Dietary Supplementation of Genistein Alleviates Liver Inflammation

Apr 17, 2015 - Nonalcoholic fatty liver disease is a complex disorder which includes simple steatosis, steatohepatitis, fibrosis and ultimately cirrho...
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Dietary Supplementation of Genistein Alleviates Liver Inflammation and Fibrosis Mediated by a Methionine-Choline-Deficient Diet in db/ db Mice Na-young Yoo,† Sookyoung Jeon,† Yerim Nam,† Youn-Jin Park,† Sae Bom Won,† and Young Hye Kwon*,†,‡ †

Department of Food and Nutrition and ‡Research Institute of Human Ecology, Seoul National University, Seoul 151-742, Republic of Korea S Supporting Information *

ABSTRACT: Nonalcoholic fatty liver disease is a complex disorder which includes simple steatosis, steatohepatitis, fibrosis and ultimately cirrhosis. Previous studies have reported that genistein, a soy phytoestrogen, attenuates steatohepatitis induced in obese and type 2 diabetic models. Here we investigated the effect of dietary genistein supplementation (0.05%) on nonalcoholic steatohepatitis (NASH) development induced by a methionine-choline-deficient (MCD) diet in db/db mice. MCD-diet-fed mice exhibited a significantly lower body weight and a higher degree of steatohepatitis with increased oxidative stress, steatosis, inflammation, stellate cell activation, and mild fibrosis. Although genistein did not inhibit hepatic steatosis, we observed that oxidative stress, endoplasmic reticulum stress, and AMP-dependent kinase inactivation were alleviated by genistein. Genistein also down-regulated the augmented gene expressions associated with hepatic inflammation and fibrosis. Therefore, these results suggest that genistein may protect MCD-diet-mediated NASH development by suppressing lipid peroxidation, inflammation, and even liver fibrosis in db/db mice. KEYWORDS: db/db mouse, fibrosis, genistein, methionine−choline-deficient diet, nonalcoholic steatohepatitis

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INTRODUCTION Nonalcoholic fatty liver disease (NAFLD) includes a wide variety of clinical conditions ranging from asymptomatic hepatic steatosis to nonalcoholic steatohepatitis (NASH) and fatty liver-associated cirrhosis, which is linked to liver-related morbidity and mortality.1 NAFLD may occur in 25−30% of the population worldwide2 and is known to be strongly associated with obesity, insulin resistance dyslipidemia, and hypertension.1 Although the precise mechanism of the progression from hepatic steatosis to NASH is not well known, a two-hit model has been proposed to explain the pathogenesis of NASH. The first hit is intrahepatic lipid accumulation or steatosis. Oxidative stress, lipid peroxidation, and proinflammatory cytokines have been considered to be the second hit leading to NASH.3 Understanding the pathogenesis of NASH development has been difficult because a suitable experimental animal model is limited. One of the available nutritional animal models is the methionine-choline-deficient (MCD) model.4,5 Mice fed an MCD diet develop steatohepatitis, which has the histologic features of human NASH.6 The MCD diet induces significantly greater reactive oxygen species (ROS) production, oxidative DNA damage, and apoptotic cell death than many of the other animal NAFLD models.7 However, extrahepatic metabolic abnormalities, including dyslipidemia, obesity, and peripheral insulin resistance observed in humans with NASH, are not typically observed in the MCD model. Several attempts to overcome these limitations include feeding an MCD diet to genetically obese and diabetic mice. Although an MCD diet still reduces body weight and improves insulin resistance, db/db mice fed an MCD diet develop greater fibrosis as well as © XXXX American Chemical Society

steatosis, inflammation, and ballooning degeneration, which makes this animal model a better fit for investigating the pathogenesis of human NASH.8 Previous studies have reported an increased prevalence of NAFLD after menopause9 and in aromatase knockout mice, which cannot synthesize endogenous estrogen.10 Estrogens are known to be powerful antioxidants and to play an important role in the inhibition of inflammation and fibrogenesis.11−13 Soybeans and soybean foods, which contain various components including bioactive peptides, isoflavone, soluble fiber, and essential omega-3 fatty acids, have generated much interest as a result of their well-documented beneficial health effects on chronic diseases.14,15 Genistein, the most abundant phytoestrogen present in soybeans,16 has diverse pharmacological features such as antioxidant, anti-inflammatory, and serum lipidlowering effects.17 In particular, genistein restores altered lipid metabolism, inhibits high-fat-diet-induced obesity, and prevents NAFLD development.18,19 Most previous studies have reported the inhibitory effects of genistein on NAFLD development using obese animal models. Therefore, the MCD model with distinct patterns of lipid accumulation between liver and adipose tissue may provide a better understanding of genistein in the regulation of hepatic and peripheral lipid accumulation and inflammatory responses. In addition, there is no study which reports the antifibrotic effect of genistein in diet-induced Received: December 24, 2014 Revised: March 20, 2015 Accepted: April 17, 2015

