Yonago Acta medica 2014;57:147–158

Original Article

Effects of Kampo Formulas on the Progression of Hypercholesterolemia and Fatty
Liver Induced by High-Cholesterol Diet in Rats
Weibin Qian,* Junichi Hasegawa,* Satoshi Tsuno,* Yusuke Endo,* Akiko Matsuda† and Norimasa Miura**Division of Pharmacotherapeutics, Department of Pathophysiological and Therapeutic Science, School of Medicine, Tottori University
Faculty of Medicine, Yonago 683-8503, Japan and †Department of Fundamental Nursing, School of Health Science, Tottori University
Faculty of Medicine, Yonago 683-8503, Japan

ABSTRACTHFABP, CFABP, MCP1 and CCR2 in liver and adipose
tissue were decreased by Boiogito and ezetimibe.
Conclusion Boiogito has a protective effect on the
progression of hypercholesterolemia and fatty liver induced by high-cholesterol diet in rats and more effective
than Bofutsushosan and Keishibukuryogan. The lipidlowering effect of Boiogito is not stronger than ezetimibe. But the anti-inflammatory (MCP1, CCR2) andanti-arteriosclerotic (ICAM-1) effects of Boiogito are
more potent than ezetimibe.

Background

Bofutsushosan is a well known Kampo,
traditional Japanese medicine, based on ancient Chinese
medicine mainly used in the treatment of hypercholesterolemia in Japan. We selected two Kampo formulas,
Boiogito and Keishibukuryogan mainly used in thetreatment of hypercholesterolemia in China to comparewith Bofutsushosan and cholesterol absorption inhibitor
ezetimibe.
Methods Hypercholesterolemia and fatty liver were
induced by high cholesterol (containing 2% cholesterol
and 0.5% cholic acid) diet in male Wistar rats for 6 and
12 weeks. Kampo formulas Boiogito, Bofutsushosan,
Keishibukuryogan and ezetimibe were added to thehigh-cholesterol diet, respectively. After 6 and 12 weeks,body and liver weights, blood chemistry, cholesterol
concentrations, fat-related and inflammatory-related factors were examined.
Results
High-cholesterol diet increased body andliver weights, and serum cholesterol concentrations.
Boiogito and ezetimibe improved them. Serum ICAM1 and RBP4 were increased in the high cholesterol dietgroup. Boiogito and ezetimibe improved them too. In
the histological examinations of liver and adipose tissues, we observed a significant improvement after treatment. Immunostaining expression of ICAM-1 in aorta
was improved by Boiogito, Bofutsushosan, Keishibukuryogan and ezetimibe. The mRNA expression of RBP4,

Key words

Bofutsushosan; Boiogito; Keishibukuryogan; fatty liver; hypercholesterolemia
Hypercholesterolemia is one of the major risk factors
for many cardiovascular diseases, such as atherosclerosis, hypertension and myocardial infarction.1, 2 Nonalcoholic fatty liver disease (NAFLD) is common in thegeneral population, and it occurs even more frequently
in patients with hypercholesterolemia.3, 4 Patients with
NAFLD have a high risk of cardiovascular disease(CVD).5 In addition, NAFLD is often associated withatherosclerotic signs including the presence of carotid
plaques6 and coronary arterial calcification.7, 8
It is generally believed that the occurrence and development of hypercholesterolemia have significantly
correlation to lipid metabolism-related genes, suchas retinol-binding protein 4 (RBP4), heart fatty acidbinding protein (HFABP), cutaneous fatty acid-binding
protein (CFABP).9, 10 RBP4, a protein secreted by hepatocytes and adipose tissue, is closely related to hypercholesterolemia and NAFLD.11 HFABP and CFABP
belongs to FABPs family which may play a broad role incellular fatty acid metabolism.12 In addition to lipid metabolism-related genes, inflammatory cytokines are associated with hypercholesterolemia. Engström et al. havesupported the view that inflammation could be a risk
factor for developing hypercholesterolemia.13 Monocyte
chemoattractant protein-1 (MCP-1) and its receptor CC
chemokine receptor 2 (CCR2) are important inflammatory chemokines linked with hypercholesterolemia.14, 15Intercellular adhesion molecule-1 (ICAM-1) can promote
the development and progression of atherosclerosis.16

