Protective Effects of Genistein and Puerarin against Chronic Alcohol

Sep 9, 2016 - This study aimed to investigate the protective effect of genistein or puerarin on chronic alcohol-induced liver injury in vivo and to ex...
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Protective Effects of Genistein and Puerarin against Chronic Alcohol-Induced Liver Injury in Mice via Antioxidant, Anti-Inflammatory, and Anti-Apoptotic Mechanisms Liang Zhao, Yong Wang, Jia Liu, Kai Wang, XiaoXuan Guo, Baoping Ji, Wei Wu, and Feng Zhou J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b02907 • Publication Date (Web): 09 Sep 2016 Downloaded from http://pubs.acs.org on September 10, 2016

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Title: Protective Effects of Genistein and Puerarin against Chronic Alcohol-Induced

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Liver Injury in Mice via Antioxidant, Anti-Inflammatory, and Anti-Apoptotic

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Mechanisms

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Short title: Hepatoprotective Effect of Genistein and Puerarin in Alcohol-Induced

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Mice

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Liang Zhao1, Yong Wang1, Jia Liu2, Kai Wang3, Xiaoxuan Guo1, Baoping Ji1, Wei

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Wu4 * and Feng Zhou1 *

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1

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Food Science & Nutritional Engineering, China Agricultural University, Beijing

Beijing Key Laboratory of Functional Food from Plant Resources, College of

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100083, People’s Republic of China

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2

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Beijing 100093, People’s Republic of China

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3

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100015, People’s Republic of China

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4

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Republic of China

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*

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F.Z. and W.W. are regarded as joint corresponding authors.

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(F.Z.) Phone: +86-10-62737129; Fax: +86-10-62347334; E-mail: [email protected]

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(W.W.)

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[email protected]

Institute of Apicultural Research, Chinese Academy of Agricultural Sciences,

China National Research Institute of Food and Fermentation Industries, Beijing

College of Engineering, China Agricultural University, Beijing 100083, People’s

Corresponding authors:

Phone:

+86-10-62736918;

Fax:

+86-10-62347334;

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E-mail:

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Abstract

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This study aimed to investigate the protective effect of genistein or puerarin on

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chronic alcohol-induced liver injury in vivo and explore the underlying mechanisms

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of hepatoprotective effects. Mice were administered with genistein or puerarin (0.3

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mmol kg−1 body weight) and gastrically infused with 50% alcohol once per day for 5

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weeks. Levels of serum transaminases, serum and hepatic lipid, hepatic antioxidant

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capacities, inflammation, apoptosis, and histopathological sections were analyzed.

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Results showed that genistein and puerarin exhibited similar effects in ameliorating

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alcohol-induced liver injury. However, genistein is more effective than puerarin in

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decreasing levels of malondialdehyde (1.05 ± 0.0947 vs. 1.28 ± 0.213 nmol/mg pro, p

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< 0.05), tumor necrosis factor α (3.12 ± 0.498 vs. 3.82 ± 0.277 pg/mg pro, p < 0.05),

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interleukin-6 (1.46 ± 0.223 vs. 1.88 ± 0.309 pg/mg pro, p < 0.05), whereas puerarin is

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more effective than genistein in ameliorating serum activities or levels of alanine

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transaminase (35.8 ± 3.95 vs. 42.6 ± 6.56 U/L, p < 0.05) and low-density lipoprotein

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cholesterol (1.12 ± 0.160 vs. 1.55 ± 0.150 mmol/L, p < 0.05). In conclusion, both

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genistein and puerarin effectively alleviates hepatic damage induced by chronic

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alcohol administration through potential antioxidant, anti-inflammatory, or anti-

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apoptotic mechanisms.

