Oryzanol Modifies High Fat Diet-Induced Obesity, Liver Gene

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Oryzanol modifies high fat dietary (HFD)-induced obesity, expression profile and inflammation response of hepatic tissues in mice Long Wang, Qinlu Lin, Tao Yang, Ying Liang, Yi Luo, Ying Nie, Junjun Shen, Xiangjin Fu, Yiping Tang, and Feijun Luo J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03230 • Publication Date (Web): 30 Aug 2017 Downloaded from http://pubs.acs.org on August 31, 2017

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Journal of Agricultural and Food Chemistry

1 Title: 2 3

Oryzanol modifies high fat diet (HFD)-induced obesity, liver gene

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expression profile and inflammation response in mice

5 †,‡

Long Wang , Qinlu Lin

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†,‡,*

†

†

†

†

Xiangjin Fu , Yiping Tang , Feijun Luo

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†

§

†

, Tao Yang , Yin Liang , Ying Nie , Yi Luo , Junjun Shen , †,*

8 9

†

Laboratory of Molecular Nutrition, College of Food Science and Engineering; The Key

10 Laboratory of Hunan Province for Special Medical Food, National Engineering 11 Laboratory for Deep Processing of Rice and Byproducts, Central South University of 12 Forestry and Technology, Changsha, Hunan 410004, China. 13

§

Department of Clinic Medicine, Xiangya School of Medicine, Central South University,

14 Changsha, Hunan 410008, China. 15 16 17 18 19 20 21 22

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ACS Paragon Plus Environment


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ABSTRACT: In Western countries and China, dietary habit of high calorie usually

24 results in hyperlipidemia, which is closely associated with cardiovascular diseases. In the 25 study, we investigated the anti-hyperlipidemic effect of oryzanol and its molecular 26 mechanism in high fat diet (HFD) mouse model. 60 ICR mice were randomly divided into 27 control group, HFD group and HFD+Ory group. The mice from HFD+Ory group were 28 additionally fed with 100 mg/kg of oryzanol by intragastric administration. Our data 29 indicated that oryzanol treatment for 10 weeks significantly reduced bodyweight, liver 30 weight and adipose tissues weight of mice; and lowered the contents of total cholesterol 31 (TC), triglycerides (TG) and low density lipoprotein–cholesterol (LDL-C) and elevated 32 high density lipoprotein–cholesterol (HDL-C) in the plasma of HFD mice. Compared with 33 HFD group, H&E staining showed that oryzanol treatment decreased size of fat droplets of 34 liver tissues and size of adipocytes. Gene chip data found that oryzanol administration 35 caused 32 genes were increased expressions while 60 genes were reduced expressions in 36 the liver tissues of HFD mice. IPA software was used to analyze the protein interaction 37 network and found that transcript factor NF-κB located in the central role of network, 38 meaning NF-κB may have important function in the lipid-lowering effect of oryzanol. 39 Western blotting and RT-qPCR confirmed that lipid metabolism-related gene expressions 40 were obviously regulated by oryzanol administration. Oryzanol also inhibited expressions 41 of inflammatory factor in the liver tissues of HDF mice. Taken together, our data indicate 42 that oryzanol treatment can regulated lipid metabolism-related gene expressions and 43 inhibit HDF-caused obesity in mice. 44 KEYWORDS: oryzanol, hyperlipidemia, gene chip, NF-κB, lipid metabolism 45 2


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INTRODUCTION Hyperlipidemia and obesity are the risk factors resulting in the different

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48 cardiovascular diseases (CVD), type 2 diabetes mellitus, hypertension and atherosclerosis . 49 Hyperlipidemia is abnormal of body fat metabolism and plasma cholesterol, high 50 triglycerides, low-density lipoprotein levels exceeds the normal level, which is a common 51 symptom of multiple cardiovascular diseases. Hyperlipidemia can cause an increase of 52 blood viscosity within the blood vessels and cause blood rheology changes; it is the 2-4

53 important factor in the atherosclerosis formation . More and more phytochemicals were 54 investigated to lower blood lipid in the obesity mice and ameliorated hyperlipidemic 55 syndrome

5-7

. Phytochemicals provide new clue to prevent hyperlipidemia-related diseases.

Oryzanol consists of a mixture of phytosteryl ferulates and cycloartenyl ferulate

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57 (CAF).