Corresponding author: Junichi Hasegawa, MD
hasegawa@med.tottori-u.ac.jp
Received 2014 October 14
Accepted 2014 November 10
Abbreviations: ALP, alkaline phosphatase; ALT, alanine aminotransferase; BOT, Boiogito; BTS, Bofutsushosan; C, control group;
CFABP, cutaneous fatty acid-binding protein; CVD, cardiovascular disease; H, high-cholesterol diet group; HA, high-cholesterol
diet with Boiogito group; HB, high-cholesterol diet with Bofutsushosan group; HC, high-cholesterol diet with Keishibukuryogangroup; HDL-C, high-density lipoprotein cholesterol; HE, highcholesterol diet with ezetimibe group; HFABP, heart fatty acidbinding protein; ICAM-1, intercellular adhesion molecule-1;
KBG, Keishibukuryogan; LDL-C, low-density lipoprotein cholesterol; MCP1, monocyte chemoattractant protein-1; NAFLD, nonalcoholic fatty liver disease; RBP4, retinol-binding protein 4; TC,
total cholesterol; VEC, vascular endothelial cells147

W. Qian et al.

Therefore, it is a simple and convenient method to detect
hypercholesterolemia by monitoring RBP4, C-FABP, HFABP, MCP1, CCR2, ICAM-1 expression.
Bofutsushosan (BTS), Boiogito (BOT) and Keishibukuryogan (KBG) are well-known Japanese Kampo
and Chinese traditional herbal medicines which are used
to improve obesity.17, 18 BTS has been reported to inhibit
atherosclerosis,19 obesity,17, 18, 20 hypertension17 and hyperglycemia.21 BOT and KBG are also Kampo preparations which have been used in patients with obesity.22, 23
However, pharmacological evidence for the effects oftreatment of NAFLD of BOT and KBG still remains
obscure. Using the well-established experimental modelof high-cholesterol diet in rats, we compared the pharmacological efficacies of BOT, BTS and KBG.

the left kidney and abdominal aorta were removed,
and then portions of the samples were stored in a 10%
formalin solution for hematoxylin-eosin staining andoil red O staining.25 The remaining samples were immediately transferred into EP tubes containing 500 μL
of RNA later (Ambion, Austin, TX), quickly frozen inliquid nitrogen, and stored at –80 ˚C. Serum levels of total cholesterol (TC), high-density lipoprotein cholesterol(HDL-C), low-density lipoprotein cholesterol (LDL-C),
triglyceride, blood sugar, creatinine, total bilirubin, alanine aminotransferase (ALT) and alkaline phosphatase(ALP) were analyzed for rats using an auto analyzer at
an accredited clinical laboratory (SRL, Tokyo).
ICAM-1 and RBP4

Serum samples were applied for an enzyme-linked
immunosorbent assay (ELISA) of ICAM-1 (R ＆ D,
Minneapolis, MN) and RBP4 (Aviscera Bioscience, CA)according to the manufacturer’s instructions.

MATERIALS AND METHODS
Rats and feeding method

Sixty male Wistar rats aged 8-weeks (purchased from
Shimizu Laboratory Supplies, Kyoto, Japan) were kept
in an air-conditioned room at 25 ˚C with 55% humidity
and given standard chow. After 3 days of acclimation,
the rats were divided into 6 groups: control group (C,n = 10), high-cholesterol diet group (H, n = 10), highcholesterol diet with Kampo formula BOT group (HA,n = 10), high-cholesterol diet with Kampo formula BTS
group (HB, n = 10), high-cholesterol diet with Kampo
formula KBG group (HC, n = 10), high-cholesterol dietwith ezetimibe group (HE, n = 10). The rats in eachgroup were numbered from 1 to 10. Group C was fed
a standard rat diet (CE-2; Japan Clea, Tokyo, Japan). A
high-cholesterol diet11, 24 was supplied for Group H; it
was prepared by adding 2% cholesterol and 0.5% cholicacid to the standard diet. The high-cholesterol diets with
BOT, BTS or KBG were made by adding 1% extract formulations of BOT, BTS or KBG to the high-cholesterol
diet, respectively. These Kampo extract formulations
were generous gift from Tsumura (Tokyo, Japan). The
high-cholesterol diet with ezetimibe was made by adding 0.0006% of ezetimibe (LKT laboratories, St Paul,
MN). The amount of feed for each rat was regulated
to 25 g/day and water was supplied ad libitum. Body
weights, systolic and diastolic blood pressure and heart
rate were measured weekly. Blood pressure and heart
rate were measured by a noninvasive computerized tailcuff method (BP-98A; Softron, Tokyo).