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DOI: 10.1021/acs.jafc.5b00398 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

SuperscriptII Reverse Transcriptase (Invitrogen, Carlsbad, CA). Hepatic mRNA levels were analyzed by qPCR with a StepOne Real Time PCR System (Applied Biosystems, Carlsbad, CA) using the SYBR Green PCR Master Mix (Applied Biosystems). Mouse ribosomal protein L19 (RPL19) was used as a reference gene, and relative gene expression levels were analyzed using the 2−ΔΔCt method. To determine mRNA levels of spliced XBP-1, semiquantitative PCR was used as previously described.24 The expression of β-actin was examined as an internal control. The primer sequences are described in Supporting Information Table S1. Statistical Analysis. All data were analyzed using SPSS software (ver. 21.0, SPSS Inc., Chicago, IL). For all experiments, one-way ANOVA followed by Duncan’s multiple range test or the Student t test was employed to assess the statistical significance. Data were expressed as the mean ± SEM, and differences were considered to be statistically significant at P < 0.05.

NASH. Therefore, in the present study, we investigated whether genistein alleviates NASH development in db/db mice fed an MCD diet.

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MATERIAL AND METHODS

Animals and Diets. At 5 weeks of age, male C57BLKS/J-db/db mice were purchased from Japan SLC, Inc. (Hamamatsu, Japan). After a 2 week acclimation period, confirmed high-glucose db/db mice were randomly divided into three groups and fed a control MCS diet (no. 518811; Dyets Inc., Bethlehem, PA), an MCD diet (no. 518810), or an MCD diet supplemented with genistein (0.5 g/kg diet; ChromaDex Inc., Irvine, CA) for 6 weeks. A previous study reported that the serum genistein concentration of C57BL/6J mice fed a genisteinsupplemented diet (0.6 g/kg diet) for 4 weeks was 3.97 μmol/L,20 which is similar to that detected in women consuming soy products.21 Body weight was recorded every week, and blood glucose levels were measured with a glucose meter (Accu-chekGo, Roche, Basel, Switzerland) after 6 h of fasting. All mice were maintained in a temperature-controlled (22 ± 3 °C), humidity-controlled (50 ± 10%) room with a 12 h dark−light cycle. All treatment protocols for this study were approved by the Seoul National University Institutional Animal Care and Use Committee (SNU-100524-5). At the end of the study, the mice fasted for 16 h, blood was collected by cardiac puncture, and tissues were removed, quickly frozen in liquid nitrogen, and stored at −80 °C until analysis. Serum and Hepatic Biochemical Analyses. Serum was analyzed using commercial colorimetric assay kits (Asan Pharmaceutical Co., Seoul, Korea) for glucose, triglycerides, total cholesterol, and alanine aminotransferase (ALT) according to the manufacturer’s protocol. Serum insulin levels were measured using an insulin ELISA kit (Millipore, Billerica, MA). Homeostasis model assessment-insulin resistance (HOMA-IR) was calculated using the following formula: HOMA-IR = [fasting glucose (mmol/L) × fasting insulin (μU/mL)]/ 22.5. Hepatic total lipids were extracted according to the method of Folch et al.22 Triglyceride and cholesterol concentrations were determined using the same commercial kits used in serum samples. Hepatic thiobarbituric acid reactive substances (TBARS) were measured according to the method of Ohkawa et al.23 The absorbance of the butanol layer was measured at 532 nm using 1,1,3,3-tetraethoxypropane as a standard. The hepatic lipid peroxide level was expressed as malondialdehyde equivalents per milligram of protein. The protein content of the homogenate was measured with a protein assay kit (Bio-Rad, Hercules, CA). Liver Histology Examination. Formalin-fixed liver tissue was processed into 4-μm-thick paraffin sections and stained with hematoxylin and eosin (H&E) for histological evaluation. Liver fibrosis was assessed with Masson’s trichrome staining for collagen fibers. The morphology was observed under an Olympus BX50 microscope with a DP-72 digital camera (Olympus, Japan), and the image was captured using the Image-Pro Plus ver. 4.5 program (Media Cybernetics Inc., USA). Immunoblotting. Liver tissues were homogenized in 10 volumes (w/v) of ice-cold protein lysis buffer. Thirty micrograms of protein was loaded into the lanes of an SDS-PAGE gel, separated, and blotted onto a PVDF membrane. After being blocked with 5% nonfat milk or bovine serum albumin in TTBS, membranes were probed with specific antibodies diluted in TTBS with 5% nonfat milk or bovine serum albumin as follows: anti-AMP kinase alpha (AMPKα; Cell Signaling, Danvers, MA), anti-p-AMPKα (Cell Signaling), anti-C/EBP homologous protein (CHOP; Santa Cruz Biotechnology, Santa Cruz, CA), anti-KDEL (Enzo Life Sciences, Farmingdale, NY), or anti-70-kDa heat shock cognate protein (HSC70; Santa Cruz Biotechnology). The membranes were then incubated with an IgG-peroxidase-conjugated secondary antibody for chemiluminescent detection. The band intensities were quantified using Quantity One software (Bio-Rad). Semiquantitative and Quantitative PCR (qPCR) Analyses. The total RNA of the liver tissues was isolated using the RNAiso Plus (Takara Bio Inc., Otsu, Japan), and cDNA was synthesized with

