Genistein Ameliorates Non-alcoholic Fatty Liver Disease by Targeting

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Bioactive Constituents, Metabolites, and Functions

Genistein Ameliorates Non-alcoholic Fatty Liver Disease by Targeting Thromboxane A2 Pathway Wenzhe Wang, Junliang Chen, Jinyan Mao, Hongling Li, Mingfu Wang, Hao Zhang, Haitao Li, and Wei Chen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01691 • Publication Date (Web): 17 May 2018 Downloaded from http://pubs.acs.org on May 17, 2018

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Genistein Ameliorates Non-alcoholic Fatty Liver Disease by Targeting Thromboxane A2 Pathway Wenzhe Wang†, ‡, Junliang Chen‡, §, Jinyan Mao‡, Hongling Li ‡, Mingfu Wangǁ, Hao Zhang†, ‡, Haitao Li*, †, ‡ and Wei Chen*, †, ‡, ¶ † State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, 214122, China ‡ School of Food Science and Technology, Jiangnan University, Wuxi, 214122, China § College of Food & Bioengineering, Henan University of Science and Technology, Luoyang, 471003, China ǁ School of Biological Sciences, The University of Hong Kong, Hong Kong, China ¶ Beijing Innovation Centre of Food Nutrition and Human Health, Beijing Technology & Business University, Beijing, 100048, China *Corresponding author: Haitao Li, Phone: +86-510-85197302, [email protected]; Wei Chen, Phone: +86-510-85912087, [email protected]

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Disclosure of Potential Conflicts of Interest No potential conflicts of interest were disclosed. Authors’ Contributions Wei Chen, Hao Zhang, Haitao Li and Mingfu Wang designed and supervised the experiments. Wenzhe Wang and Haitao Li prepared the manuscript. Wenzhe Wang, Junliang Chen, Jinyan Mao and Hongling Li performed experiments. Abbreviations: COX-1, cyclooxygenase-1; COX-2, cyclooxygenase-2; NAFLD, non-alcoholic fatty liver disease; TBXAS1, thromboxaneA2 synthase; TBXA2R, thromboxane A2 receptor; TXA2, thromboxane A2.

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ABSTRACT

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Non-alcoholic fatty liver disease (NAFLD) is now a public health issue worldwide,

3

but no drug has received approval yet. Genistein, an isoflavonoid derived from

4

soybean, ameliorates high fat diet-induced NAFLD in mice, but the molecular

5

underpinnings remain largely elusive. Arachidonic acid (AA) is a major ingredient of

6

animal fats, and AA cascade has been implicated in chronic inflammation. In this

7

study, we investigated whether genistein against NAFLD by targeting AA cascade.

8

By using a mouse model, we showed that genistein supplementation improved

9

high-fat diet induced NAFLD by normalizing hepatomegaly, liver steatosis,

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aminotransferase abnormalities and glucose tolerance. Thromboxane A2 (TXA2)

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pathway was aberrantly active in NAFLD, evidenced by an elevation of circulating

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TXA2 and hepatic thromboxane A2 receptor (TBXA2R) expression. Mechanistically,

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we found that genistein directly targeted cyclooxygenase-1 (COX-1) activity as well

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as its downstream TXA2 biosynthesis, while TXA2 pathway might mediate NAFLD

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progression by impairing insulin sensitivity. Taken together, our study revealed a

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crucial pathophysiological role of TXA2 pathway in NAFLD and provided an

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explanation as to how genistein against NAFLD progression.

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(Total 172 words)

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KEYWORDS: Aspirin, Genistein, NAFLD, TXA2

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INTRODUCTION

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Non-alcoholic fatty liver disease (NAFLD) is a global public health issue especially in

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developed countries1. For example, the prevalence rate of NAFLD is high increasing

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in Unite States and affects around a quarter of the general population. NAFLD might

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increase the risk of various chronic liver diseases such as nonalcoholic steatohepatitis,

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cirrhosis and end-stage liver failure and ever hepatocellular carcinoma2. However,

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NAFLD is usually asymptomatic at the early stage and thus is historically viewed

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with limited research interest. Despite all modern advances in medicine, yet no

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effective drug has received approval for NAFLD3. Therefore, great interest exists in

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identifying novel preventive agents against NAFLD, which is largely dependent upon

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better understanding the etiology of NAFLD.