RT-PCR

Total RNA was extracted from the liver and adipose
tissue around the left kidney using TRIzol reagent according to the manufacturer’s instructions (Promega,
Carlsbad, CA). A semiquantitative real-time PCR (RTPCR) was performed with Line-Gene (Toyobo, Tokyo)
and SYBR Green I (Roche, Basel, Switzerland). The
detection was executed at the extension reaction stage ineach cycle.
The primer sets for RBP4, HFABP, CFABP, MCP1,
CCR2 and beta-actin mRNA were all synthesized by
Hokkaido System Science (Sapporo, Japan). The sequence of each primer is listed in Table 1. Using the2-ΔΔCT method, mRNA expression was semi-quantitatively measured as a relative amount of each target RNA
to a known housekeeping gene (beta-actin) expression
level.26, 27

Table 1. The sequence of each PCR primer
Gene

PrimerForward 5'-gacaaggctcgtttctctgg-3'
Reverse 5'-gactcgtcccttggctgtag-3'
H-FABP
Forward 5'-ctagcatgagggaagcaagg-3'
Reverse 5'-tgcttcatccagacaagtgg-3'
C-FABP
Forward 5'-gggctggctcttaggaagat-3'
Reverse 5'-aaaacacggtcgtcttcacc-3'
MCP1
Forward 5'-ctgtagcatccacgtgctgt-3'
Reverse 5'-tgctgctggtgattctcttg-3'
CCR2
Forward 5'-gatcctgcccctacttgtca-3'
Reverse 5'-agatgagcctcacagcccta-3'
Beta-actin Forward 5'-gtagccatccaggctgtgtt-3'
Reverse 5'-ccctcagatgggcacagt-3'
RBP

Sample collection

On days 42 and 84, 5 rats of each group in the order of
how they were numbered were sacrificed by collecting
blood from the heart under pentobarbital anesthesia after fasting for 12 h. Liver tissue, adipose tissue around148

Kampo formulas ameliorates dyslipidemia

Fig. 1. Histopathological examination of liver.
A: Fatty degeneration (steatosis) of the liver is observed in the high-cholesterol diet-fed (H, HA, HB, HC and HE) groups, but not inGroup C in hematoxylin-eosin stained tissues. Fatty changes increase with time to a greater extent in Group H than Groups HA, HB,
HC and HE.
B: Oil red O staining reveals more lipid droplets (stained red) to be accumulated in vacuoles in Group H than other groups.
6W, 6 weeks; 12W, 12 weeks. C, control: standard diet for 6 and 12 weeks (n = 5). H: high-cholesterol diet for 6 and 12 weeks (n = 5). HA:
high-cholesterol diet with Boiogito for 6 and 12 weeks (n = 5). HB: high-cholesterol diet with Bofutsushosan for 6 and 12 weeks (n = 5).
HC: high-cholesterol diet with Keishibukuryogan for 6 and 12 weeks (n = 5). HE: high-cholesterol diet with ezetimibe for 6 and 12 weeks (n= 5). Bars express 25 μm.

149

W. Qian et al.

Fig. 2. Histopathological examination of adipose tissue.
The larger fat cells are observed in the high-cholesterol diet-fed (H, HA, HB, HC and HE) groups, but not in Group C (56.13 ± 11.51 and
64.88 ± 13.96 μm after 6 and 12 weeks, respectively) in hematoxylin-eosin stained tissues. Fat cells change with time to a greater extent inGroup H (97.00 ± 20.14 and 104.63 ± 22.56 μm after 6 and 12 weeks, respectively) than Groups HA, HB, HC and HE (55.63 ± 5.85 and78.88 ± 16.34 μm, 75.63 ± 11.53 and 78.13 ± 16.47 μm, 74.63 ± 13.94 and 88.13 ± 8.58 μm, 74.38 ± 13.21 and 83.13 ± 16.63 μm after 6 and
12 weeks, respectively). Bars express 50 μm.

Immunohistochemical studies

During the immunohistochemical analyses, 4% formaldehyde-fixed aorta tissue specimens were processed.
The following monoclonal antibody was used: antiICAM-1 (Abcam, Cambridge, United Kingdom). As anegative control, tissues were stained without the primary antibody. The optical densities were measured by Image-Pro Plus version 6.0 software (Media Cybernetics,
Rockville, MD).

changes comprising tiny and large vacuoles as well as
pleomorphic nuclei were more prominent in Groups
HA, HB, HC and HE than in Group H (Fig. 1A). Oil red
O staining revealed that the livers in all high-cholesterol
diet supplemented groups were filled with microvesicular or macrovesicular fat deposits; they were depicted as
reddish deposits (Fig. 1B). Overall, fatty liver changes
were more prominent in Group H than Groups HA, HB,
HC and HE (× 400).