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Keywords: isoflavones; liver damage; alcohol; inflammation; oxidative stress

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Introduction

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Excessive alcohol consumption can induce alcoholic liver disease (ALD), which has

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become a serious worldwide threat to human health.1 Alcohol-induced hepatic

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cirrhosis is one of the top seven diseases with the highest mortality rates, accounting

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for over 80% of liver cirrhosis cases in the USA.2 In developing countries, ALD has

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already become the second leading cause of liver damage after viral hepatitis.3 ALD

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consists of three histological stages: fatty liver (steatosis), steatohepatitis, and hepatic

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fibrosis/cirrhosis/carcinoma.4 Excessive alcohol consumption may first increase free

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fatty acid (FFA) uptake and inhibit β-oxidation and therefore induce the accumulation

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of lipid droplets and triglycerides (TG) and total cholesterol (TC) in the liver

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(alcoholic steatosis).5 Second, chronic immoderate alcohol consumption may result in

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alcoholic steatohepatitis, which is characterized by mitochondrial damage, oxidative

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stress, and the overproduction of pro-inflammatory cytokines, such as tumor necrosis

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factor α (TNF-α), interleukin-6 (IL-6), transcription factor nuclear factor kappa B

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(NF-κB), inducible NO synthase, cyclooxygenase-2 (COX-2), and monocyte

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chemoattractant protein-1 (MCP-1) in hepatocytes.6 Eventually, patients become at

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risk of developing more severe forms of ALD, including hepatic fibrosis, cirrhosis,

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and hepatocarcinoma.7

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Soybean (Glycine max) has served as a staple diet in many Asian countries for

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thousands of years; the legume contains several phytochemicals, especially

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isoflavones.8 Kudzu (Pueraria lobata), a medicinal herb in China, has been used to

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alleviate liver injury traditionally.9 The major active components of kudzu are also

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isoflavones.10 Isoflavones are beneficial for protecting against inflammation, fatty

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liver disease, atherosclerosis, cardiovascular disease, hyperlipidemia, and cancer.9, 11–

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13

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kudzu root. Genistein (1 mg/kg for 4 days) can strengthen the antioxidant system and

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shows anti-inflammatory and anti-necrotic effects on experimental liver damage

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caused by carbon tetrachloride (CCl4).14 It is also reported that treatment with

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genistein (1 mg/kg for 60 days) could activate the antioxidant profile, inhibit IL-6 and

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TNF-a levels, alleviate oxidative damage, and ameliorate fatty liver in insulin-

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resistant rats.15 Puerarin (90 mg/kg for 8 weeks) can alleviate chronic alcoholic liver

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injury in rats by inhibiting endotoxin gut leakage, Kupffer cell activation, and

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endotoxin receptors expression.16 Treatment with puerarin (60 mg/kg for 7days) can

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also improve blood circulation through partially regulating the disturbed metabolic

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pathways in blood stasis rat model.17 Puerarin (400 mg/kg for 4 weeks) is effective in

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the treatment of chemical-induced liver fibrosis in rats and mechanisms are due to

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reducing serum levels of aspartate aminotransferase (AST), alanine transaminase

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(ALT), as well as its induction of apoptosis through down-regulating bcl-2 mRNA

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expression.18 Given the compounds’ antioxidant properties and structural similarity, it

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was hypothesized that genistein and puerarin might play similar roles in protecting

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chronic alcohol-induced liver injury. So are there any differences between genistein

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and puerarin in the protective effect?

Genistein and puerarin are common isoflavones widely found in soybeans and the

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In the present study, we investigated the intervening effects of genistein or

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puerarin on hepatic injury induced by chronic alcohol intake in mice. To the best of

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our knowledge, this study is the first to compare the hepatoprotective effects of

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genistein and puerarin in vivo. The underlying mechanisms of the protective effects of

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these two isoflavones were also evaluated in terms of antioxidant, anti-inflammatory,

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and anti-apoptotic activities. We determined the levels of serum biochemical

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indicators, hepatic antioxidant capacity, pro-inflammatory cytokines, apoptotic

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proteins, and hepatic histological changes.

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Materials and Methods

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Chemicals

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Genistein (99.1%) and puerarin (98.9%) were purchased from Nanjing Jingzhu Bio-

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technology, Ltd. (Nanjing, Jiangsu, China). AST, ALT, TG, TC, high-density

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lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), and

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FFA commercial assay kits were purchased from Biosino Bio-Technology and

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Science, Inc. (Beijing, China). Malondialdehyde (MDA), catalase (CAT), superoxide

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dismutase (SOD), glutathione (GSH), and glutathione peroxidase (GSH-Px)

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commercial kits were obtained from Nanjing Jiancheng Bioengineering Institute

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(Nanjing, Jiangsu, China). Enzyme-linked immunosorbent assay (ELISA) kits were

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purchased from Keyingmei Biotechnology and Science, Inc. (Beijing, China). All

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other chemicals and reagents were purchased from Sigma–Aldrich (St. Louis, MO,

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USA).

