Original Article

Yonago Acta medica 2016;59:149–158

Effect of Daisaikoto on Expressions of SIRT1 and NF-kappaB of Diabetic Fatty
Liver Rats Induced by High-Fat Diet and Streptozotocin
Weibin Qian,*† Xinrui Cai,‡ Xinying Zhang,† Yingying Wang,§ Qiuhai Qian† and Junichi Hasegawa**Division of Pharmacotherapeutics, Department of Pathophysiological and Therapeutic Science, School of Medicine, Tottori University Faculty of Medicine, Yonago 683-8503, Japan, †Affiliated Hospital of Shandong University of Traditional Chinese Medicine, Jinan
250011, Shandong, China, ‡Shandong Academy Occupational Health and Occupational Medicine, Shandong Academy of Medical Sciences, Jinan, Shandong 250002, China and §Jinan Shizhong People’s Hospital, Jinan 250002, Shandong, China

ABSTRACTof the liver, we observed a significant improvement after
treatment. Immunostaining expression of NF-kappaB in
the liver was improved by DSKT and simvastatin. The
mRNA expressions of SIRT1 in the liver were increased
by DSKT and simvastatin.
Conclusion We have demonstrated that DSKT is capable of reversing dyslipidemia and insulin resistanceinduced by a high-fat diet and STZ. High dose DSKT
reveals a stronger effect than simvastatin on the expressions of SIRT1 and NF-kappaB. Furthermore, DSKT
has shown a strong dose-depended protective effect ondiabetic fatty liver.

Background Daisaikoto (DSKT), a classical tradition-

al Chinese herbal formula, has been used for treating
digestive diseases for 1800 years in China. Therefore,
in this study, we are going to investigate the effect of
DSKT on diabetic fatty liver rats induced by a high-fat
diet and streptozotocin (STZ), and the effects of DSKT
on silent mating type information regulation 2 homolog
1 (SIRT1) and nuclear factor kappa B (NF-kappaB).
Methods Diabetic fatty liver rat model was selected
to establish a high-fat diet and STZ. Sixty Wistar rats
were divided into six groups (n = 10): control group,
high-fat diet + STZ group, simvastatin treatment group,
DSKT low dose, medial dose and high dose treatmentgroups. After 8 weeks of drug intervention, body andliver weights, blood chemistry, blood glucose and insulin were examined. The expressions of sirtuin 1 andNF-kappaB in the liver were observed by RT-PCR and
immunohistochemistry, respectively.
Results A high-fat diet increased body, liver weights,
and serum cholesterol concentrations. Intraperitoneal
injection of STZ increased blood glucose and decreased
body weights. DSKT improved them. Homeostasis model assessment-estimated insulin resistance (HOMA-IR)
indices were increased in the high-fat diet groups.
DSKT improved them too. In histological examinations

Key words

daisaikoto; diabetes; fatty liver; nuclear
factor kappa B; silent mating type information regulation 2 homolog 1
Nonalcoholic fatty liver disease (NAFLD), the mostcommon cause of chronic liver disease, shows an increasing prevalence among adults.1 Recently, it has been
proven that NAFLD is commonly associated with many
symptoms of metabolic syndrome such as obesity, type 2
diabetes mellitus (T2DM), dyslipidemia and insulin resistance.2 The incidence of NAFLD is reported approximately in the 20–30% range of the general populationin various countries. However, it reaches nearly 70–75%
in individuals with T2DM, and is almost certainly increasing.3, 4 Accordingly, NAFLD seems to be linked tometabolic syndrome, and may precede T2DM development.5 Compared with other patients, those with T2DM
appear to have an increased risk of developing NAFLD
and certainly have a higher risk of developing fibrosis
and cirrhosis.3 Inhibition of excessive lipid synthesis anduptake is recognized to be an effective intervention for
NAFLD, such as statins and fibrates.6–8 However, theycannot meet the demands for treatment due to differences in individuals, drug dependence and some potential
side effects.9, 10
Many cytokines are associated with the progressionof NAFLD and T2DM, such as sirtuin 1 (SIRT1; silent
mating type information regulation 2 homolog 1) andnuclear factor kappa B (NF-kappaB). SIRT1, an NA-

Corresponding author: Qiuhai Qian, PhD
qianqiuhai@126.com
Received 2016 March 11
Accepted 2016 March 30
Abbreviations: ALP, alkaline phosphatase; ALT, alanine aminotransferase; C, control group; Cr, creatinine; DSKT, daisaikoto; H,
high-fat diet + STZ group; HDH, high-fat diet + STZ with high doseof DSKT treated group; HDL, high-fat diet + STZ with low dose of
DSKT treated group; HDL-C, high-density lipoprotein cholesterol; HDM, high-fat diet + STZ with medial dose of DSKT treatedgroup; HE, hematoxylin and eosin; HOMA-IR, Homeostasis model
assessment-estimated insulin resistance; HS, high-fat diet + STZ
with simvastatin treated group; LD, lactate dehydrogenase; LDL-C,
low-density lipoprotein cholesterol; NAFLD, nonalcoholic fatty
liver disease; NF-kappaB, nuclear factor kappa B; PCR, polymerase
chain reaction; ROS, reactive oxygen species; SIRT1, sirtuin 1; silent mating type information regulation 2 homolog 1; STZ, streptozotocin; TBil, total bilirubin; TC, total cholesterol; TG, triglyceride;
T2DM, type 2 diabetes mellitus

