Genistein Ameliorates Non-alcoholic Fatty Liver ... - ACS Publications

Subscriber access provided by Kaohsiung Medical University

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

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 26

Journal of Agricultural and Food Chemistry

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]

Fax:

+86-510-85197302,

Email:

Fax:

+86-510-85329081,

Email:

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.

ACS Paragon Plus Environment


1

ABSTRACT

2

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,

10

aminotransferase abnormalities and glucose tolerance. Thromboxane A2 (TXA2)

11

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

12

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

13

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

14

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

15

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

16

crucial pathophysiological role of TXA2 pathway in NAFLD and provided an

17

explanation as to how genistein against NAFLD progression.

18

(Total 172 words)

19 20

KEYWORDS: Aspirin, Genistein, NAFLD, TXA2

21


Page 2 of 26

Page 3 of 26


22

INTRODUCTION

23

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

24

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

25

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

26

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

27

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

28

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

29

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

30

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

31

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

32

better understanding the etiology of NAFLD.

33

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

34

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

35

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

36

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

37

prostanoids, leukotrienes and hydroxyeicosatetraenoic acids. Among AA derivatives

38

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

39

is capable of activating platelet, whereas platelet aggregation could conversely

40

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

41

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

42

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

43

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

44

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


AA


Page 4 of 26

45

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

46

and inflammation8. Taken together, accumulating evidence indicated that TXA2

47

pathway might participate in NAFLD.

48

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

49

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

50

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

51

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

52

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

53

TXA2 pathway.

On the basic of

54 55

MATERIALS AND METHODS

56

Materials, Chemicals and Reagents

57

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

58

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

59

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

60

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

61

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

62

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

63

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

64

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

65

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

66

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


Page 5 of 26


67

Cell Culture

68

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

69

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

70

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

71

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

72

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

73

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

74

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

75

RNA Interference

76

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

77

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

78

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

79

manufacturer’s instructions.

80

In Vitro COX Enzyme Assay

81

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

82

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

83

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

84

Plasma PG Determination

85

The measurement of plasma PGs was performed using enzyme immunoassay kits

86

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

87

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

88

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



89

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

90

Western Blot Analysis

91

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

92

C nitrocellulose membranes. The obtained membranes were further incubated with

93

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

94

the targeted protein bands were visualized by using an enhanced chemiluminescence

95

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

96

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

97

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

98

High-fat Diet-induced NAFLD Murine Model

99

All experiments were performed according to procedures approved by the animal

100

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

101

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

102

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

103

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

104

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

105

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

106

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

107

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

108

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

109

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

110

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


Page 6 of 26

Page 7 of 26


111

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

112

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

113

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

114

individual cages in the Oxymax Comprehensive Laboratory Animal Monitoring

115

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

116

18

117

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

118

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

119

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

120

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

121

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

122

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

123

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

124

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

125

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

126

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

127

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

128

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

129

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

130

biochemistry analyzer (BeckmanCX7, Chaska, MN).

131

Statistical analysis

132

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



133

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

134

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

135

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

136

statistically significant.

137 138

RESULTS

139

Involvement of the TXA2Pathway in NAFLD

140

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

141

the protein expression of cyclooxygenases as well as downstream enzymes of

142

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

143

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

144

induced NAFLD, evidenced by swollen hepatocytes, adipose infiltration and

145

hepatocellular macrovesicular vacuolationdue to lipid accumulation in liver (Figure

146

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

147

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

148

data clearly shown that both TBXA2R and TBXAS1 expression was markedly

149

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

150

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

151

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

152

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

153

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

154

progression.


Page 8 of 26

Page 9 of 26


155

Targeting TXA2 Pathway Attenuates NAFLD Progression

156

We then questioned whether TXA2 functionally mediated NAFLD. Drugs affecting

157

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

158

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

159

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

160

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

161

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

162

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

163

hepatomegaly, liver steatosis and aminotransferase abnormalities in obese mice

164

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

165

Importantly, NAFLD progression was accompanied by a pronounced elevation in

166

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

167

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

168

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

169

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

170

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

171

Implications of TXA2Pathway in Insulin Resistance

172

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

173

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

174

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

175

either aspirin or genistein greatly improved insulin sensitivity and glucose tolerance

176

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



177

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

178

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

179

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

180

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

181

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

182

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

183

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

184

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

185

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

186

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

187

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

188

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

189

TBXA2R dependence.