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Animals and treatments

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Forty male ICR mice (weight: 20-22 g) were obtained from the Beijing Vital River

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Laboratory Animal Center (Certificate No. SCXK [Beijing] 2012–0001). The mice

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were acclimatized with a daily 12 h light:12 h dark cycle at 22 ± 2 °C room

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temperature and 55% ± 5% relative humidity. After 1 week of adaption, the mice

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were randomly divided into four groups with ten mice per group. Genistein and

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puerarin were applied to the mice in sodium carboxymethyl cellulose solution (CMC–

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Na) with an equimolar concentration of 0.1 M (gastric volume: 3 mL kg−1 body

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weight). The normal control group (NG) received CMC–Na and distilled water at 1 h

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interval. The model group (MG) received CMC–Na and was orally given 50% alcohol

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(10 mL kg−1 body weight) 1 h later. Isoflavone groups were administered genistein or

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puerarin (same molar concentrations for each group, 0.3 mmol kg−1 body weight) and

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treated with 50% alcohol 1 h later. The entire experiment lasted for 5 weeks. The

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mice were weighed every 3 days, and the gastric infusion volume was adjusted on the

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basis of their body weights. At the end of the experimental period, animals were

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sacrificed after a 12 h overnight fast (with access to water only) as previously

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described19. Blood samples were obtained from the orbital plexus to determine the

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activities of serum biochemical parameters. Livers were weighed and cut into slices,

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some of which were kept in buffered formalin or liquid nitrogen for histological

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observation, and 10% of liver tissue homogenates were obtained from the remaining

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liver sections and stored at −80 °C. All the experimental protocols and procedures

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were approved by the Ethics Committee of Beijing Key Laboratory of Functional

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Food from Plant Resources and conducted in accordance with the Animal

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Management Rules of the Ministry of Health of the People’s Republic of China

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(Documentation No. 55, 2001, Ministry of Health of P.R. China).

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Determination of liver index

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The liver index of the mice was calculated as follows: liver index = liver weight/final

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body weight × 100%.

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Biochemical analysis

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Serum samples were collected by centrifugation at 4000 g for 15 min and stored at

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4 °C before biochemical parameter analysis. Activities of serum AST and ALT and

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the levels of TG, TC, HDL-C, LDL-C, and FFAs were determined using the

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corresponding kits.

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Liver tissue homogenates were collected and the lipids were extracted as

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previously described.20 Hepatic TG and TC levels were determined using the same

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method as that of serum TG and TC determination, and the results were normalized to

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the total protein. The MDA levels, as well as the activities of CAT, SOD, GSH, and

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GSH-Px in the liver homogenate, were measured using corresponding kits in

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accordance with the manufacturers’ instructions. The protein concentrations were

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ascertained through the bicinchoninic acid assay kit (Beyotime Biotechnology Inc.,

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Beijing, China).

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ELISA analysis

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Heme oxygenase 1 (HO-1), COX-2, NF-κB p65, TNF-α, IL-6, MCP-1, transforming

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growth factor β1 (TGF-β1), and caspase-3 levels were determined using the

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corresponding ELISA kits following the manufacturer’s instructions, and the results

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were normalized to the total protein.

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Histological analysis

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For the oil red O staining, hepatic sections were frozen in liquid nitrogen, sliced and

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stained with oil red O solution (0.5 g/100 ml, dissolved in isopropanol). Fresh liver

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tissues were then fixed in 10% neutral-buffered formalin, embedded in paraffin for

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hematoxylin and eosin (H&E) staining. All sections were observed under a light

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microscope (BA-9000L, Osaka, Japan).

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Statistical analysis

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Results were expressed as mean ± standard deviation. Statistical significance was

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determined by one-way ANOVA followed by Tukey’s test using SPSS 20.0 software

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(SPSS Inc., Chicago, USA). Significance was defined as p < 0.05.

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Results

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Effect of genistein and puerarin on food intake, body weight, weight gain, liver

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weight, and liver index in mice

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As shown in Table 1, no significant differences were found in food intake, initial body

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weight, and final body weight among these four groups. Mice in the MG gained less

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weight than did the NG (p < 0.05), suggesting that chronic ethanol consumption could

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affect body weight gain. Liver weight and liver index (ratio of liver weight against

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final body weight) were higher in the MG than in the NG (p < 0.05). In addition,

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genistein showed a significant effect on attenuating liver index when compared with

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MG.