Statistical analyses

Histological examination of adipose tissue

The data are expressed as the mean ± SEM. For thecontinuous variables, differences in responses amonggroups were compared using Mann-Whitney’s U test for
non-parametric variables by SPSS 11.0 J (SPSS Japan,
Tokyo). P < 0.05 was considered statistically significant.

As Fig. 2 shows, the larger fat cells were observed in
the high-cholesterol diet-fed (H, HA, HB, HC and HE)
groups, but not in Group C. It was more prominent inGroup H than Groups HA, HB, HC and HE (× 400).
Body and liver weights

RESULTSAs Table 2 shows, there were no significant differencesin the baseline of body weights at the beginning of theexperiment. The body weights had no significant differences during the experiment too. Liver weights weresignificantly lower in Group HE than Group H after 6
and 12 weeks (P < 0.05).

Histological examination of liver tissues

As Fig. 1 shows, the fatty degeneration (steatosis) of liver
was observed in the high-cholesterol diet-fed (H, HA,
HB, HC and HE) groups, but not in Group C. These
150

Kampo formulas ameliorates dyslipidemia
Table 2. Body and liver weights
Group C
Body weight (g)0W
233.1 ± 6.6
6W
402.8 ± 15.9
12W
448.6 ± 14.0
Liver weight (g)6W
10.1 ± 0.9
12W12.5 ± 3.5Group HGroup HAGroup HBGroup HCGroup HE

233.5 ± 6.6
398.5 ± 23.2
477.1 ± 15.5*

235.9 ± 6.7
390.0 ± 21.9
461.6 ± 26.1

237.2 ± 9.4
392.4 ± 8.6
457.6 ± 32.7

237.1 ± 7.8
395.9 ± 23.9
470.2 ± 32.9

236.1 ± 6.7
385.8 ± 24.6
453.3 ± 29.2

15.0 ± 0.8*
19.0 ± 3.1*

15.5 ± 2.6
18.9 ± 1.5

15.8 ± 1.4
19.9 ± 2.4

14.9 ± 1.1
19.9 ± 2.2

10.8 ± 1.0†
12.1 ± 1.1†

0W, 0 weeks; 6W, 6 weeks; 12W, 12 weeks. C, control: standard diet for 6 and 12 weeks (n = 5). H: high-cholesterol diet for 6 and 12 weeks (n= 5). HA: high-cholesterol diet with Boiogito for 6 and 12 weeks (n = 5). HB: high-cholesterol diet with Bofutsushosan for 6 and 12 weeks (n= 5). HC: high-cholesterol diet with Keishibukuryogan for 6 and 12 weeks (n = 5). HE: high-cholesterol diet with ezetimibe for 6 and 12
weeks (n = 5). *P < 0.05 versus Group C. †P < 0.05 versus Group H. Data are expressed as the mean ± SEM.

Table 3. Blood chemistry and cholesterol concentrations
TC (mg/dL)

6W
12W
6W
12W
6W
12W
6W
12W
6W
12W
6W
12W
6W
12W
6W
12W
6W
12W

HDL-C (mg/dL)
LDL-C (mg/dL)
TG (mg/dL)
BS (mg/dL)
Cr (mg/dL)
TBil (μmol/L)
ALT (U/L)
ALP (U/L)Group CGroup HGroup HAGroup HBGroup HCGroup HE55.5 ± 15.2
56.6 ± 13.0
18.0 ± 2.6
16.0 ± 2.6
5.6 ± 1.1
6.0 ± 1.4
31.5 ± 7.5
29.2 ± 9.2
109.4 ± 7.7
102.8 ± 6.1
0.30 ± 0.03
0.32 ± 0.04
0.03 ± 0.01
0.04 ± 0.02
60.0 ± 11.7
53.4 ± 3.0
413.0 ± 22.6
331.0 ± 28.5

68.2 ± 6.8
78.8 ± 5.4*
21.0 ± 2.2
22.2 ± 0.8*
8.4 ± 1.7*
11.8 ± 0.8*
34.4 ± 11.2
26.4 ± 4.7
99.6 ± 7.2
112.0 ± 11.0
0.30 ± 0.04
0.35 ± 0.03
0.03 ± 0.01
0.03 ± 0.02
72.2 ± 11.4*
148.7 ± 25.7*
497.8 ± 24.8
432.7 ± 31.7*