190 191

DISSCUSSION

192

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

193

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

194

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

195

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

196

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

197

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

198

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


Page 10 of 26

Page 11 of 26


199

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

200

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

201

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

202

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

203

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

204

hepaticprotective function of genistein might partly be explained by directly targeting

205

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

206

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

207

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

208

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

209

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

210

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

211

molecular targets and thus improve metabolic diseases across a multitude of

212

inhibitory mechanisms such as free radical scavenging, insulin sensitization,

213

anti-inflammation and modulation of steroidal hormone receptor.

214

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

215

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

216

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

217

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

218

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

219

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

220

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



221

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

222

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

223

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

224

TXA2 promoting NAFLD progression might remain unclear. How TXA2 pathway

225

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

226

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

227

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

228

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

229

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

230

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

231

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

232

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

233

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

234

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

235

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

236

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

237

still missing.

238

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

239

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

240

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

241

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

242

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


Page 12 of 26

Page 13 of 26


243

management, especially in those patients with obesity.

244

(Total 2330 words)

245 246

ACKNOWLEDGEMENTS

247

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

248

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

249

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

250

Food Science and Technology (JUFSTR20180102) and Collaborative innovation

251

center of food safety and quality control in Jiangsu Province.



252

References

253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294

1.

Angulo, P., Nonalcoholic fatty liver disease. N Engl J Med 2002, 346, 1221-31.

2.

Lin, H. Z.; Yang, S. Q.; Chuckaree, C.; Kuhajda, F.; Ronnet, G.; Diehl, A. M., Metformin reverses

fatty liver disease in obese, leptin-deficient mice. Nat Med 2000, 6, 998-1003. 3.

Wang, X.; Zhang, X.; Chu, E. S. H.; Chen, X.; Kang, W.; Wu, F.; To, K. F.; Wong, V. W. S.; Chan, H. L. Y.;

Chan, M. T. V.; Sung, J. J. Y.; Wu, W. K. K.; Yu, J., Defective lysosomal clearance of autophagosomes and its clinical implications in nonalcoholic steatohepatitis. FASEB J 2018, 32, 37-51. 4.

Wang, D.; Dubois, R. N., Eicosanoids and cancer. Nat Rev Cancer 2010, 10, 181-93.

5.

Fujita, K.; Nozaki, Y.; Wada, K.; Yoneda, M.; Endo, H.; Takahashi, H.; Iwasaki, T.; Inamori, M.; Abe,

Y.; Kobayashi, N.; Kirikoshi, H.; Kubota, K.; Saito, S.; Nagashima, Y.; Nakajima, A., Effectiveness of antiplatelet drugs against experimental non-alcoholic fatty liver disease. Gut 2008, 57, 1583-91. 6.

Yuan, M.; Konstantopoulos, N.; Lee, J.; Hansen, L.; Li, Z. W.; Karin, M.; Shoelson, S. E., Reversal of

obesity- and diet-induced insulin resistance with salicylates or targeted disruption of Ikkbeta. Science 2001, 293, 1673-7. 7.

Mihara, M.; Aihara, K.; Ikeda, Y.; Yoshida, S.; Kinouchi, M.; Kurahashi, K.; Fujinaka, Y.; Akaike, M.;

Matsumoto, T., Inhibition of Thrombin Action Ameliorates Insulin Resistance in Type 2 Diabetic db/db Mice. Endocrinology 2010, 151, 513-519. 8.

Abe, Y.; Aly, H. H.; Hiraga, N.; Imamura, M.; Wakita, T.; Shimotohno, K.; Chayama, K.; Hijikata, M.,

Thromboxane A2 synthase inhibitors prevent production of infectious hepatitis C virus in mice with humanized livers. Gastroenterology 2013, 145, 658-67 e11. 9.

