis of the protein expression levels related to lipogenesis (FAS, SCD-1, and ACC)
Figure 2.
Preventive effects of Taxifolin on hepatic steatosis in diet-induced obese mice. White square:
SD; black square: HD; dark-green square: TX-L; light-green square: TX-H. n= 6 in each group.
(A) Serum
concentrations of AST and ALT after 12 weeks of HD feeding; (
B
,
C
) hepatic triglyceride
and MDA contents; (
D
) hematoxylin and eosin (HE) staining of the liver. Insets: gross appearance of
the livers. Scale bars: 100
µ
m. (
E
,
F
) Expression levels of genes related to lipogenesis (Srebp1c,Fas,Scd1,
and Acc1) and inflammation (Tnf
α
,Il1b, and Emr1 (F4/80)) in the liver; (
G
,
H
) immunoblot analysis of
the protein expression levels related to lipogenesis (FAS, SCD-1, and ACC) and inflammation (TNF
α
)
in the liver.
β
-actin was used as a loading control. Values are presented as the means
±
SEM; n= 6;
significant differences: ** p< 0.01 vs. HD.
3.2. Molecular Mechanism Underlying the Anti-Obesity Effects of Taxifolin
Regarding the increased rectal temperature in the TX-H group, we found that the
Taxifolin treatment increased the mRNA expression of genes related to brown adipose
tissue activation such as uncoupling protein-1 (Ucp1) in brown adipose tissue (Figure 3A).
Brown adipose tissue is involved in nonshivering thermogenesis during cold exposure and
Nutrients 2023,15, 350 8 of 19
diet-induced thermogenesis, thereby contributing to whole-body energy expenditure [
25
].
Of note, the mRNA expression of Fgf21, a potent inducer of thermogenic genes in brown
adipose tissue, was increased in the liver and brown adipose tissue by the Taxifolin treat-
ment (Figure 3B). In line with this, Taxifolin treatment effectively restored the otherwise
reduced serum Fgf21 concentrations in the diet-induced obese mice (Figure 3C). These
observations led us to examine the involvement of Fgf21 in the Taxifolin-mediated anti-
obesity effects. In this study, high-dose Taxifolin was orally administered to Fgf21-deficient
and wild-type mice fed an HD for 6 weeks (Figure 3D). The suppressive effects of Taxifolin
on body weight, adipose tissue weights, and rectal temperature were partially reduced in
the Fgf21-deficient mice, whereas the treatment did not affect food intake (Figure 3E–H).
These findings suggest that Taxifolin potently suppresses the development of obesity, at
least partly, through Fgf21 production.
Nutrients 2023, 15, x FOR PEER REVIEW 8 of 20
and inflammation (TNFα) in the liver. β-actin was used as a loading control. Values are presented
as the means ± SEM; n = 6; significant differences: ** p < 0.01 vs. HD.
3.2. Molecular Mechanism Underlying the Anti-Obesity Effects of Taxifolin
Regarding the increased rectal temperature in the TX-H group, we found that the
Taxifolin treatment increased the mRNA expression of genes related to brown adipose
tissue activation such as uncoupling protein-1 (Ucp1) in brown adipose tissue (Figure 3A).
Brown adipose tissue is involved in nonshivering thermogenesis during cold exposure
and diet-induced thermogenesis, thereby contributing to whole-body energy expenditure
[25]. Of note, the mRNA expression of Fgf21, a potent inducer of thermogenic genes in
brown adipose tissue, was increased in the liver and brown adipose tissue by the Taxifolin
treatment (Figure 3B). In line with this, Taxifolin treatment effectively restored the other-
wise reduced serum Fgf21 concentrations in the diet-induced obese mice (Figure 3C).
These observations led us to examine the involvement of Fgf21 in the Taxifolin-mediated
anti-obesity effects. In this study, high-dose Taxifolin was orally administered to Fgf21-
deficient and wild-type mice fed an HD for 6 weeks (Figure 3D). The suppressive effects
of Taxifolin on body weight, adipose tissue weights, and rectal temperature were partially
reduced in the Fgf21-deficient mice, whereas the treatment did not affect food intake (Fig-
ure 3E–H). These findings suggest that Taxifolin potently suppresses the development of
obesity, at least partly, through Fgf21 production.
Figure 3. Involvement of Fgf21 in Taxifolin-mediated anti-obesity effects. (A–C) Male C57BL/6J mice
were divided into the following 4 groups: white square, SD; black square, HD; dark-green square,
TX-L; light-green square, TX-H. n = 6 in each group. (A) Expression levels of genes related to brown
adipocyte activation (Ucp1, Pgc1, Prdm16, Zfp516, and Dio2) in interscapular brown adipose tissue
of the C57BL/6J mice fed an HD with or without Taxifolin for 12 weeks. (B) Expression levels of
Fgf21 and Il6 mRNAs in the liver. (C) Serum Fgf21 concentrations after 12 weeks of HD feeding with
or without Taxifolin. Mean ± SEM; n = 6; * p < 0.05 and ** p < 0.01 vs. HD. (D–H) Male C57BL/6J mice
(wild-type, WT) and Fgf21-deficient mice (Fgf21-KO) were divided into the following 5 groups:
white square, WT/SD; black square, WT/HD; gray square, TX/H; dark-green square, Fgf21-KO/HD;
light-green square, Fgf21-KO/HD-TX-H. n = 6 in each group. (D) Experimental protocol: Fgf21-
Figure 3.