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RESULTS Effects of Genistein on Body Weight and Serum Biochemical Parameters in db/db Mice Fed an MCD Diet. We observed a gradual body weight decrease in db/db mice fed an MCD diet (Supporting Information Figure S1). Blood glucose was also significantly reduced in mice fed an MCD diet within a week. Consistently, the epididymal fat mass weight was significantly lower in db/db mice fed an MCD diet than those fed an MCS diet (Table 1). Genistein Table 1. Characteristics of db/db Mice Fed an MCS, MCD, or MCD with Genistein Dieta final body weight (g) epididymal fat (g) relative epididymal fat weight (% of body weight) liver weight (g) relative liver weight (% of body weight)

MCS

MCD

MCD + GEN

42.0 ± 1.4b 2.27 ± 0.10b 5.47 ± 0.21ab

29.1 ± 1.2a 1.47 ± 0.08a 5.01 ± 0.10a

29.8 ± 1.1a 1.69 ± 0.08a 5.67 ± 0.09b

3.47 ± 0.26b 8.19 ± 0.38b

1.62 ± 0.10a 5.55 ± 0.27a

1.69 ± 0.10a 5.64 ± 0.19a

Results are given as the mean ± SEM (n = 10). Means that do not share the same superscript are significantly different by ANOVA (P < 0.05).

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Table 2. Fasting Serum Biochemical Analyses of db/db Mice Fed an MCS, MCD, or MCD with Genistein Dieta,* MCS glucose (mg/dL) insulin (ng/mL) HOMA-IR triglyceride (mg/ dL) total cholesterol (mg/dL) ALT (IU/L)

765.9 1.59 67.86 122.8

± ± ± ±

74.7b 0.24c 12.00a 14.0

MCD 105.4 0.92 5.63 121.4

± ± ± ±

7.1a 0.13b 1.04b 12.8

MCD + GEN 127.0 0.39 2.48 108.4

± ± ± ±

14.4a 0.09a 0.34b* 2.9

230.9 ± 21.9b

126.7 ± 4.7a

128.3 ± 10.7a

180.1 ± 23.6a

332.1 ± 43.9b

296.8 ± 9.8b

Results are given as the mean ± SEM (n = 7). Means that do not share the same superscript are significantly different (P < 0.05). *MCD vs MCD + GEN by the t test (P < 0.05).