67.4 ± 13.7
73.7 ± 10.4
20.4 ± 4.5
22.7 ± 3.2
8.6 ± 2.3
9.0 ± 1.7†
28.0 ± 17.1
22.7 ± 6.9
112.8 ± 8.8†
111.3 ± 20.0
0.35 ± 0.06
0.34 ± 0.02
0.03 ± 0.01
0.03 ± 0.01
71.8 ± 18.2
79.0 ± 17.4†
377.5 ± 31.2†
334.3 ± 32.1†

65.4 ± 9.8
75.2 ± 12.0
19.4 ± 2.9
23.8 ± 3.1
8.0 ± 1.99.8 ± 2.7
18.2 ± 3.9†
24.0 ± 7.9
105.4 ± 6.6
114.8 ± 17.8
0.35 ± 0.05
0.36 ± 0.11
0.04 ± 0.01
0.03 ± 0.01
61.3 ± 10.2
104.3 ± 26.1†
399.0 ± 20.1†
337.0 ± 25.5†

68.6 ± 13.8
70.5 ± 12.0
20.0 ± 3.2
20.0 ± 3.4
8.8 ± 2.6
10.5 ± 4.4
21.8 ± 8.8
19.8 ± 5.4
110.8 ± 12.0
99.2 ± 7.6
0.34 ± 0.04
0.34 ± 0.03
0.03 ± 0.01
0.03 ± 0.01
93.6 ± 17.1
79.2 ± 18.5†
450.0 ± 35.6
283.3 ± 27.1†

78.5 ± 12.4
73.2 ± 3.5
17.5 ± 2.7
19.4 ± 1.1
8.8 ± 2.2
8.0 ± 1.2†
25.0 ± 8.0
16.4 ± 7.0†
112.6 ± 10.5
101.2 ± 9.2
0.34 ± 0.07
0.32 ± 0.03
0.03 ± 0.02
0.04 ± 0.01
111.0 ± 11.1†
109.7 ± 25.8
519.0 ± 34.9
514.8 ± 17.8†

6W, 6 weeks; 12W, 12 weeks. ALP, alkaline phosphatase; ALT, alanine aminotransferase; BS, blood sugar; Cr, creatinine; HDL-C, highdensity lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; TBil, total bilirubin; TC, total cholesterol; TG, triglyceride.
*P < 0.05 versus Group C. †P < 0.05 versus Group H. Data are expressed as the mean ± SEM.

(ng/mL)
25

*
*

20

ICAM-1††(ng/mL)

†

†

*
* †

7
6
5

RBP4

6W
12W

††

†

4

15

3
10

C

H

HA

HB

HC

HE

2

C

H

HA

HB

HC

HE

Fig. 3. Intercellular adhesion molecule-1 (ICAM-1) and retinol-binding protein 4 (RBP4) concentration in serum. ICAM-1 and RBP4
concentrations in control, standard diet group (C, n = 5), high-cholesterol diet group (H, n = 5), high-cholesterol diet with BOT group (HA,n = 5), high-cholesterol diet with BTS group (HB, n = 5), high-cholesterol diet with KBG group (HC, n = 5) and high-cholesterol diet withezetimibe group (HE, n = 5) for 6 and 12 weeks. *P < 0.05 versus Group C. †P < 0.05 versus Group H.

151

W. Qian et al.

Liver
RBP4

*

1.7

*

1.3

Adipose tissue

†

†

†

†

0.9

*

1.0

6W

RBP4

*

1.2

12W

†
†

†

†

0.8

0.5

0.6

1.5

*

HFABP

†

†

1.1

1.1

0.9

0.9

0.7

0.7

*

1.3

CFABP

*

1.1

†

†

†

CFABP

1.3

†

†

†

*

1.3

1.3

HFABP

*

1.5

1.1

†

†

0.9

†
0.9

0.7

0.7

*

1.5

MCP1

†

*

1.3

†

*

1.2

†

1.1

†
†

0.9

0.9

MCP1

*

1.5

†

†

†

†

†

†

0.6

0.7

CCR2

1.5

*

1.2

*
†
†

0.9

††

*

1.6

††

†

CCR2

*

1.3

††

1.0

0.6

†
†

†

†

0.7

C

H

HA

HB

HC

HE

C

H

HA

HB

HC

HE

Fig. 4. Changes in mRNA expression with time in liver and adipose tissue. Levels of RBP4, HFABP, CFABP, MCP1 and CCR2 against
beta-actin mRNA expression are shown in the above histograms. The left-side graphs show the expression in liver. The right-side graphs
show the expression in adipose tissue around the left kidney.