149

W. Qian et al.

D+-dependent deacetylase, is implicated in diverse cellular processes, including metabolism, inflammation andapoptosis.11–13 Increasing evidence shows that it may be
involved in the development of NAFLD.14 Deng, XQ et
al proved that SIRT1 protein levels are reduced in NAFLD induced by a high-fat diet in rats.15 It has the ability
to activate or repress many non-histone proteins such as
NF-kappaB.16 NF-kappaB, an oxidative stress sensitivetranscription factor, plays an important role in regulationof the expression of many genes involved in inflammatory responses, and NF-kappaB-induced cytokines haveappeared widely in inflammatory diseases, including
NAFLD and T2DM.17–19 However, research on SIRT1
and NF-kappaB on daisaikoto (DSKT) treatment has
not yet been included.
DSKT (“Da-chai-hu-tang” in Chinese), a classicaltraditional Chinese herbal formula, has been used fortreating digestive diseases for 1800 years in China. It
was created by the well-known Chinese clinician Zhang
Zhongjing in Eastern Han. Nowadays, it is used in Asian
countries in patients with hypertension and associatedsymptoms,20 hyperlipidemia and diabetes mellitus.21 Yamamoto et al. and Iizuka et al. have reported that DSKT
has the effect of lowering hepatic triglyceride biosynthesis in HepG2 human hepatocyte cells or experimental
hypercholesterolemic models.22, 23 However, the effect of
DSKT on diabetic fatty liver has not been proven and its
effects on SIRT1 and NF-kappaB remain unclear.
Therefore, in this study, we are going to investigate
the effect of DSKT on diabetic fatty liver in rats induced
by a high-fat diet and STZ, and the effects of DSKT on
homeostasis of SIRT1 and NF-kappaB.

cages under controlled environmental conditions (22 ± 2 °C
relative humidity 40–60%, 12 h dark/light cycles, food andwater ad libitum). Animals were used in the present study
in accordance with the guidelines of the Shandong University of Traditional Chinese Medicine Institutional Animal
Care and Use Committee. All studies were performed withapproval from the committee (2013005-KY).
Reagents
Simvastatin was purchased from Qilu Pharmaceutical(Jinan, China). Streptozotocin (STZ) was purchased from
Sigma, St. Louis, MO. Anti-NF-kappaB and goat anti-rabbit antibody were obtained from Beijing Boosen BiologicalTechnology (Beijing, China). Insulin ELISA Kit was purchased from Nanjing Jiancheng Bioengineering Institute(Nanjing, China).
Composition and preparation of DSKTDSKT consists of eight traditional Chinese herbs (Table1). Each of the herbs were purchased from Zhong-sheng
Company of Traditional Crude Drugs (Qingdao, China),
and carefully authenticated by Dr. Jun-Ying Shi, Shandong
University of Traditional Chinese Medicine (Jinan, China).
Voucher specimens (numbers were listed in Table 1) were
deposited at the Herbarium of Shandong University ofTraditional Chinese Medicine (SDU.TCM, Jinan, China).
After drying, herbs were mixed in proportion, and 84 g ofthis material was combined with 300 mL of distilled waterand boiled for 30 min at 100 °C. The extract was filtered,and the residual medicine was boiled in water following thesame procedure once more. Finally, the pool of the extracts
from two boilings and filterings was mixed and stored at 4
°C.

MATERIALS AND METHODS
Animal experiments
After 3 days adaptation, rats were randomly divided into
six groups: control group (C, n = 10), high-fat diet + STZ
group (H, n = 15), high-fat diet + STZ with simvastatin
treated group (HS, n = 15), high-fat diet + STZ with low

Animals
Eighty-five adult male Wistar rats (8 weeks, 200 ± 20 g,
No. SCXK. Lu 20130003) were purchased from Shandong
University of Traditional Chinese Medicine Laboratory Animal Center (Jinan, China). Rats were housed in individual
Table 1. Constituents of daisaikoto (DSKT) formulation
Components

Part used

Voucher specimens number Amount used (g)Bupleuri Radix (Bupleurum falcatum L.)

Root

SDU.TCM 0062

15

Scutellariae Radix (Scutellaria baicalensis Georgi)

Root

SDU.TCM 0098

9

Rhei Rhizoma (Rheum palmatumL.)