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High-fat intake increases the risk of NAFLD, but the molecular underpinnings remain

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elusive. Arachidonic acid (AA) is a major ingredient of animal fats. Although

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cascade has been implicated in chronic inflammation4, whether it contribute to

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NAFLD is still unknown. Chemically, AA metabolites are mainly classified into

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prostanoids, leukotrienes and hydroxyeicosatetraenoic acids. Among AA derivatives

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study so far, thromboxane A2 (TXA2) recently drew our attention. For example, TXA2

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is capable of activating platelet, whereas platelet aggregation could conversely

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stimulateTXA2 biosynthesis. More recently, anti-platelet therapies were reported to

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lower plasma levels of hepatic transaminase and triglyceride in obese mice and

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NAFLD patients5. Insulin resistance has been implicated in the etiology of NAFLD

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via fat accumulation in hepatocytes6, while it was greatly improved upon the

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inhibition of thrombin action7. Furthermore, thromboxane A2 synthase (TBXAS1)

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inhibitor was found to potently suppress hepatitis C virus-associated liver infection

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and inflammation8. Taken together, accumulating evidence indicated that TXA2

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pathway might participate in NAFLD.

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Genistein (4',5,7-trihydroxyisoflavone), an isoflavonoid derived from soybean, was

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recently reported against obesity as well as NAFLD9-12, but its exact molecular

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remains largely understood. Recently, genistein were reported to inhibit the TXA2

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signaling pathway both in platelets and smooth muscle cells13, 14.

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those findings, we hypothesized that genistein might attenuate NAFLD by targeting

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TXA2 pathway.

On the basic of

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MATERIALS AND METHODS

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Materials, Chemicals and Reagents

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Primary antibodies against COX-1 (#4841) and COX-2 (#12282) were from Cell

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Signaling Technology (Beverly, MA). Primary antibodies against mPGES1 (#160140),

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TBXAS1 (#160715) and the thromboxane A2 receptor (TBXA2R, #1004452) were

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obtained from Cayman Chemical Company (Ann Arbor, MI). All other primary

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antibodies were from Abcam (Cambridge, U.K.). For plasma insulin and tumor

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necrosis factor-alpha (TNF-α) determination, enzyme immunoassay kits (#M0417,

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#M0030) were purchased from SenBeiJia Biological Technology (Nanjing, Jiangsu,

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China). TBXA2R small interference RNA (siRNA) and control siRNA were obtained

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from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). All chemicals were provided

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by Sigma-Aldrich (Shanghai, China) unless otherwise specified.

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Cell Culture

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Human liver tumor cell lines (Hep-G2) were obtained from ATCC and maintained

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following their instructions. Cells were cultured in DMEM medium containing 10%

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fetal bovine serum (FBS), 100 U/mL penicillin, and 100 mg/mL streptomycin. Before

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insulin resistance study, cells were serum-starved for 16 h in phenol red-free DMEM

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containing 0.1% charcoal-stripped FBS. Cells were pretreated with I-BOP, SQ29548

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or genistein for 2 hours, and then treated with TNF-α (6nM) for 20 min, and finally

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stimulated with insulin (100 nM) for 5min at 37 °C.

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RNA Interference

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Hep-G2 cells at 40–60% confluence were transiently transfected with small

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interfering RNA (siRNA) against TBXA2R or negative control siRNA using

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Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA) according to the

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manufacturer’s instructions.

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In Vitro COX Enzyme Assay

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The inhibitory effect of genistein on COX activity was evaluated using a COX

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Inhibitor Screening Kit from Cayman Chemical Company (Ann Arbor, MI) according

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to the manufacturer's instructions. Aspirin was used as positive control in this study.

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Plasma PG Determination

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The measurement of plasma PGs was performed using enzyme immunoassay kits

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from SenBeiJia Biological Technology. Considering the fact that PGD2, PGF2α, PGI2,

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and TXA2 are unstable in vivo, their corresponding primary metabolites in plasma

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were determined as follows: 11-beta-PGF2α (#M0801), 13,14-dihydro-15-keto-PGF2α

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(#M0802), 6-keto-PGF1α (#M0803), and TXB2 (#M0585).