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Effect of genistein and puerarin on serum biochemical values

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The effect of genistein and puerarin on the modification of serum ALT, AST, HDL-C,

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LDL-C, TG, TC, and FFA alterations in mice induced by chronic alcohol

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consumption are displayed in Figure 1. Alcohol administration showed a significant

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elevation in ALT and AST activities, promoted levels of LDL-C, TG, TC, and FFAs

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(p < 0.05), and inhibited the concentration of HDL-C (p < 0.05) in mice serum

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relative to those of the NG. However, both genistein and puerarin could decrease the

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activity of AST (by 41.8% and 38.0%, respectively) and ALT (by 23.8% and 36.0%,

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respectively), reduce (p < 0.05) the levels of LDL-C, TG, TC, and FFAs and increase

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levels of HDL-C (p < 0.05) in serum compared with MG. No significant differences

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were found between the genistein group and the NG in AST activity and serum HDL-

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C level and neither between the puerarin group and the NG in ALT activity and serum

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LDL-C and TG level. However, puerarin is more effective in decreasing ALT activity

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and LDL-C level than genistein (p < 0.05). These results showed that both genistein

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and puerarin could partially protect against liver damage and dyslipidemia caused by

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alcohol consumption.

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Effect of genistein and puerarin on hepatic TG and TC levels

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The hepatic TG and TC contents in MG mice were obviously augmented by 2.3- and

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0.6-fold, respectively (vs. NG, Figures 2A and B). Both genistein and puerarin

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treatment could attenuate alcohol-induced hepatic TG accumulation in the mouse liver

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(vs. MG, p < 0.05). Genistein also reduced hepatic TC content compared with MG (p

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< 0.05). However, puerarin showed no significant decrease in hepatic TC levels

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compared with the MG.

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Effect of genistein and puerarin on lipid peroxidation and antioxidant capacities

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in mouse liver

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Chronic alcohol consumption provoked a significant elevation in hepatic MDA level

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and remarkable reductions in HO-1, CAT, SOD, GSH, and GSH-Px activities

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compared with those in the NG (Figure 3). Treatment with genistein or puerarin

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promoted the activities of SOD, GSH, and GSH-Px relative to those of the MG (p


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0.05, Figures 1E, F and G) and lipid accumulation (G vs. P, p > 0.05, Figure 2) in

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mice compared with the alcohol-only treatment. This result may be due to the

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compounds’ activation of the AMPK pathway, which could be verified in future

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research.

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The metabolism of alcohol by CYP2E1 could induce the generation of reactive

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oxygen species or free radicals, further increasing the degree of oxidative stress in the

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liver.24 Here, excessive consumption of alcohol promoted hepatic MDA content (vs.

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NG, p < 0.05, Figure 3A), which indicated enhanced lipid peroxidation and hepatic

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oxidative stress.19 HO-1, an inducible form of the rate-limiting enzyme that

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catabolizes heme into biliverdin, could regulate the content of downstream

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antioxidant enzymes and inhibit cell inflammation.25, 26 In this study, treatment with

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genistein or puerarin induced significant decrease in the hepatic MDA levels

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compared with alcohol-only treatment (Figure 3A). In addition, both genistein and

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puerarin promoted the levels or activities of SOD, GSH, and GSH-Px when compared

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with those of MG (p < 0.05, Figures 3D, E, and F). Hence, genistein and puerarin may

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scavenge free radicals and inhibit lipid peroxidation through the recruitment of the

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antioxidant defense system in alcohol-induced liver injury.5, 19, 27

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NF-κB is a master regulator of inflammation and cell death; the protein is rapidly

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increased during stress-induced cellular transformations.22 Meanwhile, TGF-β1 is a

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highly important inflammatory mediator that arbitrates fibrosis in hepatic cells.21 The

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inhibition of TGF-β1 by natural components may activate an effective means for

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combating hepatic fibrosis.28 NF-κB activation can stimulate the production of

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inflammatory mediators, such as COX-2, TGF-β1, and MCP-1, and induce the release

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of pro-inflammatory cytokines, such as TNF-α and IL-6.21, 26 In our study, treatment

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with genistein or puerarin down-regulated the levels of TGF-β1 and COX-2 compared

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with those in MG (p < 0.05, Figures 4B and C), indicating that both genistein and

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puerarin could effectively alleviate inflammatory stress and potentially inhibit hepatic

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fibrosis. However, treatment with genistein could attenuate the contents of NF-κB p65,

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IL-6, and TNF-α relative to those in MG (p < 0.05, Figures 4A, E, and F). Meanwhile,

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genistein is more effective in decreasing levels of IL-6 and TNF-α in mice compared

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with puerarin (p < 0.05, Figures 4E and F).