RhizomeSDU.TCM 0076

6

Aurantii Fructus Immaturus (Citrus aurantium L. var. daidai Makino) FruitSDU.TCM 0203

9Paeoniae Radix (Paeonia lactiflora Pallas)

Root

SDU.TCM 0046

9

Pinnelliae Tuber (Pinellia ternata Breit)

Tuber

SDU.TCM 0057

9

Zingiberis Rhizoma (Zingiber officinale Roscoe)

RhizomeSDU.TCM 0132

15

Zizyphi Fructus (Zizyphus jujuba Mill. var. inermis Rehder)

Fruit

SDU.TCM 0080

12

150

Daisaikoto ameliorates diabetic fatty liver

dose of DSKT treated group (HDL, n = 15), high-fat diet
+ STZ with medial dose of DSKT treated group (HDM, n= 15), high-fat diet + STZ with high dose of DSKT treatedgroup (HDH, n = 15). Group C was fed a standard rat diet.
A high-fat diet was supplied for the other groups; it wasmade by adding 10% lard oil, 20% sucrose, 10% egg yolk
powder and 0.5% cholic acid to the standard diet (purchasedfrom Keao Xieli Feed, Beijing, China). After 6 weeks, STZ(30 mg/kg, 2% citrate buffer solution, pH 4.2) was supplied
by intraperitioneal injection for rats in Groups H, HS, HDL,
HDM, HDH. After 72 h of injection, rats with a level offasting blood glucose over 11.1 mmol/L were regarded as
successful models. Ten successful model rats in each groupwere selected for the following experiment. Rats in Groups
C (n = 10) and H (n = 10) were treated with 0.9% saline byintragastric administration (10 mL/kg body weight per day);rats in Group HS (n = 10) were treated with simvastatin by
intragastric administration (0.9 mg/10 mL/kg body weightper day); rats in Groups HDL (n = 10), HDM (n = 10) andHDH (n = 10) were treated with DSKT by intragastric administration (3.75, 7.5, 15 g/10 mL/kg body weight per day,respectively). Dosages of drugs were calculated according
to body surface area. The drug dosage for rats was equally
proportionate to adult humans who take simvastatin (10 mg)
and DSKT (84 g) per day, respectively. The body surface
area conversion coefficient from humans to rats is 0.018.
Dosage of drug for adult humans × 0.018 = dosage of drugfor rats weighing 200 g. So the dosage of simvastatin for
rats can be calculated according to the above formula: 10
mg × 0.018 = 0.18 mg for rats weighing 200 g. This resultsin the dosage for rats weighing 1 kg as 0.9 mg. The dosageof DSKT for rats can also be calculated similarly according
to the above formula: 84 g × 0.018 = 1.5 g for rats weighing
200 g where the dosage for rats weighing 1 kg is 7.5 g, a
medial dose; 3.75 g is defined as a low dose; 15 g is defined
as a high dose. The drug intervention period is continued for
8 weeks. The amount of feed for each rat was regulated to
25 g/day and water was supplied ad libitum. Body weights
were measured at 0, 6, 10 and 14 weeks. Blood glucose wasmeasured at 0 week, 6 weeks, 6 weeks + 3 days, 8 weeks,
10 weeks, 12 weeks and 14 weeks.

lesterol), LDL-C (low-density lipoprotein cholesterol), ALT(total bilirubin, alanine aminotransferase), ALP (alkaline
phosphatase) and TG (triglyceride) levels were analyzed forrats using an auto analyzer at an accredited clinical laboratory (Affiliated Hospital of Shandong University of Traditional Chinese Medicine, Jinan, China).
Insulin linked immunosorbent assay (ELISA)
Serum samples were applied for an ELISA of Insulin (Nanjing Jiancheng Bioengineering Institute, Nanjing, China)
according to the manufacturer’s instructions. Insulin resistance was evaluated according to the homeostasis model
assessment-estimated insulin resistance (HOMA-IR) index.
The HOMA-IR index was calculated according to the following equation reported by Matthews et al.24, 25 HOMA-IR
index = fasting plasma insulin (mIU/L) × fasting plasma
glucose (mmol/L)/22.5.
RT-PCR of SIRT1
The tissues were quickly immersed in RNA later and stored
at –80 °C. The total RNA was sequentially extracted using
TRIZOL Reagent (Sigma-Aldrich, St. Louis, MO) according to the manufacturer’s instructions. The RNA was treated with DNase (DNAfree, Ambion, Austin, TX) in order toremove contaminating genomic DNA, followed by phenol,
chloroform extraction and ethanol precipitation. Total RNA
was assessed for purity using the NanoDrop system. Total
RNA was performed in a two-step procedure as describedby Power SYBR Green PCR Master Mix kits (Applied
Biosystems, Waltham, MA). Briefly, in the first step, cDNA
was prepared from 500 ng RNA by reverse transcription
in a final volume of 20 μL in a thermal cycler (Tgradient96, Whatman Biometra, Niedersachsen, Germany). The
samples were incubated at 37 °C for 60 min and 95 °C for
5 min. The cDNA were stored at –20 °C. The rat-specificprimers for the genes of PPT, NEP and beta-actin were
designed using Primer Premier 5.0 (Premier Biosoft, Palo
Alto, CA). The primer sequences are shown in Table 2.
Primers were synthesized by BioAsia (Shanghai, China). In
the second step, quantitative real-time PCR was performed
on a LightCycler apparatus (Roche Diag Diagnostics,
Mannheim, Germany) using Power SYBR Green. PCR
Master Mix kits (Applied Biosystems). The reaction was
conducted with an initial denaturing at 95 °C for 10s, andthen involved 40 cycles of 55 °C 10 s, 72 °C 15 s, and terminated by a cooling step 30 s at 40 °C. A melting-curve analysis was performed to confirm the absence of primer dimers in specific PCR products. The efficiency of PCR wasassessed with serial dilutions of a sample of cDNA fromthe normal control group. Each experiment was performedin duplicate and the data were analyzed using Light Cycler
Software 4.0 (Roche Diagnostics, Basel, Switzerland). Be-