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supplementation significantly increased the relative epididymal fat mass weight. Although MCD feeding significantly reduced the relative liver weight, the effect of genistein on liver weight was not observed. We observed significantly reduced serum glucose and insulin levels and HOMA-IR in mice fed an MCD diet compared to mice fed an MCS diet (Table 2). Supplementation of genistein B

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supplementation of an MCD diet with genistein did not alleviate the accumulation of hepatic triglyceride in db/db mice. There was no significant difference in hepatic cholesterol levels among the groups. The microscopic examination of H&E staining of liver tissues was consistent with hepatic lipid biochemical characteristics (Figure 1B). We observed the microvascular steatosis in db/db mice fed an MCS diet. Interestingly, the MCD diet induced macrovascular steatosis and inflammatory foci, which were less observed in db/db mice fed a genistein-supplemented diet. Effects of Genistein on Oxidative Stress and ER Stress in db/db Mice Fed an MCD Diet. The increased generation of ROS by microsomal CYP2E1 enzyme has been proposed as an initiator of oxidative stress associated in MCD-diet-mediated steatohepatitis in mice.25 To determine the oxidative stress, we measured hepatic malondialdehyde levels and hepatic enzyme levels involved in oxidative stress. Mice on an MCD diet exhibited significantly higher TBARS levels, which were alleviated by genistein supplementation (Figure 2A). Accordingly, the mRNA levels of heme oxygenase (HO-1) were significantly induced in mice fed an MCD diet (Figure 2B). Genistein supplementation significantly reduced the mRNA levels of HO-1. In addition to oxidative stress, endoplasmic reticulum (ER) stress has been shown to be induced in an MCD-diet-induced NASH model. To determine the effects of genistein on ER stress, we examined the cellular markers of ER stress including glucose-regulated protein 78 (Grp78/BiP) and CHOP proteins

significantly reduced serum insulin levels and HOMA-IR (P = 0.014, t test). MCD feeding significantly reduced serum total cholesterol levels but not serum triglyceride levels. Serum ALT levels were significantly increased in db/db mice compared to those in mice fed an MCD diet, but there was no significant effect of genistein on serum ALT levels. Effects of Genistein on Liver Steatosis in db/db Mice Fed an MCD Diet. The MCD diet significantly increased hepatic triglyceride levels in db/db mice (Figure 1A). The

Figure 1. (A) Hepatic triglycerides and cholesterol contents in db/db mice fed an MCS diet, MCD diet, or MCD with genistein diet. Each bar represents the mean ± SEM (n = 7), and bars that do not share the same letter are significantly different by ANOVA (P < 0.05). (B) Representative H&E staining of liver tissue sections (n = 4). Black arrows indicate inflammatory foci.

Figure 2. Oxidative stress and ER stress markers in db/db mice fed an MCS diet, MCD diet, or MCD with genistein diet. (A) Hepatic TBARS levels (n = 5). (B) Relative mRNA expression of HO-1/RPL19 determined by qPCR (n = 3−4). (C) Relative BiP/HSC70 and (D) CHOP/HSC70 protein levels determined by immunoblotting (n = 3). (E) Relative mRNA expression levels of the spliced form of XBP-1/β-actin determined by semiquantitative PCR (n = 3−4). Each bar represents the mean ± SEM, and bars that do not share the same letter are significantly different by ANOVA (P < 0.05). C

DOI: 10.1021/acs.jafc.5b00398 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry and the spliced form of XBP-1 mRNA.26 As shown in Figure 2C,D, protein expression levels of Grp78/BiP and CHOP were significantly increased in db/db mice fed an MCD diet compared to those in db/db mice fed an MCS diet. Genistein supplementation significantly alleviated Grp78/BiP and CHOP induced in mice fed an MCD diet. We also observed a significant induction of the spliced form of XBP-1 mRNA in mice fed an MCD diet. The suppressive effect of genistein on the induction of the spliced form of XBP-1 mRNA was significant (Figure 2E). Effects of Genistein on Hepatic Inflammation in db/db Mice Fed an MCD Diet. Consistent with increased inflammatory foci, the MCD diet significantly induced the mRNA levels of tumor necrosis factor alpha (TNF-α), monocyte chemoattractant protein 1 (MCP-1), toll-like receptor 4 (TLR4), and interleukin 1 beta (IL-1β) (Figure 3A). These inflammatory gene expressions were significantly alleviated by genistein supplementation. The hepatic adiponectin resistance has been suggested to be involved in steatohepatitis progression by changing peroxisome proliferator-activated receptor alpha (PPARα) activity and ROS accumulation.27 Therefore, we determined whether genistein modulates the AMPK pathway in the MCD-mediated steatohepatitis model. Feeding an MCD diet significantly inhibited AMPK activation, which was alleviated by genistein supplementation (Figure 3B). Furthermore, mRNA levels of PPARα, a downstream transcription factor of adiponectin signaling, were significantly reduced in mice fed an MCD diet (Figure 3C). Although genistein supplementation tended to increase PPARα mRNA levels, the difference was not significant. As previously reported,28 adiponectin resistance was not associated with an altered adiponectin receptor expression in the livers of MCD-diet-fed mice (data not shown). Effects of Genistein on Liver Fibrosis in db/db Mice Fed an MCD Diet. To examine the effects of genistein supplementation on liver fibrosis, sections of liver were stained with Masson’s trichrome. Mild periportal fibrosis in the absence of apparent hepatocellular ballooning was observed in MCDdiet-fed mice (Figure 4A). Genistein apparently reduced Masson’s trichrome-stained fibrils induced by an MCD diet. Parallel to the changes in histological fibrosis, increased hepatic transforming growth factor β (TGF-β), procollagen, type I, alpha 1 type I (COL1A1), and tissue inhibitors of metalloproteinase 1 (TIMP1) mRNA levels in MCD-diet-fed db/db mice were significantly diminished by genistein treatment (Figure 4B).