Amanat, S.; Eftekhari, M. H.; Fararouei, M.; Bagheri Lankarani, K.; Massoumi, S. J., Genistein

supplementation improves insulin resistance and inflammatory state in non-alcoholic fatty liver patients: A randomized, controlled trial. Clin Nutr 2017. 10. Jeon, S.; Park, Y. J.; Kwon, Y. H., Genistein alleviates the development of nonalcoholic steatohepatitis in ApoE(-/-) mice fed a high-fat diet. Mol Nutr Food Res 2014, 58, 830-41. 11. Kim, M. H.; Kang, K. S.; Lee, Y. S., The inhibitory effect of genistein on hepatic steatosis is linked to visceral adipocyte metabolism in mice with diet-induced non-alcoholic fatty liver disease. British Journal of Nutrition 2010, 104, 1333-1342. 12. Miller, C. N.; Yang, J. Y.; Avra, T.; Ambati, S.; Della-Fera, M. A.; Rayalam, S.; Baile, C. A., A Dietary Phytochemical Blend Prevents Liver Damage Associated with Adipose Tissue Mobilization in Ovariectomized Rats. Obesity 2015, 23, 112-119. 13. Guerrero, J. A.; Lozano, M. L.; Castillo, J.; Benavente-Garcia, O.; Vicente, V.; Rivera, A., Flavonoids inhibit platelet function through binding to the thromboxane A(2) receptor. Journal of Thrombosis and Haemostasis 2005, 3, 369-376. 14. Nakashima, S.; Koike, T.; Nozawa, Y., Genistein, a Protein Tyrosine Kinase Inhibitor, Inhibits Thromboxane-A2-Mediated Human Platelet Responses. Molecular Pharmacology 1991, 39, 475-480. 15. Tatsumi, Y.; Cho, Y. Y.; He, Z.; Mizuno, H.; Seok Choi, H.; Bode, A. M.; Dong, Z., Involvement of the paxillin pathway in JB6 Cl41 cell transformation. Cancer Res 2006, 66, 5968-74. 16. Chen, Y. K.; Cheung, C.; Reuhl, K. R.; Liu, A. B.; Lee, M. J.; Lu, Y. P.; Yang, C. S., Effects of green tea polyphenol (-)-epigallocatechin-3-gallate on newly developed high-fat/Western-style diet-induced obesity and metabolic syndrome in mice. J Agric Food Chem 2011, 59, 11862-71. 17. Choi, H. M.; Kim, H. R.; Kim, E. K.; Byun, Y. S.; Won, Y. S.; Yoon, W. K.; Kim, H. C.; Kang, J. G.; Nam, K. H., An age-dependent alteration of the respiratory exchange ratio in the db/db mouse. Lab Anim


Page 14 of 26

Page 15 of 26


295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317

Res 2015, 31, 1-6. 18. Marvyn, P. M.; Bradley, R. M.; Mardian, E. B.; Marks, K. A.; Duncan, R. E., Data on oxygen consumption rate, respiratory exchange ratio, and movement in C57BL/6J female mice on the third day of consuming a high-fat diet. Data Brief 2016, 7, 472-5. 19. Nakahata, N., Thromboxane A2: physiology/pathophysiology, cellular signal transduction and pharmacology. Pharmacol Ther 2008, 118, 18-35. 20. Li, H.; Liu, K.; Boardman, L. A.; Zhao, Y.; Wang, L.; Sheng, Y.; Oi, N.; Limburg, P. J.; Bode, A. M.; Dong, Z., Circulating Prostaglandin Biosynthesis in Colorectal Cancer and Potential Clinical Significance. EBioMedicine 2015, 2, 165-171. 21. Li, H.; Zhu, F.; Chen, H.; Cheng, K. W.; Zykova, T.; Oi, N.; Lubet, R. A.; Bode, A. M.; Wang, M.; Dong, Z.,

6-C-(E-phenylethenyl)-naringenin

suppresses

colorectal

cancer

growth

by

inhibiting

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.

318 319



320

Figure Legends:

321 322

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

323

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

324

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

325

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

326

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

327

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

328

representative experiment performed in triplicate.

329 330

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

331

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

332

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

333

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

334 335

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

336

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

337

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

338

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

339

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

340

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

341


Page 16 of 26

Page 17 of 26


342

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

343

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

344

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

345

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

346

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

347

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

348

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

349

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

350

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

351

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

352

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

353

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

354

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

355

asterisks indicate a significant difference compared with each respective vehicle

356

group (***, p < 0.001).

357 358

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

359

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

360

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

361

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

362

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

363

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



364

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

365

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

366 367

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

368

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

369

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

370

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

Genistein Ameliorates Non-alcoholic Fatty Liver ... - ACS Publications

Recommend Documents