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Apoptosis is a highly recognized feature of ALD.29 The induction of apoptosis is

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related to the increase in caspase activity.30 Accordingly, in this study, treatment with

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genistein could decrease the hepatic level of caspase-3 compared with MG (p < 0.05,

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Figure 5). Puerarin also decreased the caspase-3 content relative to that of the MG,

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although no statistically significant difference was noted. It is reported that treatment

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with naringin (30 mg/kg) for seven consecutive days could protect against CCl4-

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induced apoptosis in mice by decreasing the expression of caspase-3.26 Zeaxanthin

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dipalmitate (25 mg/kg for 10 weeks) could also attenuate hepatic apoptosis induced

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by alcohol administration in rats through decreasing the activities of caspase-3/7. 30

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These data supported the protective effects of genistein and puerarin against alcohol-

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induced hepatocellular apoptosis by suppressing caspase-3 levels.

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In conclusion, treatment with genistein or puerarin could effectively alleviate

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chronic alcohol-induced liver injury in mice by antioxidant (HO-1, SOD, CAT, GSH,

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and GSH-Px), anti-inflammatory (NF-κB, COX-2, TGF-β1, MCP-1, TNF-α, and IL-6)

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and anti-apoptotic (caspase-3) pathways. Genistein is more effective than puerarin in

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decreasing levels of MDA, TNF-α and IL-6, whereas puerarin is more effective than

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genistein in ameliorating serum ALT activity and LDL-C level.

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Abbreviations

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The following abbreviations are used in this manuscript:

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ALD: Alcoholic liver disease

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TG: Triglycerides

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TC: Total cholesterol

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TNF-α: Tumor necrosis factor α

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IL-6: Interleukin-6

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NF-κB: Nuclear factor kappa B

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COX-2: Cyclooxygenase-2

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MCP-1: Monocyte chemoattractant protein-1

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NG: Normal control group

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MG: Model group

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AST: Aspartate aminotransferase

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ALT: Alanine transaminase

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HDL-C: High-density lipoprotein cholesterol

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LDL-C: Low-density lipoprotein cholesterol

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FFAs: free fatty acids

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MDA: Malondialdehyde

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CAT: Catalase

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SOD: Superoxide dismutase

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GSH: Glutathione

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GSH-Px: Glutathione peroxidase

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HO-1: Heme oxygenase-1

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TGF-β1: Transforming growth factor-β1

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H&E: Hematoxylin and eosin

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AMPK: Adenosine monophosphate-activated protein kinase

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SREBP: Sterol regulatory element-binding protein

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Acknowledgments

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This work was financially supported by the National Natural Science Foundation of

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China (Grant No. 31571831) and Natural Science Foundation of Shandong Province

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of China (Grant No. ZR2015BQ015).

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Conflicts of Interest

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The authors declare no conflict of interest.