Involvement of Fgf21 in Taxifolin-mediated anti-obesity effects. (
A
–
C
) Male C57BL/6J mice
were divided into the following 4 groups: white square, SD; black square, HD; dark-green square,
TX-L; light-green square, TX-H. n= 6 in each group. (
A
) Expression levels of genes related to brown
adipocyte activation (Ucp1,Pgc1,Prdm16,Zfp516, and Dio2) in interscapular brown adipose tissue
of the C57BL/6J mice fed an HD with or without Taxifolin for 12 weeks. (
B
) Expression levels of
Fgf21 and Il6 mRNAs in the liver. (
C
) Serum Fgf21 concentrations after 12 weeks of HD feeding
with or without Taxifolin. Mean
±
SEM; n= 6; * p< 0.05 and ** p< 0.01 vs. HD. (
D
–
H
) Male
C57BL/6J mice (wild-type, WT) and Fgf21-deficient mice (Fgf21-KO) were divided into the following
5 groups: white square, WT/SD; black square, WT/HD; gray square, TX/H; dark-green square,
Fgf21-KO/HD; light-green square, Fgf21-KO/HD-TX-H. n= 6 in each group. (
D
) Experimental
protocol: Fgf21-deificient and wild-type mice were fed an HD with or without Taxifolin for 6 weeks;
(
E
) growth curve; (
F
) food intake; (
G
) rectal temperature; (
H
) tissue weights: liver, liver-to-body
weight ratio, epididymal fat, subcutaneous fat, and interscapular brown adipose tissue. Values are
presented as the means; n= 5–6; significant differences: * p< 0.05 and ** p< 0.01.
Nutrients 2023,15, 350 9 of 19
Moreover, we investigated the direct effects of Taxifolin on brown adipocytes. In
addition to body temperature, the expression of “BATokines”, secreted factors from mature
brown adipocytes, is useful for evaluating brown adipose tissue activity [
26
]. In this study,
human iPS cell-derived brown adipocytes (hiPSCdBAs) were treated with Taxifolin for 48 h
and then subjected to mRNA expression experiments (Figure 4A). The Taxifolin treatment
significantly increased the mRNA expression of UCP1 and brown adipocyte-specific genes,
such as Epithelial V-like antigen 1 (EVA1) and Elongation of very long chain fatty acid elongase
3(ELOVL3) (Figure 4B). The Taxifolin treatment also significantly increased the mRNA
expression of BATokines (FGF21 and IL6) (Figure 4C). Collectively, these findings suggest
that Taxifolin exerts its anti-obesity effects through at least two different pathways: directly
acting on brown adipocytes and inducing Fgf21 expression in the liver.
Nutrients 2023, 15, x FOR PEER REVIEW 9 of 20
deificient and wild-type mice were fed an HD with or without Taxifolin for 6 weeks; (E) growth
curve; (F) food intake; (G) rectal temperature; (H) tissue weights: liver, liver-to-body weight ratio,
epididymal fat, subcutaneous fat, and interscapular brown adipose tissue. Values are presented as
the means; n = 5–6; significant differences: * p < 0.05 and ** p < 0.01.
Moreover, we investigated the direct effects of Taxifolin on brown adipocytes. In ad-
dition to body temperature, the expression of “BATokines”, secreted factors from mature
brown adipocytes, is useful for evaluating brown adipose tissue activity [26]. In this study,
human iPS cell-derived brown adipocytes (hiPSCdBAs) were treated with Taxifolin for 48
h and then subjected to mRNA expression experiments (Figure 4A). The Taxifolin treat-
ment significantly increased the mRNA expression of UCP1 and brown adipocyte-specific
genes, such as Epithelial V-like antigen 1 (EVA1) and Elongation of very long chain fatty acid
elongase 3 (ELOVL3) (Figure 4B). The Taxifolin treatment also significantly increased the
mRNA expression of BATokines (FGF21 and IL6) (Figure 4C). Collectively, these findings
suggest that Taxifolin exerts its anti-obesity effects through at least two different path-
ways: directly acting on brown adipocytes and inducing Fgf21 expression in the liver.
Figure 4. Direct action of Taxifolin on brown adipocytes: (A) experimental protocol: human iPS cell-
derived brown adipocytes (hiPSCdBAs) were differentiated and then treated with Taxifolin at 100
μM for 48 h; (B) expression levels of genes related to brown adipocyte markers (UCP1, PRDM16,
EVA1, and ELOVL3); (C) expression levels of FGF21 and IL6 mRNAs in the hiPSCdBAs. Values are
presented as the means ± SEM; n = 3; significant differences: ** p < 0.05 vs. hiPSCdBAs without Tax-
ifolin treatment.
3.3. Therapeutic Effects of Taxifolin on Hepatic Steatosis in Diet-Induced Obese Mice
We next examined the therapeutic effects of Taxifolin after the mice developed obe-
sity and hepatic steatosis. The mice fed an HD for 12 weeks were divided into the follow-
ing three groups: HD/SD group with an SD; HD/HD group with an HD; and HD/TX-H
group with an HD containing high-dose Taxifolin. Each group was then fed the respective
diet for an additional 12 weeks (Figure 5A). Unlike the preventive protocol (Figure 1),
Taxifolin treatment did not suppress body weight gain in the therapeutic protocol (Figure
5B). In contrast, Taxifolin was still effective for metabolic parameters, rectal temperature,
hepatic steatosis, and mRNA expression in brown adipose tissue and liver (Figure 5C–T).