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Western Blot Analysis

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Protein samples (20 µg) were resolved by SDS-PAGE and then transferred to Hybond

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C nitrocellulose membranes. The obtained membranes were further incubated with

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primary antibodies overnight at 4°C. After hybridization with a secondary antibody,

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the targeted protein bands were visualized by using an enhanced chemiluminescence

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reagent (GE Healthcare Biosciences, Pittsburgh, PA).The bands were quantified by

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scanning densitometry, and finally normalized with β-actin protein level and

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quantified by comparison with non-treated control as described previously15.

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High-fat Diet-induced NAFLD Murine Model

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All experiments were performed according to procedures approved by the animal

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experimentation ethics committee of Jiangnan University (protocol number: JN. No

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20170418-0930). A high-fat diet-induced NAFLD mouse model was adopted as

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described previously16. 7-week-old male C57BL/6 mice were divided into five groups

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(n = 10 per group) and ad libitum fed with control diet (1022, Beijing HFK

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Bioscience CO., LTD), high-fat diet (HFD, DIO-H10060, Beijing HFK Bioscience

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CO., LTD ) or high-fat diet supplemented with various dosages of genistein for 22 wk

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(Supplementary Table 1). Based on the food intake and genistein content in different

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groups, mice in Group1-5 were consumed genistein approximately at dose of 1.5, 0,

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32, 64, 0 mg·kg-1 body weight, respectively. For Group 5, mice were given aspirin

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(120mg·kg-1 body weight) in drinking water, taking into account mouse body weight

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and the volume of water consumed each day. Body weights, food intake and fasting

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blood glucose were measured once a week. The oral glucose tolerance test (OGTT)

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and insulin tolerance test (ITT) were performed at 19 and 20 wk respectively. At the

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last week of experiment, the metabolic rate and activity of mice were tested in

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individual cages in the Oxymax Comprehensive Laboratory Animal Monitoring

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System (CLAMS) for 24h through the light and dark cycle as described previously17,

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18

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for the estimation of oxygen consumption (VO2) and carbon dioxide production

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(VCO2). Respiratory exchange ratio (RER, the ratio of VCO2 to VO2) was adopted to

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estimate the fuel source for energy production, while activity was calculated by

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summing the X-axis and Z-axis movement counts.

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At the end of experiment, all mice were euthanatized after 12h fasting. Their organs

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and blood samples were collected. After gross examination, half of liver tissues was

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fixed in 10% buffered formalin for 24 h for histology studies, while the other half was

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kept in liquid nitrogen for western blot and cytokine determination. For histology

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study, fixed tissues were embedded in paraffin, sectioned at 6 µm, and stained with

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haematoxylin and eosin (H&E). Blood were centrifuged at 2,000×g for 15 minutes,

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and the supernatant fraction was designated as plasma. Plasma biomarkers such as

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total cholesterol(TG), triglyceride(TC), alanine aminotransferase (ALT), aspartate

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aminotransferase (AST) and creatinine (CREA) were determined by anautomatic

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biochemistry analyzer (BeckmanCX7, Chaska, MN).

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Statistical analysis

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Statistical analysis was performed by using the Prism 5.0 statistical package. Tukey’s

. Briefly, this system was sampled for 1 min and passed through O2 and CO2 sensors

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t-test was used to compare data between 2 groups. One-way ANOVA and the

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Bonferroni correction were used to compare data between 3 or more groups. Values

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are expressed as mean values ± SEM and a p value of < 0.05 was considered

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statistically significant.

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RESULTS

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Involvement of the TXA2Pathway in NAFLD

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To clarify the possibility of TXA2 pathway involved in NAFLD, we firstly examined

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the protein expression of cyclooxygenases as well as downstream enzymes of

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thromboxane metabolism. To mimic NAFLD progression in human being, a high-fat

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diet mouse model was adopted. As expected, high-fat intake for 22 wk successfully

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induced NAFLD, evidenced by swollen hepatocytes, adipose infiltration and

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hepatocellular macrovesicular vacuolationdue to lipid accumulation in liver (Figure

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1A). TXA2 is well-known to function through the activation of TBXA2R19, while

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TBXAS1 is a rate-limiting enzyme coupled with COXs in biosynthesis of TXA2. Our

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data clearly shown that both TBXA2R and TBXAS1 expression was markedly

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up-regulated in the fatty liver tissues compared with health ones (Figure 1B).