Figure 3. Hepatic inflammatory markers in db/db mice fed an MCS diet, MCD diet, or MCD with genistein diet. (A) Relative mRNA expression of genes involved in inflammation determined by qPCR and normalized to RPL19 (n = 3−4). (B) Relative p-AMPK/AMPK protein levels determined by immunoblotting (n = 3). (C) Relative mRNA expression of PPARα/RPL19 determined by qPCR (n = 3). Each bar represents the mean ± SEM, and bars that do not share the same letter are significantly different by ANOVA (P < 0.05).

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DISCUSSION We observed that genistein inhibited oxidative stress and ER stress, allowing for the alleviation of liver inflammation and fibrosis in the MCD-diet-mediated NASH model. Although this animal model has its own limitations, including the lack of obesity and peripheral insulin resistance,29 it is a useful model for clarifying whether genistein could alleviate the progression of steatohepatitis to liver fibrosis, which is not easily induced in dietary NASH models. In addition, the present study allows us to clarify whether genistein could exhibit the anti-inflammatory effect in the NASH model which is not accompanied by obesity. In the MCD model, the “first hit” is the impaired mitochondrial β-oxidation, and the “second hit” is increased oxidative stress and lipid peroxidation,30 which induce

proinflammatory gene expression, neutrophil chemotaxis, and hepatic stellate cell activation.31−33 The protective action of genistein may be due to the inhibition of the pathway involved in the second hit because we did not observe a significant decrease in hepatic triglyceride levels by genistein supplementation. Indeed, genistein attenuated the oxidative stress in the liver of mice fed an MCD diet. In support of this view, we observed the antioxidative effect of genistein in the previous study, in which genistein supplementation (0.5 g/kg diet) in a D

DOI: 10.1021/acs.jafc.5b00398 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Figure 4. Hepatic fibrosis markers in db/db mice fed an MCS diet, MCD diet, or MCD with genistein diet. (A) Representative Masson’s trichrome staining of liver tissue sections (n = 4). (B) Relative mRNA expression of genes involved in fibrosis determined by qPCR and normalized to RPL19 (n = 3−4). Each bar represents the mean ± SEM, and bars that do not share the same letter are significantly different by ANOVA (P < 0.05).

feeding an MCD diet induces hepatic adiponectin resistance, which fails to activate AMPK or PPARα,28 and that the administration of a PPARα agonist reduces MCD-induced steatohepatitis in part by limiting lipid peroxidation.31 Adiponectin has been shown to increase gene expressions of antioxidant enzymes and regulate the recruitment and phenotype polarization of Kupffer cells.41 The anti-inflammatory effects of adiponectin in macrophages, although still not clearly understood, are likely to involve the inactivation of the TLR4 signaling pathway.42 Additionally, the enhancement of adiponectin signaling reduced the hepatic expression of TGFβ1, a profibrotic factor involved in hepatic stellate cell activation in mice fed an MCD diet.43 Although some studies reported that hepatic steatosis is critical to the pathogenesis of liver injury in MCD-diet-fed mice,32 we observed that hepatic inflammation and subsequent fibrosis could be regulated independently of hepatic steatosis in the MCD model. These data are in keeping with a previous study in which the treatment of MCD-diet-fed mice with a pancaspase inhibitor decreased liver inflammation and fibrosis. However, serum ALT levels and hepatic triglyceride levels were comparable between the control and treated mice. 44 Polaprezinc, a zinc-carnosine chelate compound, also significantly alleviated MCD-mediated liver fibrosis without the inhibition of steatosis,45 suggesting that liver steatosis is not