Among others, the most striking effects were observed in the hepatic mRNA expression
of genes related to lipogenesis and inflammation (Figure 5S,T).
Figure 4.
Direct action of Taxifolin on brown adipocytes: (
A
) experimental protocol: human iPS
cell-derived brown adipocytes (hiPSCdBAs) were differentiated and then treated with Taxifolin at
100
µ
M for 48 h; (
B
) expression levels of genes related to brown adipocyte markers (UCP1,PRDM16,
EVA1, and ELOVL3); (
C
) expression levels of FGF21 and IL6 mRNAs in the hiPSCdBAs. Values are
presented as the means
±
SEM; n= 3; significant differences: ** p< 0.05 vs. hiPSCdBAs without
Taxifolin treatment.
3.3. Therapeutic Effects of Taxifolin on Hepatic Steatosis in Diet-Induced Obese Mice
We next examined the therapeutic effects of Taxifolin after the mice developed obesity
and hepatic steatosis. The mice fed an HD for 12 weeks were divided into the following
three groups: HD/SD group with an SD; HD/HD group with an HD; and HD/TX-H group
with an HD containing high-dose Taxifolin. Each group was then fed the respective diet
for an additional 12 weeks (Figure 5A). Unlike the preventive protocol (Figure 1), Taxifolin
treatment did not suppress body weight gain in the therapeutic protocol (Figure 5B). In
contrast, Taxifolin was still effective for metabolic parameters, rectal temperature, hepatic
steatosis, and mRNA expression in brown adipose tissue and liver (Figure 5C–T). Among
others, the most striking effects were observed in the hepatic mRNA expression of genes
related to lipogenesis and inflammation (Figure 5S,T).
We next investigated the direct effects of Taxifolin on hepatocytes using human HepG2
cells. After confirming the cell viability treated with less than 50
µ
M Taxifolin, HepG2
cells were treated with Taxifolin for 24 h in the presence of palmitate (Figure 6A,B). Oil
Red O staining revealed that palmitate-induced lipid accumulation was suppressed by
Taxifolin in a dose-dependent manner (Figure 6C,D). Taxifolin also effectively suppressed
the otherwise increased mRNA expression of lipogenic genes in this experimental setting
(Figure 6E,F). Collectively, these
in vivo
and
in vitro
data strongly suggest that Taxifolin
can directly act on hepatocytes to ameliorate hepatic steatosis, in addition to its effects on
systemic energy expenditure.
Nutrients 2023,15, 350 10 of 19
Nutrients 2023, 15, x FOR PEER REVIEW 10 of 20
Figure 5. Therapeutic effects of Taxifolin on hepatic steatosis in diet-induced obese mice. (A) Exper-
imental protocol: after being fed an HD for 12 weeks, C57BL/6J mice were divided into the following
3 groups and then fed the respective diets for an additional 12 weeks—HD/SD group with an SD,
HD/HD group with an HD, and HD/TX-H group with an HD containing 3% (wt/wt) Taxifolin. The
mice were also fed an HD for 12 weeks as the pretreatment HD group and an SD for 24 weeks as
the control SD/SD group. n = 6 in each group. (B–D) Time course of body weight (B), fasting blood
glucose levels (C), and rectal temperature (D). E-P: Metabolic parameters and tissue weights of the
HD, HD/SD, HD/HD, and HD/TX-H groups. Serum concentrations of insulin (E), triglyceride (G),
total cholesterol (H), NEFA (I), MDA (J), AST (N), and ALT (O). (F) HOMA-IR. Liver (K) and epi-
didymal fat (L) tissue weights. Hepatic MDA (M) and triglyceride (P) contents. (Q–T) Four groups:
white square, SD/SD; gray square, HD/SD; dark-red square, TX-L; light-red square: TX-H. (Q) Ex-
pression levels of genes related to brown adipocyte markers (Ucp1, Pgc1, Prdm16, Zfp516, and Dio2)
in the interscapular brown adipose tissue. (R) HE staining of the liver. Scale bars: 200 µ m. (S,T)
Expression levels of genes related to lipogenesis (Srebp1c, Fas, Scd1, and Acc1) and inflammation
(Tnfα, Il1b, and Emr1 (F4/80)) in the liver. Values are presented as the means ± SEM; n = 6; significant
differences: * p < 0.05 and ** p < 0.01 vs. HD/HD; # p < 0.05 vs. HD.
Figure 5.
Therapeutic effects of Taxifolin on hepatic steatosis in diet-induced obese mice.
(A) Experimental
protocol: after being fed an HD for 12 weeks, C57BL/6J mice were divided into
the following 3 groups and then fed the respective diets for an additional 12 weeks—HD/SD group
with an SD, HD/HD group with an HD, and HD/TX-H group with an HD containing 3% (wt/wt)
Taxifolin. The mice were also fed an HD for 12 weeks as the pretreatment HD group and an SD for
24 weeks as the control SD/SD group. n= 6 in each group. (
B
–
D
) Time course of body weight (
B
),
fasting blood glucose levels (
C
), and rectal temperature (
D
). E-P: Metabolic parameters and tissue
weights of the HD, HD/SD, HD/HD, and HD/TX-H groups. Serum concentrations of insulin (
E
),
triglyceride (
G
), total cholesterol (
H
), NEFA (
I
), MDA (
J
), AST (
N
), and ALT (
O
). (
F
) HOMA-IR.