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Although widely accepted as a housekeeping gene, we noticed COX-1 was

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dramatically up-regulated in fatty liver tissues. By contract, COX-2, an immediate

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early response gene upon inflammation, only slightly increased in fatty liver. Overall,

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these findings indicated that the TXA2 pathway was aberrantly active during NAFLD

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progression.

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Targeting TXA2 Pathway Attenuates NAFLD Progression

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We then questioned whether TXA2 functionally mediated NAFLD. Drugs affecting

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TXA2 pathway is normally classified into three categories as following: COX

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inhibitors, TBXAS1 inhibitors and TBXA2R antagonists19. It is wide accepted that

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aspirin is a classic COX-1 inhibitor and exerts its anti-thrombosis activity by targeting

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COX-1/TXA2 biosynthesis20. Our data clearly indicated that aspirin potently

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suppressed high-fat diet-induced obesity, hyperlipidemia as well hyperglycemia (Figure

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2). Aspirin also effectively improved experimental NAFLD, evidenced by normalizing

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hepatomegaly, liver steatosis and aminotransferase abnormalities in obese mice

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(Figure 3). Phenotypically, similar results were obtained with genistein treatment.

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Importantly, NAFLD progression was accompanied by a pronounced elevation in

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circulating TXA2 levels, whereas it was normalized by either aspirin or genistein

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(Figure 4A). COX-1, but not COX-2, is thought to account for TXA2 biosynthesis in

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vivo21, 22. In this regards, our data indicated that genistein might share a similar

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mechanism of action with aspirin and preferentially target COX-1 rather than COX-2

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activity, especially at low doses (Figure 4B).

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Implications of TXA2Pathway in Insulin Resistance

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We next explored how TXA2 pathway drover NAFLD progression. Insulin resistance

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has been implicated in the etiology of NAFLD via fat accumulation in hepatocytes1.

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During this study performing, we noticed that switching off TXA2 biosynthesis by

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either aspirin or genistein greatly improved insulin sensitivity and glucose tolerance

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(Figure 5). Inspired by findings above, we wondered TXA2 pathway might be

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involved in insulin resistance (Figure 6). TNF-α is well-known as a key mediator of

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insulin resistance23. By using a cultured cell model, we showed that TNF-α

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pretreatment induced a insulin resistance effect, evidenced by a decrease in the

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insulin-stimulated phosphorylation of insulin receptor (IR) as well as insulin receptor

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substrate 1(IRS1). Importantly, such “insulin resistance” effect was greatly

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aggravated by TBXA2R agonist (I-BOP), whereas was reversed by TBXA2R

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antagonist (SQ29548), aspirin as well as genistein (Figure 6C and Supplementary

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Figure 1). To investigate whether the effects of genistein were mediated directly

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through TXA2 pathway, we further compared the effects of cells transfected with a

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control or si-TBXA2R plasmid. As shown in Figure 6C, genistein reversed TNF-α

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induced insulin resistance, but had less effect in si-TBXA2R cells. These data

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suggested that reversal of TNF-α-induced insulin resistance with genistein might be

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TBXA2R dependence.

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DISSCUSSION

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Non-alcoholic fatty liver disease (NAFLD) is now a public health issue worldwide,

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but no drug has received approval yet. This study revealed for the first time the

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critical role of the TXA2 pathway in NAFLD and laid the groundwork for introducing

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TXA2-targeting strategy against NAFLD. Mechanically, we provided direct evidence

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supporting the involvement of TXA2 pathway in insulin resistance,which acted as not

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only an integral feature but also a causal factor for NAFLD24. As such, TXA2 pathway

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might serve as a promising target for NAFLD management in the future.