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H.; Lee, S. J., Restoration of autophagy by puerarin in ethanol-treated hepatocytes via the activation of AMP-activated protein kinase. Biochem. Bioph. Res. Co. 2011, 414, 361-366. 14. Kuzu, N.; Metin, K.; Dagli, A. F.; Akdemir, F.; Orhan, C.; Yalniz, M.; Ozercan, I. H.; Sahin, K.; Bahcecioglu, I. H., Protective Role of Genistein in Acute Liver Damage Induced by Carbon Tetrachloride. Mediators Inflamm. 2007, 2007, 36381. 15. Mohamed Salih, S.; Nallasamy, P.; Muniyandi, P.; Periyasami, V.; Carani Venkatraman, A., Genistein improves liver function and attenuates non-alcoholic fatty liver disease in a rat model of insulin resistance. J. Diabetes 2009, 1, 278287. 16. Peng, J. H.; Cui, T.; Huang, F.; Chen, L.; Zhao, Y.; Xu, L.; Xu, L. L.; Feng, Q.; Hu, Y. Y., Puerarin Ameliorates Experimental Alcoholic Liver Injury by Inhibition of Endotoxin Gut Leakage, Kupffer Cell Activation, and Endotoxin Receptors Expression. J. Pharmacol. Exp. Ther. 2013, 344, 646-654. 17. Zou, Z. J.; Liu, Z. H.; Gong, M. J.; Han, B.; Wang, S. M.; Liang, S. W., Intervention effects of puerarin on blood stasis in rats revealed by a 1H NMR-based metabonomic approach. Phytomedicine 2015, 22, 333-343. 18. Zhang, S.; Ji, G.; Liu, J., Reversal of chemical-induced liver fibrosis in Wistar rats by puerarin. J. Nutr. Biochem. 2006, 17, 485-491. 19. Han, Y.; Xu, Q.; Hu, J. N.; Han, X. Y.; Li, W.; Zhao, L. C., Maltol, a food flavoring agent, attenuates acute alcohol-induced oxidative damage in mice. Nutrients 2015, 7, 682-696. 20. Wang, O.; Liu, J.; Cheng, Q.; Guo, X.; Wang, Y.; Zhao, L.; Zhou, F.; Ji, B. P., Effects of Ferulic Acid and gamma-Oryzanol on High-Fat and High-Fructose DietInduced Metabolic Syndrome in Rats. PloS one 2015, 10, e0118135. 21. Lim, J. D.; Lee, S. R.; Kim, T.; Jang, S.-A.; Kang, S. C.; Koo, H. J.; Sohn, E.; Bak, J. P.; Namkoong, S.; Kim, H. K., Fucoidan from Fucus vesiculosus Protects against Alcohol-Induced Liver Damage by Modulating Inflammatory Mediators in Mice and HepG2 Cells. Mar. Drugs 2015, 13, 1051-1067. 22. Koneru, M.; Sahu, B. D.; Kumar, J. M.; Kuncha, M.; Kadari, A.; Kilari, E. K.; Sistla, R., Fisetin protects liver from binge alcohol-induced toxicity by mechanisms including inhibition of matrix metalloproteinases (MMPs) and oxidative stress. J. Funct. Foods 2016, 22, 588-601. 23. You, Y.; Yuan, X.; Lee, H. J.; Huang, W.; Jin, W.; Zhan, J., Mulberry and mulberry wine extract increase the number of mitochondria during brown adipogenesis. Food Funct. 2015, 6, 401-408. 24. Chang, Y. Y.; Lin, Y. L.; Yang, D. J.; Liu, C. W.; Hsu, C. L.; Tzang, B. S.; Chen, Y. C., Hepatoprotection of Noni Juice against Chronic Alcohol Consumption: Lipid Homeostasis, Antioxidation, Alcohol Clearance, and Anti-inflammation. J. Agric. Food Chem. 2013, 61, 11016-11024. 25. Cho, J. H.; Kwon, J. E.; Cho, Y.; Kim, I.; Kang, S. C., Anti-Inflammatory Effect of Angelica gigas via Heme Oxygenase (HO)-1 Expression. Nutrients 2015, 7, 4862-

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4874. 26. Dong, D.; Xu, L.; Yin, L.; Qi, Y.; Peng, J., Naringin prevents carbon tetrachlorideinduced acute liver injury in mice. J. Funct. Foods 2015, 12, 179-191. 27. Lin, Y. L.; Chang, Y. Y.; Yang, D. J.; Tzang, B. S.; Chen, Y. C., Beneficial effects of noni (Morinda citrifolia L.) juice on livers of high-fat dietary hamsters. Food Chem. 2013, 140, 31-38. 28. Go, J.; Kim, J. E.; Koh, E. K.; Song, S. H.; Sung, J. E.; Lee, H. A.; Lee, Y. H.; Lee, Y.; Hong, J. T.; Hwang, D. Y., Protective Effect of Gallotannin-Enriched Extract Isolated from Galla Rhois against CCl₄-Induced Hepatotoxicity in ICR Mice. Nutrients 2016, 8, 107. 29. Lu, Y.; Wu, D.; Wang, X.; Ward, S. C.; Cederbaum, A. I., Chronic alcohol-induced liver injury and oxidant stress are decreased in cytochrome P4502E1 knockout mice and restored in humanized cytochrome P4502E1 knock-in mice. Free Radic. Biol. Med. 2010, 49, 1406-1416. 30. Xiao, J.; Wang, J.; Xing, F.; Han, T.; Jiao, R.; Liong, E. C.; Fung, M. L.; So, K. F.; Tipoe, G. L., Zeaxanthin Dipalmitate Therapeutically Improves Hepatic Functions in an Alcoholic Fatty Liver Disease Model through Modulating MAPK Pathway. PLoS One 2014, 9, e95214.