Liver (
K
) and epididymal fat (
L
) tissue weights. Hepatic MDA (
M
) and triglyceride (
P
) contents.
(Q–T) Four
groups: white square, SD/SD; gray square, HD/SD; dark-red square, TX-L; light-red
square: TX-H. (
Q
) Expression levels of genes related to brown adipocyte markers (Ucp1,Pgc1,Prdm16,
Zfp516, and Dio2) in the interscapular brown adipose tissue. (
R
) HE staining of the liver. Scale bars:
200
µ
m. (
S
,
T
) Expression levels of genes related to lipogenesis (Srebp1c,Fas,Scd1, and Acc1) and
inflammation (Tnf
α
,Il1b, and Emr1 (F4/80)) in the liver. Values are presented as the means
±
SEM;
n= 6; significant differences: * p< 0.05 and ** p< 0.01 vs. HD/HD; # p< 0.05 vs. HD.
Nutrients 2023,15, 350 11 of 19
Nutrients 2023, 15, x FOR PEER REVIEW 11 of 20
We next investigated the direct effects of Taxifolin on hepatocytes using human
HepG2 cells. After confirming the cell viability treated with less than 50 µ M Taxifolin,
HepG2 cells were treated with Taxifolin for 24 h in the presence of palmitate (Figure 6A,B).
Oil Red O staining revealed that palmitate-induced lipid accumulation was suppressed
by Taxifolin in a dose-dependent manner (Figure 6C,D). Taxifolin also effectively sup-
pressed the otherwise increased mRNA expression of lipogenic genes in this experimental
setting (Figure 6E,F). Collectively, these in vivo and in vitro data strongly suggest that
Taxifolin can directly act on hepatocytes to ameliorate hepatic steatosis, in addition to its
effects on systemic energy expenditure.
Figure 6. Direct action of Taxifolin on hepatocytes. (A) Experimental protocol: HepG2 cells were
treated with Taxifolin (0.01 and 10 µM) for 24 h in the presence of palmitate (400 µM). (B) Cell
viability after treatment with Taxifolin (1, 10, 50, and 100 µM and 1 mM) for 24 h. (C,D) Representa-
tive image of Oil Red O staining (C) and its quantitative evaluation measuring the absorbance at
540 nm (D). HepG2 cells were treated with vehicle (a), palmitate 400 µ M (b), and palmitate with
0.01 µM (c) or 10 µM (d) Taxifolin for 24 h. Scale bars: 100 µm. E and F: Expression levels of SREBP1
(E) and FAS (F) mRNAs in the HepG2 cells. Values are presented as the means ± SEM; n = 3; signif-
icant differences: ** p < 0.01 vs. palmitate.
3.4. Preventive Effects of Taxifolin on the Development of NASH in a Murine Model
Next, we investigated the effects of Taxifolin on the development of NASH in a mu-
rine model. As a preventive protocol, genetically obese Mc4r-deficient mice were on a WD
with or without high-dose Taxifolin for 20 weeks (Figure 7A). Consistently with the prior
experiments shown in Figure 1, the Taxifolin treatment significantly suppressed the in-
crease in body weight, liver weight, and hepatic lipid contents in a NASH model (Figure
7B–D). The Taxifolin treatment also decreased the serum concentrations of ALT, AST, and
total cholesterol, whereas the serum triglyceride and blood glucose levels were not af-
fected in this model (Supplementary Materials Table S3). After 20 weeks of WD feeding,
the Mc4r-deficient mice showed histological features similar to human NASH, including
Figure 6.
Direct action of Taxifolin on hepatocytes. (
A
) Experimental protocol: HepG2 cells were
treated with Taxifolin (0.01 and 10
µ
M) for 24 h in the presence of palmitate (400
µ
M). (
B
) Cell viability
after treatment with Taxifolin (1, 10, 50, and 100
µ
M and 1 mM) for 24 h. (
C
,
D
) Representative image
of Oil Red O staining (
C
) and its quantitative evaluation measuring the absorbance at 540 nm (
D
).
HepG2 cells were treated with vehicle (
a
), palmitate 400
µ
M (
b
), and palmitate with 0.01
µ
M (
c
)
or
10 µM
(
d
) Taxifolin for 24 h. Scale bars: 100
µ
m. Eand F: Expression levels of SREBP1 (
E
) and
FAS
(F) mRNAs
in the HepG2 cells. Values are presented as the means
±
SEM; n= 3; significant
differences: ** p< 0.01 vs. palmitate.
3.4. Preventive Effects of Taxifolin on the Development of NASH in a Murine Model
Next, we investigated the effects of Taxifolin on the development of NASH in a
murine model. As a preventive protocol, genetically obese Mc4r-deficient mice were on
a WD with or without high-dose Taxifolin for 20 weeks (Figure 7A). Consistently with
the prior experiments shown in Figure 1, the Taxifolin treatment significantly suppressed
the increase in body weight, liver weight, and hepatic lipid contents in a NASH model
(Figure 7B–D). The Taxifolin treatment also decreased the serum concentrations of ALT,
AST, and total cholesterol, whereas the serum triglyceride and blood glucose levels were not
affected in this model (Supplementary Materials Table S3). After 20 weeks of WD feeding,
the Mc4r-deficient mice showed histological features similar to human NASH, including
micro–macro vesicular steatosis, ballooning degeneration (indicating hepatocyte damages),
and massive infiltration of inflammatory cells (Figure 7E), as previously described [
5
].