Sample collection
At the end of 14 weeks, rats in each group were sacrificed
by collecting blood from the heart under pentobarbital anesthesia after fasting for 12 h. Liver tissues were removed,
and then portions of the samples were stored in 10% formalin solution for hematoxylin and eosin (HE) staining.
The remaining samples were immediately transferred into
EP tubes containing 500 μL of RNA later, quickly frozen
in liquid nitrogen, and stored at –80°C. Serum levels of TC(total cholesterol), HDL-C (high-density lipoprotein cho-151

W. Qian et al.

Five fields from every section were randomly selected for examination using an Olympus CKX41-32PH
microscope equipped with an imaging system (Olympus
Optical, Tokyo, Japan). The distribution and staining of
positive expression of liver tissue were observed under a
400 x light microscope. The optical densities were measured by Image-Pro Plus v 6.0 software (Media Cybernetics, Rockville, MD).

ta-actin mRNA was used as the housekeeping gene, and alldata are represented using the 2−ΔΔCT method .
Immunohistochemistry of NF-kappaB
The samples were fixed in 10% buffered formalin at 4 °C
for 24–48 h. The samples were sectioned (4 μm thick) using
a freezing microtome (ASpZr 35) and standard immunohistochemical procedures were used to visualize NF-kappaB
protein. Sections were washed 4 × 10 min in PBS (pH 7.4) atroom temperature and were blocked with 10% normal goat
serum. Sections were then incubated with anti- NF-kappaB(Beijing Bioss Biological Technology, Beijing, China) overnight on a shaker at room temperature. Following washing(4 × 10 min) with PBS, sections were incubated for 2 h withgoat anti-rabbit antibody. Finally, sections were washed (3× 10 min) in PBS, transferred onto gelatin-coated slides.
Tissue processed without the primary NF-kappaB antibody served as a negative control. Adjacent sections were
mounted onto plus-coated slides and stained using Neutral
Red (0.5%), washed through a series of increasing alcohol
concentrations, cleared with xylene and coverslipped with
Neutral Blasam.

Statistical analysis

All statistical analyses were performed using SPSS 17.0
software (SPSS, Chicago, IL). Data were expressed asmean ± S.E. Analysis of variance (ANOVA) was performed when more than two groups were compared.
Values of P < 0.05 were considered statistically significant.
RESULTS
Body and liver weights

As Table 3 shows, there were no significant differencesin the baseline of body weights at the beginning of theexperiment. High-fat diet increased body weights inTable 2. The sequence of each PCR primer
Gene

Forward

ReverseBeta-actin

5’-GAGGGAAATCGTGCGTGAC-3’

5’- GGACTCATCGTACTCCTGCTTG-3’

SIRT15’-CCAGAACAGTTTCATAGAGCC-3’

5’TCTTACTTTCAGAGAAGACCCAATA-3’

PCR, polymerase chain reaction; SIRT1, sirtuin 1; silent mating type information regulation 2 homolog 1.

Table 3. Body and liver weights
Group C

Body weights (g)0W 208.30 ± 11.95
6W 355.60 ± 27.37
10W 398.60 ± 20.92
14W 452.70 ± 15.30
Liver weights (g)14W 14.79 ± 1.29

Group HGroup HSGroup HDLGroup HDM

Group HDH

206.10 ± 12.58
427.40 ± 27.59*
432.90 ± 31.07*
388.90 ± 34.55*

205.40 ± 11.69
425.20 ± 26.60
428.40 ± 22.10
390.10 ± 24.78

208.80 ± 7.84
422.70 ± 23.86
446.30 ± 25.32
423.40 ± 21.43†‡

207.90 ± 11.29
428.80 ± 17.25
453.00 ± 25.28
428.90 ± 26.77†‡

207.70 ± 10.31
430.40 ± 15.64
469.50 ± 25.01†
450.60 ± 27.92†‡§

21.39 ± 2.52*

15.14 ± 1.69†

16.83 ± 1.29†‡

16.41 ± 1.35†

15.02 ± 2.12†§

C: normal control group, standard rat diet for 14 weeks and treated with 0.9% saline 10 mL/kg every day (n = 10); H: high-fat diet + STZ
group, high-fat diet for 14 weeks and treated with 0.9% saline 10 mL/kg every day (n = 15 at 0 week and n = 10 at 6, 10 and 14 weeks);
HS: high-fat diet + STZ with simvastatin treated group, high-fat diet for 14 weeks and treated with simvastatin 0.9 mg/10 mL/kg every day (n= 15 at 0 week and n = 10 at 6, 10 and 14 weeks); HDL: high-fat diet + STZ with low dose of DSKT treated group, high-fat diet for 14
weeks and treated with DSKT 3.75 g/10 mL/kg every day (n = 15 at 0 week and n = 10 at 6, 10 and 14 weeks); HDM: high-fat diet + STZ
with medial dose of DSKT treated group, high-fat diet for 14 weeks and treated with DSKT 7.5 g/10 mL/kg every day (n = 15 at 0 week
and n = 10 at 6, 10 and 14 weeks); HDH: high-fat diet + STZ with high dose of DSKT treated group, high-fat diet for 14 weeks and treatedwith DSKT 15 g/10 mL/kg every day (n = 15 at 0 week and n = 10 at 6, 10 and 14 weeks). *P < 0.05 vs. Group C, †P < 0.05 vs. Group H,‡P < 0.05 vs. Group HS, §P < 0.05 vs. Group HDL, ||P < 0.05 vs. Group HDM. Data are expressed as the means ± standard deviation.
0W, 0 week; 6W, 6 weeks; 10W, 10 weeks; 14W, 14 weeks; DSKT, daisaikoto; STZ, streptozotocin.