high-fat diet significantly lowered hepatic lipid peroxidation levels in ApoE knockout mice.18 Antioxidants including vitamin E and α-lipoic acid have been shown to prevent the development and progression of NASH in mice fed an MCD diet.34,35 ROS is shown to be involved in TLR4-mediated inflammasome activation in NASH,36 which has been shown to be involved in hepatocyte pyroptosis, liver inflammation, and fibrosis.37 Furthermore, we observed that dietary supplementation of genistein attenuates liver fibrosis via the inhibition of collagen synthesis and the activation of extracellular matrix degradation. Several experimental studies have shown the protective effect of estrogen against liver fibrosis. Estradiol inactivated transcription processes involved in TGF-β1 expression by suppressing ROS generation and the mitogen-activated protein kinase pathways in hepatic stellate cells from rats,13 suggesting that the estrogenic effect of genistein may be associated with the observed antifibrogenesis. Previous studies reported that genistein had positive effects on liver fibrosis induced by CCl4 administration in rats38,39 and by chronic alcohol administration in rats.40 However, this is the first study to report the antifibrotic effect of genistein in dietary models of NASH. We also demonstrated that genistein alleviated the impaired adiponectin signaling pathway. Previous studies reported that E

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IR, homeostasis model assessment-insulin resistance; IL-1β, interleukin-1 beta; MCD, methionine-choline deficient; MCP1, monocyte chemoattractant protein 1; MCS, methioninecholine sufficient; NAFLD, nonalcoholic fatty liver disease; NASH, nonalcoholic steatohepatitis; PPARα, peroxisome proliferator-activated receptor alpha; ROS, reactive oxygen species; TBARS, thiobarbituric acid reactive substances; TGFβ, transforming growth factor beta; TIMP-1, tissue inhibitor of metalloproteinase 1; TLR4, toll-like receptor 4; TNFα, tumor necrosis factor alpha; XBP-1, X-box binding protein 1

correlated with liver injury in the MCD model. In another setting, treating MCD-diet-fed mice with diacylglycerol Oacyltransferase 2 antisense oligonucleotide alleviated hepatic steatosis but increased lipid peroxidation, inflammation, and fibrosis.25 Several biopsies in pediatric-type NASH (type 2 NASH) show prominent zone 3 steatosis and minimal zone 1 steatosis with the confined fibrosis in the portal area, suggesting that steatosis in the portal area may not be required for fibrogenesis.46 Further studies are needed to investigate the molecular mechanism in the development of portal NASH in the MCD model. Contradictory to the previous studies using obese animal models, the effects of genistein on either body weight change or serum profiles were not significant in the present study using the NASH model associated with lipodystophy. These minimal effects of genistein on metabolic profiles may be attributed to the overwhelming effect of the MCD diet per se. Previous observations reported that inhibitory effects of genistein on the development of NASH in obese mice are accompanied by a significant decrease in body weight. However, our present results clearly show that genistein could alleviate the development of NASH without reducing the adipose tissue and body weights of the subjects. Indeed, we observed that genistein activated adiponectin signaling in the liver of mice fed an MCD diet, suggesting that improved adiponectin resistance may be involved, at least in part, in alleviation of NASH progression. When these results are taken together, our study shows that genistein supplementation alleviated liver inflammation and fibrosis in the MCD-diet-induced NASH model. The beneficial effects of genistein on NASH development seem to be mediated by its ability to inhibit oxidative stress and ER stress, to diminish proinflammatory cytokine expression, and to modulate impaired PPARα-mediated signaling even without affecting hepatic TG accumulation at least in the MCD model.