The histological evaluation using the NAFLD Activity Score (NAS) system revealed that
the Taxifolin treatment significantly reduced the scores of steatosis, inflammation, and
ballooning degeneration (Figure 7E), suggesting the preventive effects of Taxifolin on the
development of NASH. Previously, we found a unique histological structure termed CLS,
where macrophages aggregate around dead hepatocytes with large lipid droplets and
engulf the dead cells and residual lipids [
27
]. We also provided evidence that CLS is a
driver that promotes liver fibrosis during the development of NASH [
28
]. In this study,
the histological analysis revealed CLS formation and liver fibrosis (pericellular fibrosis) in
Nutrients 2023,15, 350 12 of 19
Mc4r-deficient mice fed a WD, which was markedly suppressed by the Taxifolin treatment
(Figure 7F,G). The measurement of hydroxyproline contents of the liver confirmed the data
on liver fibrosis (Figure 7H). Consistently, the Taxifolin treatment inhibited the upregulation
of the mRNA levels related to inflammation, fibrosis, and lipid metabolism in this NASH
model (Figure 7I). In particular, Itgax (Cd11c) was selectively expressed in the macrophages
within the CLS, which possess profibrotic properties [
28
]. Taken together, these results
indicate that Taxifolin can prevent the development of hepatic steatosis and subsequent
liver fibrosis in a NASH model.
Nutrients 2023, 15, x FOR PEER REVIEW 13 of 20
Figure 7. Preventive effects of Taxifolin on the development of NASH in a mouse model. (A) Exper-
imental protocol: genetically obese melanocotin-4 receptor (Mc4r)-deficient mice on a WD with or
without 3% Taxifolin for 20 weeks (MC/WD or MC/WD-TX, respectively). Wild-type mice on a
standard diet for 20 weeks (WT/SD) were used as a control. (B) Growth curve: C-I 3 groups–white
square, WT/SD; black square, MC/WD; light-red square, MC/WD-TX. (C) Liver and epididymal fat
weights. (D) Hepatic triglyceride and total cholesterol contents. (E) HE staining of the liver. Histo-
logical analysis using the nonalcoholic fatty liver disease (NAFLD) activity score (NAS) system. (F)
F4/80 immunostaining. The arrows indicate the crown-like structures (CLS). (G) Sirius red staining.
(H) Hydroxyproline contents of the liver. (I) Expression levels of genes related to inflammation
(Emr1, Itgax, and Tnfα), fibrosis (Tgfb1, Timp1, and Col1a1), and lipid metabolism (Ppara, Cpt1a, Acox,
Srebp1, and Fas). Scale bars: 100 µm. Values are presented as the means ± SEM; n = 11–12; significant
differences: * p < 0.05 and ** p < 0.01.
3.5. Therapeutic Effects of Taxifolin during the Progression from NASH to Liver Tumors
We next assessed whether Taxifolin has a therapeutic potential for NASH. The Mc4r-
deficient mice were on a WD for 16 weeks to develop NASH-like liver phenotypes, and
the mice were then further fed a WD with or without Taxifolin for an additional 8 weeks
(Figure 8A). Similar to the preventive protocol (Figure 7), the Taxifolin treatment
Figure 7.
Preventive effects of Taxifolin on the development of NASH in a mouse model.
(A) Experimental
protocol: genetically obese melanocotin-4 receptor (Mc4r)-deficient mice on a
WD with or without 3% Taxifolin for 20 weeks (MC/WD or MC/WD-TX, respectively). Wild-type
mice on a standard diet for 20 weeks (WT/SD) were used as a control. (
B
) Growth curve: C-I
3 groups
–white square, WT/SD; black square, MC/WD; light-red square, MC/WD-TX. (
C
) Liver
and epididymal fat weights. (
D
) Hepatic triglyceride and total cholesterol contents. (
E
) HE staining
of the liver. Histological analysis using the nonalcoholic fatty liver disease (NAFLD) activity score
(NAS) system. (
F
) F4/80 immunostaining. The arrows indicate the crown-like structures (CLS).
(G) Sirius
red staining. (
H
) Hydroxyproline contents of the liver. (
I
) Expression levels of genes related
to inflammation (Emr1,Itgax, and Tnf
α
), fibrosis (Tgfb1,Timp1, and Col1a1), and lipid metabolism
(Ppara,Cpt1a,Acox,Srebp1, and Fas). Scale bars: 100
µ
m. Values are presented as the means
±
SEM;
n= 11–12; significant differences: * p< 0.05 and ** p< 0.01.