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Figure legends

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Figure. 1 Effect of genistein and puerarin on hepatic function and serum biochemical

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values. (A): Aspartate aminotransferase activity; (B): Alanine aminotransferase

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activity; (C): Serum high density lipoprotein-cholesterol level; (D): Serum low

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density lipoprotein-cholesterol level; (E): Serum total triglyceride level; (F): Serum

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total cholesterol level; (G): Serum free fatty acids level. Values are expressed as mean

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± SD (n =10). Labeled means without a common letter difference. p < 0.05 by one-

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way ANOVA followed by Tukey’s test. NG, normal group; MG, model group; G,

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genistein; P, puerarin.

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Figure 2. Effect of genistein and puerarin on hepatic lipid profiles. (A): Hepatic total

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triglyceride level; (B): Hepatic total cholesterol level. Values are expressed as mean ±

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SD (n =10). Labeled means without a common letter difference. p < 0.05 by one-way

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ANOVA followed by Tukey’s test. NG, normal group; MG, model group; G,

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genistein; P, puerarin.

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Figure 3. Effect of genistein and puerarin on hepatic lipid peroxidation and

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antioxidant capacity. (A): Malondialdehyde level; (B): Heme oxygenase 1 level; (C):

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Activity of catalase; (D): Activity of superoxide dismutase; (E): Hepatic content of

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glutathione; (F): Activity of glutathione peroxidase. Values are expressed as mean ±

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SD (n =10). Labeled means without a common letter difference. p < 0.05 by one-way

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ANOVA followed by Tukey’s test. NG, normal group; MG, model group; G,

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genistein; P, puerarin.

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Figure 4. Effect of genistein and puerarin on hepatic inflammatory stress. (A): Level

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of nuclear factor-κB p65; (B): Transforming growth factor β1 level; (C):

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Cyclooxygenase 2 level; (D): Monocyte chemoattractant protein 1 level; (E):

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Interleukin-6 level; (F): Tumor necrosis factor α level. Values are expressed as mean

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± SD (n =10). Labeled means without a common letter difference. p < 0.05 by one-

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way ANOVA followed by Tukey’s test. NG, normal group; MG, model group; G,

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genistein; P, puerarin.

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Figure 5. Effect of genistein and puerarin on the content of caspase-3. Values are

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expressed as mean ± SD (n =10). Labeled means without a common letter difference.

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p < 0.05 by one-way ANOVA followed by Tukey’s test. NG, normal group; MG,

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model group; G, genistein; P, puerarin.

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Figure 6. Histological examination of liver sections (original magnification: ×200, the

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bars represent 100 µm). Representative samples of liver tissues were stained with

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hematoxylin and eosin (A) and oil red O (B). NG, normal group; MG, model group; G,

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genistein; P, puerarin.

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Table 1. Effect of genistein and puerarin on food intake, body weight, weight gain, liver weight, and liver index in mice. Variable

NG

MG

G

P

Food intake, g/day

4.13±0.56a

3.71±0.57a

3.78±0.58a

3.92±0.55a

Initial body weight, g

22.6±0.93a

22.6±1.02a

22.9±0.81a

22.8±0.83a

Final body weight, g

30.3±2.77a

28.9±1.35a

29.7±2.71a

30.3±1.72a

Weight gain, g

8.56±1.64a

6.14±1.08b

7.55±1.95ab

7.51±2.05ab

Liver weight, g

1.09±0.09a

1.28±0.07b

1.16±0.17ab

1.24±0.13ab

Liver index, %

3.60±0.18a

4.38±0.13b

4.10±0.22c

4.18±0.38bc

Values represent the mean ± SD (n=10). Labeled means without a common letter difference. p < 0.05 by one-way ANOVA followed by Tukey’s test. NG, normal group; MG, model group; G, genistein; P, puerarin.

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