Blood chemistry and cholesterol concentrations

13.0 mg/dL) (P < 0.05). The concentration of HDL-C in
Group H (22.2 ± 0.8 mg/dL) was significantly elevatedcompared with it in Group C (16.0 ± 2.6 mg/dL) (P < 0.05).
The concentration of LDL-C elevated to a greater extent
in Group H (11.8 ± 0.8 mg/dL) than that in Groups HA
and HE (9.0 ± 1.7 and 8.0 ± 1.2 mg/dL, respectively) (P< 0.05). The concentration of ALT in Group H (148.7 ±After 6 weeks of the experiment, the concentration of
LDL-C in Group H (8.4 ± 1.7 mg/dL) was significantly
elevated compared to the concentration in Group C (5.6
± 1.1 mg/dL) (P < 0.05). After 12 weeks, the concentration of TC in Group H (78.8 ± 5.4 mg/dL) was significantly elevated compared with it in Group C (56.6 ±152

Kampo formulas ameliorates dyslipidemiaGroup CGroup HGroup HAGroup HBGroup HCGroup HE*

Optical density

0.026
0.022

†
†

†

†

0.018
0.014
0.010

C

H

HA

HB

HC

HE

Fig. 5. Immunohistochemistry expression of ICAM-1 in aorta.
A: Seldom ICAM-1 immunostaining (red-brown deposits indicate positive staining) is found on the whole layers of abdominal aortas inGroup C. Bars express 25 μm.
B: Mean optical density values of ICAM-1. The photographs generated were quantitatively analyzed the optical density of ICAM-1 with
Image-Pro Plus version 6.0 software. *P < 0.05 versus Group C. †P < 0.05 versus Group H.

25.7 U/L) was significantly elevated compared with it inGroup C (53.4 ± 3.0 U/L) (P < 0.05). It was decreased inGroups HA, HB and HC (79.0 ± 17.4, 104.3 ± 26.1, 79.2
± 18.5 U/L, respectively) than in Group H (P < 0.05).
The concentration of ALP in Group H (432.7 ± 31.7 U/L) was significantly elevated compared with it in GroupC (331.0 ± 28.5 U/L) (P < 0.05). It was lower in Groups
HA, HB and HC (334.3 ± 32.1, 337.0 ± 25.5, 283.3 ± 27.1
U/L, respectively) than in Group H (P < 0.05) (Table 3).

for 6 and 12 weeks.
RBP4 mRNA expression in liver in Group H wassignificantly increased compared to that in Groups C,
HA and HE after 6 and 12 weeks (P < 0.05). HFABPmRNA expression in Group H was significantly increased compared to that in Groups C, HA and HE after
12 weeks (P < 0.05). Compared with the other groups,
the mRNA expression of CFABP in liver in Group H
were higher than that in Groups C, HA, HB and HE at
12 weeks (P < 0.05), whereas they were not obvious at
6 weeks. Compared with the other groups, the mRNA
expression of MCP1 in liver in Group H remained highduring the entire experiment, especially after 12 weeks(P < 0.05). The expression of CCR2 in liver in Groups
HA, HB, HC (at 6 and 12 weeks) and HE (at 12 weeks)
were lower than that in Group H (P < 0.05).
A down-regulation of RBP4 mRNA expression inGroup H appeared in adipose tissue around the left kidney after 12 weeks; even it was higher in Group H thanthat in Groups C, HA and HE (P < 0.05). The mRNA
expression of HFABP was higher in Group H than that

ICAM-1 and RBP4 in serum

ICAM-1 concentrations were increased in Group H
compared to Groups C, HA, HB and HE after 12 weeks(Fig. 4, P < 0.05). RBP4 concentrations were increasedin Group H compared to the other groups including Groups C, H, HA and HE after 6 and 12 weeks(Fig. 3, P < 0.05).
mRNA expression in liver and adipose tissue

Figure 4 demonstrates the changes of mRNA expression
in liver and adipose tissue round the left kidney of rats153

W. Qian et al.

in Groups C, HA, HE (at 6 and 12 weeks) and HB (at12 weeks) (P < 0.05). CFABP mRNA expression had
no significant difference during the whole experimentamong all the groups. The expression of MCP1 in adipose tissue in Groups HA, HB, HE (at 6 and 12 weeks)and HC (at 12 weeks) was lower than that in Group H (P< 0.05). The CCR2 mRNA expression in Group H was
higher than that in Groups HA, HB and HC (P < 0.05).