152

Daisaikoto ameliorates diabetic fatty liverFig. 1. Histological examination of the liver.
Fatty degeneration (steatosis) of the liver is observed in high-fat
diet-fed groups (H, HS, HDL, HDM and HDH), but not in GroupC in hematoxylin-eosin stained tissues. C: normal control group,
standard rat diet for 14 weeks and treated with 0.9% saline 10 mL/kg every day (n = 10); H: high-fat diet + STZ group, high-fat dietfor 14 weeks and treated with 0.9% saline 10 mL/kg every day(n = 10); HS: high-fat diet + STZ with simvastatin treated group,
high-fat diet for 14 weeks and treated with simvastatin 0.9 mg/10
mL/kg every day (n = 10); HDL: high-fat diet + STZ with lowdose of DSKT treated group, high-fat diet for 14 weeks and treatedwith DSKT 3.75 g/10 mL/kg every day (n = 10); HDM: high-fat
diet + STZ with medial dose of DSKT treated group, high-fat dietfor 14 weeks and treated with DSKT 7.5 g/10 mL/kg every day (n= 10); HDH: high-fat diet + STZ with high dose of DSKT treatedgroup, high-fat diet for 14 weeks and treated with DSKT 15 g/10
mL/kg every day (n = 10). Bars express 25 μm. DSKT, daisaikoto;
STZ, streptozotocin.

Table 4. Blood chemistry and cholesterol concentrations

LDL-C(mmol/L)
HDL-C(mmol/L)
TC(mmol/L)TG(mmol/L)
ALT
(U/L)
ALP
(U/L)

Group CGroup HGroup HSGroup HDLGroup HDM

Group HDH

0.65 ± 0.08

2.09 ± 0.18*

0.96 ± 0.08†

1.88 ± 0.16†‡

1.35 ± 0.14†‡§1.00 ± 0.10†§||1.09 ± 0.22

0.54 ± 0.13*

0.91 ± 0.14†

0.67 ± 0.08†‡

0.84 ± 0.09†§

0.89 ± 0.14†§

1.18 ± 0.26

2.49 ± 0.51*

1.32 ± 0.4†

1.98 ± 0.4†‡

1.70 ± 0.44†‡

1.38 ± 0.44†§

0.8 ± 0.27

2.29 ± 0.42*

1.79 ± 0.34†

1.93 ± 0.49†

1.70 ± 0.32†

1.36 ± 0.43†‡§

39.11 ± 2.55

61.75 ± 5.18*

63.4 ± 6.96

55.14 ± 3.79†‡

49.96 ± 3.48†‡§45.14 ± 4.00†‡§||

100.45 ± 9.95

154.69 ± 17.54*

168.8 ± 7.05†

127.38 ± 9.59†‡

120.37 ± 10.33†‡ 112.47 ± 6.57†‡§

C: normal control group, standard rat diet for 14 weeks and treated with 0.9% saline 10 mL/kg every day (n = 10); H: high-fat diet + STZ
group, high-fat diet for 14 weeks and treated with 0.9% saline 10 mL/kg every day (n = 10); HS: high-fat diet + STZ with simvastatin
treated group, high-fat diet for 14 weeks and treated with simvastatin 0.9 mg/10 mL/kg every day (n = 10); HDL: high-fat diet + STZ withlow dose of DSKT treated group, high-fat diet for 14 weeks and treated with DSKT 3.75 g/10 mL/kg every day (n = 10); HDM: highfat diet + STZ with medial dose of DSKT treated group, high-fat diet for 14 weeks and treated with DSKT 7.5 g/10 mL/kg every day (n= 10); HDH: high-fat diet + STZ with high dose of DSKT treated group, high-fat diet for 14 weeks and treated with DSKT 15 g/10 mL/kg every day (n = 10). *P < 0.05 vs. Group C, †P < 0.05 vs. Group H, ‡P < 0.05 vs. Group HS, §P < 0.05 vs. Group HDL, ||P < 0.05 vs.
Group HDM. Data are expressed as the means ± standard deviation. ALP, alkaline phosphatase; ALT, alanine aminotransferase; DSKT,
daisaikoto; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; STZ, streptozotocin; TC, total cholesterol; TG, triglyceride.