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In the present study, we established that genistein, an isoflavonoid derived from

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soybean, effectively attenuated high fat diet-induced NAFLD progression. It is

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consistent with previous findings by others. Although several targets (such as AMPK,

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PPARγ, and the metabolic rate of resting muscle) have been proposed9-12, the direct

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evidence is still missing. Herein we provided direct evidence supporting that the

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hepaticprotective function of genistein might partly be explained by directly targeting

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COX-1 as well as its downstream TXA2 pathway. However, it should be pointed out that

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alternate mechanisms might also at play. For example, genistein greatly lowered the

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weight of the epididymal compartment(Figure 2B), a finding suggested that genistein

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might affect lipid metabolic pathway such as lipid mobilization and oxidation12.

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Genistein also affect the systemic energy balance (Figure 7), but the interpretation for

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such phenomenon is still missing25. Most likely, genisteinmight have multiple

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molecular targets and thus improve metabolic diseases across a multitude of

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inhibitory mechanisms such as free radical scavenging, insulin sensitization,

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anti-inflammation and modulation of steroidal hormone receptor.

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Although our findings in this study are exciting, several issues must to be addressed.

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For example, our data were mainly derived from animal studies. To verify whether it

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better serve the interest of NAFLD patients, more rigorous clinical studies need to be

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performed. We also observed that upon long-term intake aspirin, mice plasma TXA2

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levels were even less than control group (Figure 4A). Aspirin is well-known to exert

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its cardio-protective activity by inhibiting platelet-derived COX-1/TXA2 biosynthesis.

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Most likely, platelets might be a major source of TXA2 in the blood. Considering its

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key roles in platelet aggregation and wound repair, interfering TXA2 pathway had

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better be applied judiciously at discrete stages as a short-term intermittent therapy by

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monitoring platelet counts. Another issue is that the molecular mechanism underlying

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TXA2 promoting NAFLD progression might remain unclear. How TXA2 pathway

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exactly functionally mediated insulin resistance remains unknown. We also did not

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know whether a crosstalk between TXA2 and TNF-α pathway in insulin resistance

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exists. Notably, we observed that knockdown of TBXA2R greatly amplified insulin

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signaling. It strongly indicated that TXA2 pathway itself might contribute to insulin

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resistance by impinging on insulin signaling, and thus TXA2 pathway might represent

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as a novel target for insulin sensitization. To gain a deeper insight into the role of the

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TXA2 pathway in NAFLD, further studies examining susceptibility to lipotoxicity in

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mice with liver deletions in specific TXA2 synthase and/or receptor are greatly needed.

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Furthermore, the potential contribution of other prostaglandins such as PGD2, PGE2 and

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PGF2α in NAFLD remains unexplored. In addition, we noticed that aspirin and

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genistein lowered plasma levels of insulin and TNF-α in this study (Figure 6). It

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pointed to alternate mechanisms at play, but the interpretation for such phenomenon is

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still missing.

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In summary, this study provided direct evidence supporting that TXA2, an AA

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metabolite, might link high-fat intake and NAFLD pathophysiology. Genistein, an

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isoflavonoid derived from soybean, might improve NAFLD, at least partially, by

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targeting TXA2 pathway. Considering its low price and widespread sources in nature,

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dietary supplement of genistein might have a strong potential in practical use in NAFLD

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management, especially in those patients with obesity.

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(Total 2330 words)

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ACKNOWLEDGEMENTS

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

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(81773064, 31601450 and 31530056), National Youth 1000 Talents Plan, the Jiangsu

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Specially-Appointed Professor Program, National first-class discipline program of

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Food Science and Technology (JUFSTR20180102) and Collaborative innovation

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center of food safety and quality control in Jiangsu Province.

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6-C-(E-phenylethenyl)-naringenin

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colorectal

cancer

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by

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cyclooxygenase-1. Cancer Res 2014, 74, 243-52. 22. Pedersen, A. K.; FitzGerald, G. A., Dose-related kinetics of aspirin. Presystemic acetylation of platelet cyclooxygenase. N Engl J Med 1984, 311, 1206-11. 23. Hotamisligil, G. S.; Murray, D. L.; Choy, L. N.; Spiegelman, B. M., Tumor necrosis factor alpha inhibits signaling from the insulin receptor. Proc Natl Acad Sci U S A 1994, 91, 4854-8. 24. Axelsson, A. S.; Tubbs, E.; Mecham, B.; Chacko, S.; Nenonen, H. A.; Tang, Y.; Fahey, J. W.; Derry, J. M. J.; Wollheim, C. B.; Wierup, N.; Haymond, M. W.; Friend, S. H.; Mulder, H.; Rosengren, A. H., Sulforaphane reduces hepatic glucose production and improves glucose control in patients with type 2 diabetes. Sci Transl Med 2017, 9. 25. Nogara, L.; Naber, N.; Pate, E.; Canton, M.; Reggiani, C.; Cooke, R., Piperine's mitigation of obesity and diabetes can be explained by its up-regulation of the metabolic rate of resting muscle. Proc Natl Acad Sci U S A 2016, 113, 13009-13014.