of liver and hypertrophy of fat cells caused by highcholesterol diet could be alleviated by BOT, BTS andKBG as shown by histopathological examinations.
Retinol-binding protein 4 (RBP4), a protein secreted by hepatocytes (80%) and adipose tissue (20%), is a
21-kDa protein that facilitates the transport of retinol
through the circulation to peripheral tissues.11 It plays
a key role in the control of metabolic and proliferativecell functions,31 including steatogenesis.32 Recently,
the role of adipokines, specifically RBP4 in the pathogenesis of obesity-related diseases is widely being
discussed.33–36 Many studies have reported the relationship between RBP4 and obesity as well as its related complications.35–37 Recent studies demonstrated
that RBP4 levels were increased in obese and insulinresistant humans and mouse models.38–41 Stefan et al.
found a direct relation between hepatic fat content andblood levels of RBP4 in healthy subjects, too.42 It has
been reported that RBP4 mRNA expression can be
up-regulated in liver and adipose tissue of rats withhigh cholesterol diet for 12 weeks.11 In our experiment,
RBP4 expression in liver and adipose tissue of rats
fed a high-cholesterol diet as well as the expression inserum were up-regulated. BOT and ezetimibe shows
greater effect to decrease the level of RBP4 mRNA
expression in liver and adipose tissue, the serum expression of RBP4 can also be decreased by BOT andezetimibe.
HFABP is a member of a family of binding proteins with distinct tissue distributions and diverseroles in fatty acid metabolism, trafficking, and signaling.43 It is a low molecular-weight cytoplasmic proteinthat is abundant in the myocardium44 and producedby skeletal muscle,45 cardiomyocytes, kidney distal
tubular cells46 and specific parts of the brain.47, 48
HFABP mRNA expression has been reported to be
up-regulated in livers of rats fed high-cholesterol dietfor 28 days.11 In our study, this phenomenon was also
demonstrated. HFABP mRNA expression in the liver
and adipose tissue of high-cholesterol diet groups was
significantly increased compared to the control group
after 12 weeks.
CFABP was originally identified as being overexpressed in the psoriatic skin.49, 50 It is typical of the
FABP family and binds long-chain fatty acids withhigh affinity49, 51 and were thought to play a importantrole in the storage and transport of fatty acids.50, 52, 53
CFABP was reported to be up-regulated after 12
weeks in liver and adipose tissue of high-cholesterol
diet rats.11 Here, the mRNA expression of CFABP inliver of high-cholesterol diet rats were higher after 12
weeks while there were no significant differences be-

ICAM-1immunostaining expression in aorta

As shown in Fig. 5, after 12 weeks, little ICAM-1
immunostaining (red-brown deposits indicate positive staining) was found on the whole layers of abdominal aortas in Group C. Significantly positive
ICAM-1 immunostaining was observed in Group H
and mainly localized on the endothelial layers. Positive ICAM-1 immunostaining was also observed inGroups HA, HB, HC and HE, but less than Group H.
While ICAM-1 immunoreactivities were very weakin Groups HA, HB, HC and HE, the positive area andthe strength of ICAM-1 stainings was markedly lower
than those in Group H (× 400).
Blood pressure and heart rate

No significant changes in systolic or diastolic bloodpressure and heart rates were observed during the experiment.
DISCUSSION
Hypercholesterolemia is one of major risk factors
contributing to cardiovascular diseases.3, 4 Patientswith NAFLD have a high risk of CVD, too.5 Elevatedconcentrations of plasma TC and LDL-C as well as
reduced concentration of HDL-C are negative risk factors for CVD is well documented.28–30 Lipid-loweringdrugs, such as statins, hydroxyl-methylglutaryl-coenzyme A reductase inhibitors can well reduce LDL-C
and increase HDL-C. Adverse reactions such as rhabdomyolysis and hepatitis were sometimes caused bythem. Therefore, minimizing the side effects of lipidlowering drugs is also very important. The mechanismof Kampo formula is to restore the balance of the body.
It is a safe way to achieve the balance between LDL-C
and HDL-C concentrations. In this study, we observed
the effect of Kampo formulas (BOT, BTS, KBG) on
the progression of hypercholesterolemia and fatty
liver induced by high-cholesterol diet in rats. LDL-C,
ALT and ALP concentrations in the serum of rats fed
high-cholesterol diet with BOT decreased. ALT (at 6weeks) and ALP (at 12 weeks) concentrations in Group
HE was significantly elevated compared with that inGroup H. The reason is still unknown. The steatosis
154Kampo formulas ameliorates dyslipidemia