Nutrients 2023,15, 350 13 of 19
3.5. Therapeutic Effects of Taxifolin during the Progression from NASH to Liver Tumors
We next assessed whether Taxifolin has a therapeutic potential for NASH. The Mc4r-
deficient mice were on a WD for 16 weeks to develop NASH-like liver phenotypes, and
the mice were then further fed a WD with or without Taxifolin for an additional 8 weeks
(Figure 8A)
. Similar to the preventive protocol (Figure 7), the Taxifolin treatment signifi-
cantly suppressed the liver weight and hepatic lipid contents, whereas the treatment did not
affect body weight gain in the therapeutic protocol (Figure 8B–D). The Taxifolin treatment
also reduced the serum concentrations of ALT and AST, whereas the serum concentrations
of total cholesterol and triglyceride and blood glucose levels were not affected in this
model (Supplementary Materials Table S4). The histological examinations revealed that the
Taxifolin treatment significantly ameliorated hepatic steatosis, CLS formation, and liver
fibrosis (Figure 8E–H). The data were confirmed by mRNA expression (Supplementary
Materials Figure S2).
Nutrients 2023, 15, x FOR PEER REVIEW 15 of 20
Figure 8. Therapeutic effects of Taxifolin on the progression of NASH in a mouse model. (A) Exper-
imental protocol: the Mc4r-deficient mice were on a WD for 16 weeks to develop NASH and then
treated with or without 3% Taxifolin for an additional 8 weeks (MC/WD/WD or MC/WD/TX, re-
spectively). (B) Growth curve; (C) liver and epididymal fat weights; (D) hepatic triglyceride and
total cholesterol contents. (E–H) Two groups: dark-red square, MC/WD/WD; light-red square,
MC/WD/WD-TX. (E) HE staining of the liver. Histological analysis using the NAS. Scale bars: 100
µm. (F) F4/80 immunostaining of the liver. The arrows indicate the CLS. (G) Immunostaining for
Figure 8.
Therapeutic effects of Taxifolin on the progression of NASH in a mouse model.
(A) Experimental
protocol: the Mc4r-deficient mice were on a WD for 16 weeks to develop NASH and then treated with or
Nutrients 2023,15, 350 14 of 19
without 3% Taxifolin for an additional 8 weeks (MC/WD/WD or MC/WD/TX, respectively).
(B) Growth
curve; (
C
) liver and epididymal fat weights; (
D
) hepatic triglyceride and total cholesterol
contents. (
E
–
H
) Two groups: dark-red square, MC/WD/WD; light-red square, MC/WD/WD-TX.
(E) HE
staining of the liver. Histological analysis using the NAS. Scale bars:
100 µm
. (
F
) F4/80 im-
munostaining of the liver. The arrows indicate the CLS. (
G
) Immunostaining for collagen type III of the
liver. (
H
) Hydroxyproline contents of the liver. Scale bars: 100
µ
m;
n= 10–11
; * p< 0.05 and
** p< 0.01
.
(
I
) experimental protocol: the Mc4r-deficient mice were fed a WD for
20 weeks
to develop NASH and
then treated with or without 3% Taxifolin for an additional 30 weeks (MC/WD/WD or MC/WD/TX,
respectively). (
J
) Representative image of the gross appearance of the livers.
(K–M) Two
groups:
black square, MC/WD/WD; light-red square, MC/WD/WD-TX. (
K
,
L
) Incidence and multiplicity of
foci (
K
) and tumors (
L
) in the liver. (
M
) Representative images of the HE staining of the macroscopic
tumoral (left) and nontumoral (right) lesions. The areas defined by yellow and red lines indicate a
grossly detectable tumor and an HCC-like lesion that can only be detected by histological examina-
tion, respectively. a–c: A higher magnification view of the HCC-like lesion (a), background tumor (b),
and dysplastic nodule (c). (
N
): Expression levels of genes related to inflammation (Emr1,Itgax, and
Tnf
α
), fibrosis (Tgfb1,Timp1, and Col1a1), and lipid metabolism (Ppar
α
,Cpt1a,Acox,Srebp1, and Fas)
in nontumorous lesions of the liver. Values are presented as the means
±
SEM; n= 13 and 12, for
MC/WD/WD and MC/WD/TX, respectively; significant differences: ** p< 0.01.
Finally, we investigated whether Taxifolin can prevent the progression from NASH
to liver tumors. The Mc4r-deficient mice were on a WD for 20 weeks to develop NASH,
and then the mice were further fed a WD with or without Taxifolin for an additional
30 weeks
(Figure 8I). As we previously reported [
3
], these mice developed multiple liver
tumors, which could also be detected by macroscopic observations of the surface of the
liver (Figure 8J). We grossly examined the number of foci and tumors according to their
size and found that the Taxifolin treatment markedly suppressed the number of foci and
tumors (Figure 8K,L, Supplementary Materials Figure S1). Interestingly, close histological
examination of the tissue sections obtained from the grossly observed tumors revealed
that they were histologically heterogeneous, with some areas resembling human HCC and
others being composed of proliferative dysplastic hepatocytes with steatosis (Figure 8M,
left). In the HCC-like lesions, the tumor cells exhibited a uniform morphology with enlarged
and hyperchromatic nuclei, and they formed irregular and thick trabeculae consisting of
two or more cells, accompanied by the loss of normal liver architecture (Figure 8Ma,b).
Although not reaching a statistically significant difference, the Taxifolin treatment tended
to decrease the area of HCC-like lesions (Figure 8M, left). Furthermore, our histological
observations also detected small dysplastic nodules with diameters between 0.5 and 2 mm
in the macroscopically nontumoral NASH liver, where we found proliferation of atypical
hepatocytes with enlarged and hyperchromatic nuclei (Figure 8M, right). There were fewer
microscopic dysplastic nodules in the Taxifolin treatment group than the control groups
(Figure 8M, right). Finally, we found that the Taxifolin treatment significantly reduced
the mRNA expression of genes related to inflammation and fibrosis in the tumorous
lesions of the liver, without affecting Cd206, a representative marker for tumor-associated
macrophages (Supplementary Materials Figure S3). Taken together, these observations
suggest that Taxifolin potently prevents the progression from NASH to liver tumors in a
murine model.