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Groups H, HS, HDL, HDM and HDH compared withGroup C after 6 weeks (P < 0.05). Intraperitoneal injection of STZ decreased body weights in Group H compared with Group C after 10 and 14 weeks (P < 0.05).
DSKT ameliorated body weights in a dose dependent
manner (P < 0.05). Liver weights were significantly
higher in Group H than Group C after 14 weeks (P <0.05). Groups HS, HDL, HDM and HDH had decreased
liver weights after 14 weeks (P < 0.05). Groups HS andHDH had a better effect than HDL (P < 0.05).
Histological examination of liver

From hematoxylin-eosin staining we could find that the
fatty degeneration (steatosis) of the liver was observedin the high-fat-diet-fed groups, but not in Group C. The
tiny and large vacuoles as well as pleomorphic nuclei
were more conspicuous in the treatment groups than inGroup H (Fig. 1). Groups HS and HDH seem to show abetter effect (× 400).

Fig. 2. Blood glucose level.
Blood glucose increased greatly 3 days after intraperitioneal injection of STZ and decreased after treatment in Groups HDL, HDM
and HDH. C: normal control group, standard rat diet for 14 weeks
and treated with 0.9% saline 10 mL/kg every day (n = 10); H:
high-fat diet + STZ group, high-fat diet for 14 weeks and treatedwith 0.9% saline 10 mL/kg every day (n = 15 at 0 and 6 weeks
and n = 10 at 6 weeks + 3 days, 8 weeks, 10 weeks, 12weeks and
14 weeks; HS: high-fat diet + STZ with simvastatin treated group,
high-fat diet for 14 weeks and treated with simvastatin 0.9 mg/10
mL/kg every day (n = 15 at 0 and 6 weeks and n = 10 at 6 weeks+ 3 days, 8 weeks, 10 weeks, 12weeks and 14 weeks); HDL: highfat diet + STZ with low dose of DSKT treated group, high-fat dietfor 14 weeks and treated with DSKT 3.75 g/10 mL/kg every day (n= 15 at 0 and 6 weeks and n = 10 at 6 weeks + 3 days, 8 weeks, 10
weeks, 12weeks and 14 weeks); HDM: high-fat diet + STZ withmedial dose of DSKT treated group, high-fat diet for 14 weeks
and treated with DSKT 7.5 g/10 mL/kg every day (n = 15 at 0
and 6 weeks and n = 10 at 6 weeks + 3 days, 8 weeks, 10 weeks,
12weeks and 14 weeks); HDH: high-fat diet + STZ with high doseof DSKT treated group, high-fat diet for 14 weeks and treated withDSKT 15 g/10 mL/kg every day (n = 15 at 0 and 6 weeks andn = 10 at 6 weeks + 3 days, 8 weeks, 10 weeks, 12weeks and 14
weeks). 0W, 0 week; 2W, 2 weeks; 4W, 4weeks; 6W, 6weeks; 8W,
8 weeks; 10W, 10 weeks; 12W, 12 weeks; 14W, 14 weeks. 6 weeks+ 3 days means 3 days after intraperitioneal injection of STZ.
DSKT, daisaikoto; STZ, streptozotocin.

Blood chemistry and cholesterol concentrations

As Table 4 shows, the high-fat diet increased the concentrations of LDL-C, TC, TG, ALT, ALP and reduced theconcentrations of HDL-C in Group H compared withGroup C after 14 weeks in serum (P < 0.05). Comparedwith Group H, the concentrations of LDL-C, TC, TG,
ALT and ALP decreased in Groups HS, HDL, HDM
and HDH (P < 0.05), while the concentration of HDL-C
had an opposite trend (P < 0.05). Groups HS and HDH
had a better effect (P < 0.05). DSKT regulated blood
chemistry and cholesterol concentrations in a dose dependent manner (P < 0.05).
Blood glucose level

As shown in Fig. 2, there were no significant differencesin the baseline of blood glucose level at the beginningof the experiment. The high fat diet increased blood
glucose level slightly in Groups H, HS, HDL, HDM andHDH than in Group C after 6 weeks, but there were no
statistical differences. Blood glucose increased greatly
3 days after intraperitoneal injection of STZ in Groups
H, HS, HDL, HDM and HDH. After treatment, blood
glucose obviously decreased in Groups HDL, HDM andHDH compared with Group H, but not in Group HS after 8, 10, 12 and 14 weeks.

Groups HS, HDL, HDM and HDH (P < 0.05). DSKT
decreased HOMA-IR in a dose dependent manner (P <0.05).
SIRT1 mRNA expression in the liver
As Fig. 4 shows, SIRT1 mRNA expression in the liver inGroup H was significantly decreased compared to that inother groups (P < 0.05). Groups HS, HDL, HDM and HDH
increased SIRT1 mRNA expression in the liver to a various
degree (P < 0.05). Group HDH had a better effect. DSKT
increased SIRT1 mRNA expression in the liver in a dose
dependent manner (P < 0.05).