tween groups in adipose tissue. BOT, BTS and ezetimibe can down-regulate the CFABP mRNA expres-unknown.
ICAM-1, a member of the immunoglobulin superfamily, is one of the markers of endothelial cell activation. It plays an important role in neutrophil migration
and adhesion of endothelial cells64 and is partly involvedin the whole process of monocyte adhesion, migrationand transformation.16 This migration is one of the earliest
events in the atherosclerotic process.65 ICAM-1 was upregulated in neointimal and medial smooth muscle cells
after vascular injury.66 Sekiya et al. reported that KBG
could prevent the progression of atheromatous plaque, 67
but no literature could prove the effect of BOT and BTS
on preventing atherosclerosis. In our experiment, serum
ICAM-1 concentration and immunostaining expression
were increased in Group H after 12 weeks. BOT, BTS,KBG and ezetimibe can down-regulate it. We speculatethat down-regulated ICAM-1 may be one of the mechanisms of BOT, BTS and KBG to prevent atherosclerosis.
Overall, BOT has a protective effect on the progression of hypercholesterolemia and fatty liver induced byhigh-cholesterol diet in rats and more effective than BTS
and KBG. Ezetimibe has the similar effect on lipid related factors, such as LDL-C, RBP4, HFABP and CFABP.
However, the anti-inflammatory (MCP1, CCR2) andanti-arteriosclerotic (ICAM-1) effects of BOT are morepotent than ezetimibe. This may be a guide on clinicaluse. The mechanism is still uncertain and further studies
should be designed.

sion in liver.

BOT, BTS and KBG are all used as anti-obesity
medications in Japan.54 BOT is used for people with asolid build and thick abdominal subcutaneous fat.54 It
could decrease the mouse body weight, fat accumulation, TC and triglyceride level, but cannot influence
fasting blood glucose levels or insulin levels.22 BTS isused for patients with flabby constitution and proneness to fatigue.54 It can decrease food intake, bodyweight, blood pressure, white adipose tissue weight
and ameliorate the adipocytokine dysregulation inwhite adipose tissue.55 KBG is commonly used for
women with sudden weight gain in menopause andcould led to a reduction in blood cholesterol.54, 56 According to these reports, even BOT, BTS and KBG are
all used in the treatment of obesity, they are suitablefor different situations. In our study, BOT has a better effect on the progression of hypercholesterolemia
and fatty liver induced by high-cholesterol diet in rats.
Ougi and Ginger are the key components of BOT,
and it has been reported that Ougi and Ginger of BOT
contribute greatly to the beneficial effects on abnormal
lipid metabolism.22 Matsuda et al. also found that Ginger tends to improve lipid metabolism.24 Wang et al.
reported that Ougi could significantly reduced plasma
levels of TC and LDL-C and improved the atherosclerosis profile.57 These results partly explain the present
findings. We speculate that inhibition of lipid absorption might contribute to the effect of BOT.22 However,
the mechanisms of BTS and KBG are still unknown.
Inflammatory factors play important roles in
NAFLD and atherosclerosis progression.8 MCP1 is
an important inflammatory chemokine that can beproduced by a variety of cells including vascular endothelial cells, vascular smooth muscle cells, monocytes
and other cells. It is a member of the CC chemokine
family.58, 59 Plasma concentration of MCP1 increases
with obesity.60, 61 CCR2 is a receptor of MCP1 andcan help MCP1 to accomplish its effect.62 Dietary cholesterol can induce the MCP1 gene expression,14 andCCR2 expression was increased in hypercholesterolemic patients compared with normocholesterolemic
controls.15, 63 In our experiment, compared with theother groups, the mRNA expression of MCP1 andCCR2 in both liver and adipose tissue in Group H remained high level during the entire experiment, especially after 12 weeks. BOT, BTS, KBG and ezetimibe
can down-regulate the MCP1 and CCR2 expression.
This effect of BOT, BTS and KBG is direct or indirect
through inhibition of cholesterol metabolism is still

Acknowledgments: We would like to express our sincere appreciation for the fellowship and research grant from the Japan Research
Foundation of Clinical Pharmacology (JRFCP).
A preliminary report has appeared in abstract form inJapanese.68
The authors declare no conflict of interest.
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