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(1) Anderson, N.; Borlak, J. Molecular mechanisms and therapeutic targets in steatosis and steatohepatitis. Pharmacol Rev. 2008, 60, 311− 57. (2) Lazo, M.; Clark, J. M. The epidemiology of nonalcoholic fatty liver disease: a global perspective. Semin. Liver Dis. 2008, 28, 339−50. (3) Day, C. P.; James, O. F. Steatohepatitis: a tale of two ″hits″? Gastroenterology 1998, 114, 842−5. (4) Larter, C. Z.; Yeh, M. M. Animal models of NASH: getting both pathology and metabolic context right. J. Gastroenterol. Hepatol. 2008, 23, 1635−48. (5) Varela-Rey, M.; Embade, N.; Ariz, U.; Lu, S. C.; Mato, J. M.; Martinez-Chantar, M. L. Non-alcoholic steatohepatitis and animal models: understanding the human disease. Int. J. Biochem. Cell Biol. 2009, 41, 969−76. (6) Leclercq, I. A.; Farrell, G. C.; Field, J.; Bell, D. R.; Gonzalez, F. J.; Robertson, G. R. CYP2E1 and CYP4A as microsomal catalysts of lipid peroxides in murine nonalcoholic steatohepatitis. J. Clin. Invest. 2000, 105, 1067−75. (7) Gao, D.; Wei, C.; Chen, L.; Huang, J.; Yang, S.; Diehl, A. M. Oxidative DNA damage and DNA repair enzyme expression are inversely related in murine models of fatty liver disease. Am. J. Physiol Gastrointest Liver Physiol 2004, 287, G1070−7. (8) Sahai, A.; Malladi, P.; Pan, X.; Paul, R.; Melin-Aldana, H.; Green, R. M.; Whitington, P. F. Obese and diabetic db/db mice develop marked liver fibrosis in a model of nonalcoholic steatohepatitis: role of short-form leptin receptors and osteopontin. Am. J. Physiol Gastrointest Liver Physiol 2004, 287, G1035−43. (9) Hashimoto, E.; Tokushige, K. Prevalence, gender, ethnic variations, and prognosis of NASH. J. Gastroenterol. 2011, 46 (Suppl 1), 63−9. (10) Jones, M. E.; Thorburn, A. W.; Britt, K. L.; Hewitt, K. N.; Wreford, N. G.; Proietto, J.; Oz, O. K.; Leury, B. J.; Robertson, K. M.; Yao, S.; Simpson, E. R. Aromatase-deficient (ArKO) mice have a phenotype of increased adiposity. Proc. Natl. Acad. Sci. U. S. A. 2000, 97, 12735−40. (11) Helisten, H.; Hockerstedt, A.; Wahala, K.; Tiitinen, A.; Adlercreutz, H.; Jauhiainen, M.; Tikkanen, M. J. Accumulation of high-density lipoprotein-derived estradiol-17beta fatty acid esters in low-density lipoprotein particles. J. Clin. Endocrinol. Metab. 2001, 86, 1294−300. (12) Uzui, H.; Sinha, S. K.; Rajavashisth, T. B. 17beta-estradiol inhibits oxidized low-density lipoprotein-induced increase in matrix metalloproteinase-9 expression in human macrophages. J. Investig. Med. 2011, 59, 1104−1108. (13) Itagaki, T.; Shimizu, I.; Cheng, X.; Yuan, Y.; Oshio, A.; Tamaki, K.; Fukuno, H.; Honda, H.; Okamura, Y.; Ito, S. Opposing effects of oestradiol and progesterone on intracellular pathways and activation processes in the oxidative stress induced activation of cultured rat hepatic stellate cells. Gut 2005, 54, 1782−9. (14) Omoni, A. O.; Aluko, R. E. Soybean foods and their benefits: potential mechanisms of action. Nutr. Rev. 2005, 63, 272−83. (15) Messina, M.; Messina, V. The role of soy in vegetarian diets. Nutrients 2010, 2, 855−88. (16) Fang, N.; Yu, S.; Badger, T. M. Comprehensive phytochemical profile of soy protein isolate. J. Agric. Food Chem. 2004, 52, 4012−20.

ASSOCIATED CONTENT

S Supporting Information *

Primer sequences for qPCR and semiquantitative PCR. Changes in body weight and fasting blood glucose levels of db/db mice fed an MCS diet, MCD diet, or MCD with genistein diet. MCS vs MCD and MCD + GEN (P < 0.05 by ANOVA). This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +82-2-880-6833. Fax: +82-2884-0305. Funding

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology, Republic of Korea (no. 2009-0069120 and no. 2012-014736). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS ALT, alanine aminotransferase; AMPK, AMP-dependent kinase; CHOP, C/EBP homologous protein; COL1A1, collagen, type I, alpha 1; CYP2E1, cytochrome P450 2E1; ER, endoplasmic reticulum; HO-1, heme oxygenase 1; HOMAF

DOI: 10.1021/acs.jafc.5b00398 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

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DOI: 10.1021/acs.jafc.5b00398 J. Agric. Food Chem. XXXX, XXX, XXX−XXX