Insulin in serum and HOMA-IR Index

Insulin concentrations were increased in Group H compared with Groups C and HDH after 14 weeks (Fig. 3,
P < 0.05). HOMA-IR obviously increased in Groups
H, HS, HDL, HDM and HDH compared with GroupC (P < 0.05). After treatment, HOMA-IR decreased in154

Daisaikoto ameliorates diabetic fatty liverFig. 4. Changes of mRNA expression of SIRT1 in liver after 14
weeks.
Levels of SIRT1 against beta-actin mRNA expression are shownin the above histogram. C: normal control group, standard rat dietfor 14 weeks and treated with 0.9% saline 10 mL/kg every day (n= 10); H: high-fat diet + STZ group, high-fat diet for 14 weeks andtreated with 0.9% saline 10 mL/kg every day (n = 10); HS: highfat diet + STZ with simvastatin treated group, high-fat diet for 14
weeks and treated with simvastatin 0.9 mg/10 mL/kg every day (n= 10); HDL: high-fat diet + STZ with low dose of DSKT treatedgroup, high-fat diet for 14 weeks and treated with DSKT 3.75 g/10
mL/kg every day (n = 10); HDM: high-fat diet + STZ with medialdose of DSKT treated group, high-fat diet for 14 weeks and treated with DSKT 7.5 g/10 mL/kg every day (n = 10); HDH: high-fat
diet + STZ with high dose of DSKT treated group, high-fat dietfor 14 weeks and treated with DSKT 15 g/10 mL/kg every day (n= 10). *P < 0.05 vs. Group C, †P < 0.05 vs. Group H, ‡P < 0.05 vs.
Group HS, §P < 0.05 vs. Group HDL, ||P < 0.05 vs. Group HDM.
DSKT, daisaikoto; SIRT1, sirtuin 1, silent mating type information regulation 2 homolog 1; STZ, streptozotocin.

Fig. 3. Insulin in serum and HOMA-IR Index.
C: normal control group, standard rat diet for 14 weeks and treatedwith 0.9% saline 10 mL/kg every day (n = 10); H: high-fat diet
+ STZ group, high-fat diet for 14 weeks and treated with 0.9%
saline 10 mL/kg every day (n = 10); HS: high-fat diet + STZ withsimvastatin treated group, high-fat diet for 14 weeks and treatedwith simvastatin 0.9 mg/10 mL/kg every day (n = 10); HDL: highfat diet + STZ with low dose of DSKT treated group, high-fat dietfor 14 weeks and treated with DSKT 3.75 g/10 mL/kg every day(n = 10); HDM: high-fat diet + STZ with medial dose of DSKT
treated group, high-fat diet for 14 weeks and treated with DSKT
7.5 g/10 mL/kg every day (n = 10); HDH: high-fat diet + STZ withhigh dose of DSKT treated group, high-fat diet for 14 weeks andtreated with DSKT 15 g/10 mL/kg every day (n = 10). HOMA-IR
index = fasting plasma insulin (mIU/L) × fasting plasma glucose(mmol/L)/22.5. *P < 0.05 vs. Group C, †P < 0.05 vs. Group H,‡P < 0.05 vs. Group HS, §P < 0.05 vs. Group HDL, ||P < 0.05 vs.
Group HDM. DSKT, daisaikoto; HOMA-IR, Homeostasis model
assessment-estimated insulin resistance; STZ, streptozotocin.

DISCUSSION
NAFLD is commonly associated with many symptomsof metabolic syndrome such as obesity and T2DM.2
Patients with T2DM appear to have an increased riskof developing NAFLD and certainly have a higher riskof developing fibrosis and cirrhosis.3 Individuals always
present simultaneously with characteristics of high blood
glucose, insulin resistance, hyperlipidemia and fatty liver. It has been reported that the abnormal serum levels of
HDL-C and LDL-C are associated with NAFLD.26 And
NAFLD with T2DM patients always show up-regulated
TC and LDL-C and down-regulated HDL-C levels.27, 28
Statins, one kind of lipid-lowering drug, can inhibit cholesterol synthesis. They can definitely decrease LDL-C
and increase HDL-C. However, there is a risk that theysometimes are instrumental in causing rhabdomyolysis

NF-kappaB immunostaining expression in liver

As shown in Fig. 5A, after 14 weeks, NF-kappaB immunostaining (Yellow-brown deposits indicate positive
staining) was seldom found in hepatic cells in Group C.
Significantly positive NF-kappaB immunostaining wasobserved in Group H and mainly localized in cell cytoplasm and the nucleus. Positive NF-kappaB immunostaining was also observed in Groups HS, HDL, HDM
and HDH, but less than in Group H (× 400). Mean optical density values of NF-kappaB are shown in Fig. 5B.
The photographs generated quantitatively analyzed theoptical density of NF-kappaB by Image - Pro Plus. After
treatment, the expressions in Groups HS, HDL, HDM
and HDH decreased more than in Group H. Also, GroupHDH was significantly lower compared with Groups
HS, HDL and HDM (P < 0.05).
155

Fig 5.
W. Qian et al.

AA

Fig 5.