4. Discussion
In this study, we demonstrated that the Taxifolin treatment markedly prevents the
development of hepatic steatosis, NASH, and liver tumors in a mouse model. In particular,
Taxifolin is effective on lipid accumulation, chronic inflammation, and fibrosis in the liver
when the treatment starts after the mice develop hepatic steatosis and NASH. Although the
precise mechanisms of action of Taxifolin remain to be fully elucidated, our data suggest that
Taxifolin directly acts on hepatocytes and brown adipocytes to suppress lipogenesis and
activate energy expenditure, respectively. Moreover, we found that the Taxifolin treatment
Nutrients 2023,15, 350 15 of 19
effectively prevents the progression from NASH to liver tumors. To date, numerous clinical
studies have been conducted for NASH in which the primary propositions include the
resolution of NASH without the worsening of fibrosis or the improvement of fibrosis
without the resolution of NASH [
29
]. Considering the long-term outcome, there is a need to
investigate the effects of novel medicines on liver tumorigenesis. However, it is technically
difficult because of a lack of appropriate animal models that develop hepatic steatosis,
NASH, and liver tumors, sequentially. In this regard, using our unique animal model, we
provided evidence of Taxifolin’s therapeutic potential in hepatic steatosis, NASH, and liver
tumors, with high safety and long-term efficacy, because it is already widely used as a
health supplement.
In this study, we confirmed the anti-obese and antidiabetic effects of Taxifolin using
two different mouse models (i.e., diet-induced and genetically obese mice). For its underly-
ing mechanism, we found that Taxifolin increases the activity of brown adipose tissue at
least through two distinct pathways: direct action on brown adipocytes and indirect action
via FGF21 production. Since there are species differences in the cellular functions and
markers of brown adipocytes, we employed human iPS cell-derived brown adipocytes to
examine the direct effects of Taxifolin. In addition to the genes related to differentiation and
thermogenesis, BATokines are supposed to play a key role in energy homeostasis. Indeed,
while the contribution of brown adipose tissue to whole-body energy expenditure is not
evident in humans, accumulating evidence supports the significant role of brown adipose
tissue in ameliorating systemic metabolic conditions [
30
–
33
]. In contrast, the activation
of brown adipose tissue may be involved in cancer cachexia [
34
,
35
]. In this regard, our
data show that Taxifolin prevents tumorigenesis in the liver without inducing cachexia,
suggesting the appropriate brown adipose tissue activation. Collectively, this study pro-
vides novel insight into the clinical translation of Taxifolin for the treatment of obesity and
its complications.
It is important to discuss the potential mechanisms of Taxifolin-mediated antitumor
effects in a mouse model of NASH. Accumulating evidence indicates that tumor-associated
macrophages with anti-inflammatory properties enhance tumor growth and induce resis-
tance against conventional antitumor therapies. On the other hand, sustained low-grade
inflammation has been implicated in the pathogenesis of liver fibrosis, which plays a piv-
otal role in carcinogenesis. In this study, Taxifolin effectively suppressed hepatic steatosis,
inflammation, and fibrosis in our NASH model. Similar anti-inflammatory and antifibrotic
effects were observed in tumorous lesions of the liver. Therefore, it is conceivable that
Taxifolin suppresses tumor development of the liver, mainly through inhibiting chronic
inflammation in nontumorous lesions. In line with this, several studies reported that
Taxifolin inhibits pro-inflammatory cytokine expression and NF-
κ
B activation in cultured
macrophages [
36
,
37
]. Further studies are required to evaluate the direct effect of Taxifolin
on the growth of tumor cells.
As the body-weight-lowering effects of Taxifolin were relatively mild when Taxifolin
was administered to obese mice, it is important to determine how Taxifolin regulates
NASH-like liver phenotypes. Indeed, the Taxifolin treatment markedly suppressed hep-
atic expression of genes related to lipogenesis, suggesting a direct action of Taxifolin on
hepatocytes. Consistently, Taxifolin significantly inhibited the palmitate-induced lipid
accumulation and upregulation of lipogenic genes in cultured hepatocytes. Of note, based
on previous studies [
38
], the dose of Taxifolin used in our
in vitro
study is considered to be
within the physiological range, although we did not determine its serum concentrations in
our mouse models. Taxifolin is also known to suppress proinflammatory cytokine expres-
sion in cultured macrophages
in vitro
and chemically induced liver fibrosis
in vivo
[
18
,
39
].
Several studies suggest the involvement of Nrf-2 (nuclear factor erythroid 2-related
factor 2
),
HO-1 (heme oxygenase 1), and AMPK (AMP-activated protein kinase) as the underlying
mechanism of the antioxidative properties of Taxifolin [
8
,
40
,
41
]. Given that the NASH
pathogenesis is heterogenous and diverse in humans, these pleiotropic effects of Taxifolin
may be advantageous for clinical practice. In this respect, recent studies developed a novel
Nutrients 2023,15, 350 16 of 19
nanocomplex of selenium with sorafenib [
42
] or Taxifolin [
43
], which exhibits more benefi-
cial effects than those of single molecules
in vitro
in terms of anticancer or neuroprotective
effects. Accordingly, the use of the nanocomplex would be a valuable therapeutic option
for NASH. Our next step will be to explore the carcinogenic mechanism of Taxifolin on
NASH using different animal models, although there are few animal models suitable for
investigating the NASH continuum.