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Figure Legends:

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Figure 1. TXA2 pathway was aberrantly active during NAFLD progression. A,

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Hepatic histology. Histological studies were based on H&E staining as described.

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Original magnification: 200 ×. B, Effect of high-fat intake on COXs/TXA2 cascade in

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liver tissues. Age-matched wild-type C57BL/6 were grouped and fed with control

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normal chow (NC) or high-fat diet (HFD)for total 22 wk. The expression pattern of

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COXs/ TXA2 cascade in liver was examined by Western blot. Data are presented a

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representative experiment performed in triplicate.

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Figure 2. Effects of genistein on high-fat diet induced metabolic syndrome. A, Body

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weight. B, Epididymis adipose weight. C, Plasma cholesterol. D, Plasma triglyceride.

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Data are presented as mean ± SEM (n=10). The asterisks indicate a significant

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difference compared with each respective vehicle group (*, p < 0.05; ***, p < 0.001).

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Figure 3. Effects of genistein on high-fat diet induced fatty liver diseases. A, Liver

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weight. B, Liver index. C, Plasma alanine aminotransferase (ALT) activity. D, Plasma

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aspartate aminotransferase (AST) activity. Data are presented as mean ± SEM (n =

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10). The asterisks indicate a significant difference compared with each respective

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vehicle group (**, p < 0.01; ***, p < 0.001). E, Hepatic histology. Histological

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studies were based on H&E staining as described. Original magnification: 200 ×.

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Figure 4. Effect of genistein on arachidonic acid cascade. A, Genistein affects on the

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profile of circulating PG biosynthesis in obese mice. Data are presented as mean ±

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SEM (n=10). The asterisks indicate a significant difference compared with each

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respective vehicle group (***, p < 0.001). B, Genistein inhibits COX activity in vitro.

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The inhibitory activity of genistein was evaluated using a COX Inhibitor Screening

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Kit (Cayman) according to the manufacturer's instructions. Data are presented as

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mean values ± SEM (n = 4). The asterisks indicate a significant difference compared

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with each respective vehicle group (*, p < 0.05;**, p < 0.01; ***, p < 0.001).

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Figure 5. Effects of genistein on high-fat diet induced hyperglycemia. A, Fasting

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blood glucose. B, Oral glucose tolerance test. For oral glucose tolerance test (GTT),

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2g/kg glucose was administered by gavage after an overnight fasting. C, Insulin

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tolerance test. For insulin tolerance test (ITT), 0.4U/kg insulin was injected

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intraperitoneally after 8h fasting. Data are presented as mean ± SEM (n=10). The

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asterisks indicate a significant difference compared with each respective vehicle

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group (***, p < 0.001).

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Figure 6. TXA2 pathway functionally mediates insulin resistance. A, Effects of

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genistein on high-fat diet induced hyperinsulinemia. B, Plasma TNFα concentrations.

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Data are presented as mean ± SEM (n=10). The asterisks indicate a significant

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difference compared with each respective vehicle group (***, p < 0.001). C,

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Involvement of TXA2 pathway in insulin resistance. After RNA interference, Hep-G2

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cells were serum starved for 16h, and then were treated with TNF-α (6nM) with

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I-BOP (1uM), SQ29548 (1uM) and genistein (100uM) for 5h. Cells were then

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stimulated with insulin (5uM) for 30min, and finally subjected to Western blot.

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Figure 7. Effects of genistein on systemic energy balance. A, Food intake. B,

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Respiratory exchange rate (RER). C, Daily physical activity counts. Data are

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presented as mean ± SEM (n=10). The asterisks indicate a significant difference

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compared with each respective vehicle group (*, p < 0.05;**, p < 0.01; ***, p