B
B

B

Fig. 5. NF-kappaB immunostaining expression in liver after 14 weeks.
A: Immunohistochemistry expression of NF-kappaB in liver. Arrows show the positive NF-kappaB immunostaining. B: Mean opticaldensity values of NF-kappaB. The photographs generated were quantitatively analyzed the optical density of NF-kappaB with Image Pro Plus. C: normal control group, standard rat diet for 14 weeks and treated with 0.9% saline 10 mL/kg every day (n = 10); H: high-fat
diet + STZ group, high-fat diet for 14 weeks and treated with 0.9% saline 10 mL/kg every day (n = 10); HS: high-fat diet + STZ with simvastatin treated group, high-fat diet for 14 weeks and treated with simvastatin 0.9 mg/10 mL/kg every day (n = 10); HDL: high-fat diet +STZ with low dose of DSKT treated group, high-fat diet for 14 weeks and treated with DSKT 3.75 g/10 mL/kg every day (n = 10); HDM:
high-fat diet + STZ with medial dose of DSKT treated group, high-fat diet for 14 weeks and treated with DSKT 7.5 g/10 mL/kg every day(n = 10); HDH: high-fat diet + STZ with high dose of DSKT treated group, high-fat diet for 14 weeks and treated with DSKT 15 g/10 mL/kg every day (n = 10). *P < 0.05 vs. Group C, †P < 0.05 vs. Group H, ‡P < 0.05 vs. Group HS, §P < 0.05 vs. Group HDL, ||P < 0.05 vs.
Group HDM. Bars express 25 μm. DSKT, daisaikoto; NF-kappaB, nuclear factor kappa B; STZ, streptozotocin.

156

Daisaikoto ameliorates diabetic fatty liver

way.33 NF-kappaB, an oxidative stress sensitive transcription factor, plays an important role in regulation of
the expression of many genes involved in inflammatory
responses, and NF-kappaB-induced cytokines appear
widely in inflammatory diseases, including NAFLD and
T2DM.17–19 Andrade JM et al proved that NF-kappaB
was increased in high-fat diet mice.34 Group H in ourstudy displayed higher hepatic NF-kappaB activity ascompared to Group C. According to Morgan,35 this activation could have been triggered, at least partially, byreactive oxygen species (ROS), which occurs in excess
in the liver. However, Group HDH showed the strongest
effect to prevent liver NF-kappaB activation and thisreveals a dose-depend trend. The antioxidant effects ofa high dose of DSKT likely contributed to the inhibition
of NF-kappaB activation.
In conclusion, we have demonstrated that DSKT iscapable of reversing blood lipid and blood glucose disorders induced by high-fat diet and STZ. DSKT improves
insulin resistance as well as liver oxidative and inflammatory status, which are closely linked to the development of NAFLD with T2DM. Similarly, the antioxidant
properties of DSKT contribute, at least partially, toimproving the inflammatory status of the liver. Molecular targets involved in these beneficial effects include
NF-kappaB inhibition and the involvement of SIRT1.
These results may be a guide for clinical use in the treatment of T2DM with fatty liver.

and hepatitis. Traditional Chinese medicine is always
considered a safe way to harmonize the abnormal serum
levels of HDL-C and LDL-C, to improve high blood
glucose as well as minimize side effects. In a previous
study of ours, we found fatty liver, up-regulated TC andLDL-C as well as down-regulated HDL-C in high-cholesterol diet rats.29, 30 In this experiment, fatty steatosis
and high blood glucose were observed after administration of a high-fat diet and STZ in Group H. Body
weights decreasing after intraperitoneal injection of STZ
may be associated with the elevated blood glucose; it
could be ameliorated by DSKT. DSKT and simvastatin
could decrease the concentrations of LDL-C and TC
and increase HDL-C. However, ALT and ALP concentrations in Group HS were significantly elevated than in
the DSKT groups. The reason may be that DSKT has
fewer side effects than simvastatin. On the other hand,
DSKT can reduce the high blood glucose levels, whilesimvastatin does not have very stable effects and cannot
improve high blood glucose effectively. Insulin secretion
increasing in Group H compared with Group C may be
a compensatory response against the increased blood
glucose and impaired insulin function. HOMA-IR increased obviously in Groups H, HS, HDL, HDM andHDH compared with Group C. This shows that the body
is not sensitive to insulin, and leads to difficulty in decreased blood glucose. DSKT also obviously inhibits insulin resistance. Furthermore, DSKT has shown a strong
dose-dependent effect on diabetic fatty liver.
SIRT1, an NAD + -dependent deacetylase, is implicated in diverse cellular processes, including metabolism, inflammation and apoptosis.11–13 Increasing
evidence has shown that it may be involved in the development of NAFLD.14 Deng, XQ et al have proven that
SIRT1 protein levels are reduced in NAFLD inducedby high-fat diet in rats.15 SIRT1 target multiple cellular
proteins take part in metabolic homeostasis, oxidative
stress as well as in inflammation, such as NF-kappaB.31
It is reported that hepatic SIRT1 is a key regulator of
lipid homeostasis, in particular fatty acid oxidation. Lossof SIRT1 in hepatocytes can lead to a decrease in fatty
acid oxidation, resulting in the development of hepatic
steatosis and inflammation on high-fat diet mice.32 In
this study, hepatic mitochondrial SIRT1 expression was
significantly reduced in Group H, as previously shownby others. DSKT tended to increase hepatic SIRT1
expression, explaining in part its beneficial effect withrespect to high-fat diet induced disorders.
An additional mechanism related to DSKT could
elicit beneficial effects through NF-kappaB. As is wellknown, long-time intake of a high-fat diet could resultin low-grade inflammation. This inflammation state ischaracterized by the activation of the NF-kappaB path-Acknowledgments: This work was supported by the Natural Science Foundation of Shandong Province, China (ZR2012HM093)
and the Science and Technology Research Project of Traditional
Chinese Medicine in Shandong Province, China (2011-038).
The authors declare no conflict of interest.

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