5. Conclusions
In summary, we demonstrated the novel therapeutic potentials of Taxifolin, a unique
bioactive flavonoid, for obesity-induced hepatic steatosis, fibrogenesis, and tumorigenesis
in mice. Previous studies have pointed to the anti-obesity, antidiabetic, anti-inflammatory,
and antitumor effects of Taxifolin in various
in vitro
and
in vivo
models [
8
]. In addition,
this study provides evidence that Taxifolin is effective on the NASH continuum. Our data
also provide insight into the novel mechanisms of action of Taxifolin and collectively may
pave the way for the clinical translation of Taxifolin.
Supplementary Materials:
The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/nu15020350/s1, Figure S1: Representative image of the gross
appearance of livers; Figure S2: Expression levels of genes related to inflammation, fibrosis, and
lipid metabolism in the liver of a NASH model treated with Taxifolin; Figure S3: Expression levels of
genes related to inflammation, fibrosis, and tumor-associated macrophages in tumorous lesions of
the liver in a mouse model of NASH treated with Taxifolin; Table S1: Primers for mice used in the
present study; Table S2: Primers for humans used in the present study; Table S3: Preventive effects
of Taxifolin on serum parameters in Mc4r-deficient mice fed a Western diet; Table S4: Therapeutic
effects of Taxifolin on serum parameters in Mc4r-deficient mice fed a Western diet after the mice
developed NASH.
Author Contributions:
Conceptualization, M.T. (Miyako Tanaka), N.S.-A. and T.S.; methodology,
M.T. (Miyako Tanaka), N.S.-A. and T.S.; validation, M.T. (Miyako Tanaka), N.S.-A. and T.S.; formal
analysis, T.I., B.F., M.T. (Miyako Tanaka), M.N., and T.S.; investigation, T.I., B.F., M.N., H.K., K.O.
and S.I.; resources, H.N. and A.K.; data curation, M.T. (Miyako Tanaka), N.S.-A. and T.S.; writing—
original draft preparation, T.I. and B.F.; writing—review and editing, H.Y., A.I., S.S., M.I. (Masafumi
Ihara), M.T. (Miyako Tanaka), H.K., N.S.-A. and T.S.; visualization, T.I., B.F., M.T. (Miyako Tanaka),
M.N., H.K., Y.S., A.E., N.S.-A. and T.S.; supervision, M.T. (Masashi Tanaka), M.I. (Michiko Itoh).
and K.S.; project administration, M.T. (Miyako Tanaka), N.S.-A. and T.S.; funding acquisition, M.T.
(Miyako Tanaka), N.S.-A. and T.S. All authors have read and agreed to the published version of
the manuscript.
Funding:
This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry
of Education, Culture, Sports, Science, and Technology of Japan (18H02737, 18K19769, 19K07905,
20H03447, 21H02835, 21K08526, 22H04806, 22K11720, 22K19524, and 22K19723); Japan Agency for
Medical Research and Development (CREST (JP22gm1210009s0104), Research Program on Hepatitis
(JP22fk0210082s0102 and 22fk0210094s0202), and Research Program on Rare and Intractable Diseases
(JP22ek0109488)). This study was also supported by research grants from the SEI Group CSR Founda-
tion, ONO Medical Research Foundation, KOSE Cosmetology Research Foundation, Smoking Re-
search Foundation, Daiko Foundation, Kobayashi Foundation, Takeda Medical Research Foundation,
Gout and Uric Acid Research Foundation of Japan, and TERUMO LIFE SCIENCE FOUNDATION.
Institutional Review Board Statement:
All animal experiments were conducted in accordance with
the guidelines for the care and use of laboratory animals of the National Hospital Organization
Kyoto Medical Center and Nagoya University. The protocols were approved by the Animal Care
and Use Committee, Clinical Research Institute, National Hospital Organization Kyoto Medical
Center (approval number: 2-19-2), and the Animal Care and Use Committee, Research Institute of
Environmental Medicine, Nagoya University (approval number: R210097).
Informed Consent Statement: Not applicable.
Data Availability Statement:
The data that support the findings of this study are available from the
corresponding author upon reasonable request.
Nutrients 2023,15, 350 17 of 19
Acknowledgments:
We thank Joel K. Elmquist (University of Texas Southwestern Medical Center)
and Nobuyuki Itoh (Kyoto University, Kyoto, Japan) for their generous gifts of Mc4r-deficient mice
and Fgf21-deficient mice, respectively. We also thank the members of the Suganami and Satoh-
Asahara laboratories for their helpful discussions.
Conflicts of Interest:
The authors declare no conflict of interest. The funding agencies had no role
in the design of the study; in the collection, analyses, or interpretation of data; in the writing of
the manuscript; or in the decision to publish the results. Seaknit Biotechnology Co., Ltd., donated;
Department of Metabolic Syndrome and Nutritional Science, Research Institute of Environmental
Medicine, Nagoya University.
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