Medical

functions

of

oryzanol

include

antioxidant,

anti-ulcerogenic,

58 anti-neoplastic, anti-diabetic and anti-allergic role, but the molecular mechanism is not 8-10

59 clear

. Several publications indicated that oryzanol and oryzanol-rich food could have

60 anti-hyperlipidemic effect. Son MJ et al. showed that addition of oryzanol or ferulic acid 61 in the diet inhibited high fat diet (HFD)-induced hyperlipidemia and oxidative stress in 62 mice, and oryzanol and ferulic acid could be effective in decreasing the risk of 11

63 HFD-induced obesity . Oryzanol and ferulic acid also significantly decreased the blood 64 glucose

level,

enzyme

activities

of

glucose-6-phosphatase

(G6pase)

and

65 phosphoenolpyruvate carboxykinase (PEPCK) in the HFD mice, but the mechanism is not 12

66 known . In hypercholesterolemic diet (HCD) hamster model, oral administration of 67 ferulic acid and oryzanol diets obviously decreased lipid content of plasma comparing

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68 with the control diet mice . All of those evidences show that oryzanol has lowering-lipid 69 effect, but the molecular mechanisms need to further explore. 70

So far, only few of publications were referred to the molecular mechanisms of

71 oryzanol. In HFD rat model, germinated brown rice (GBR) and GBR-derived gamma 72 oryzanol-rich extract increased insulin resistance of rat. The global DNA methylation of 73 genomics was lower in the mice from GBR groups and epigenetic modifications were 14

74 occurred in the pregnant rats and their offsprings . Oryzanol treatment significantly 75 lowered hepatic TG content, serum levels of C-reactive protein and interleukin 6 (IL-6), 76 and increased serum concentration of adiponectin. Oryzanol also decreased the 77 expressions of stearoyl coenzyme-A desaturase-1 in the oleic acid-stimulated HepG2 15

78 cells . Our recent data approved that oryzanol decreased expressions of inflammatory 79 factors interleukin 1β (IL-1β), tumor necrosis factor α (TNF-α), IL-6 and iNOS in 16

80 lipopolysaccharide (LPS)-stimulated macrophage , and it also inhibited dextran sulphate 17

81 sodium (DSS)-induced colitis in mice . In this study, HFD mouse model was used to 82 assess the anti-hyperlipidemic effect

of oryzanol and if oryzanol can inhibit

83 obesity-induced chronic inflammation. Gene chip was used to analyze the gene expression 84 profile alterations of liver tissues after oryzanol treatment in the HFD mice. 85 Bioinformatics was used to analyze the possible signal pathways participated in the 86 anti-hyperlipidemia effect of oryzanol and lipid metabolism-related key gene expressions 87 were also assessed by Western blotting and RT-qPCR. Our experiments provide a series of 88 new clues to explain the molecular mechanism of the anti-hyperlipidemia function of 89 oryzanol.

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MATERIALS AND METHODS Feed of Control and HFD Mice. Basic diet for mice (SLAC, Changsha, Hunan,

93 China) included wheat 38.0%, maize 20.0%, soybean powder 18.0%, fish powder 10.0%, 94 wheat bran 5.0%, soybean oil 3.0%, maltodextrin 2.0%, and other 2.0% of minerals and 95 vitamins. HFD was included 79.6% basic diet adding 1.0% cholesterol, 0.2% bile acid 96 sodium, 0.2% propylthiouracil, 10.0% lard, 5.0% egg yolk powder, 4.0% whole milk 97 powder, which contains about 54.5% carbohydrates, 19.5% protein and 24% fat in the 98 high fat diet. 99

Animal Experiments. All experiments involving animals were reviewed and

100 approved by the Guidelines for the Care and Use of Experimental Animals, the School of 101 Medicine, Hunan Normal University (SYXK-Xiang, 2014-0008). All mice used in the 102 experiment were housed in an air-conditioned animal room under specific pathogen-free 103 conditions with a 12h: 12h light-dark phase and free access to water and food. Wild-type 104 littermates of ICR male mice (about 7-8 weeks of age) were housed in different cages (2-3 105 mice per cage) and maintained at 23 ± 2℃ environment. After about 7-10 day adaption 106 process in animal unit, mice were steady in eating food and water and then the mice were 107 used to do experiment. 108

Oryzanol Treatment. Oryzanol was purchased from OSHA Haz Company, Japan

109 and the purity is more than 99%. Oryzanol was completely dissolved in 95℃ corn oil 110 (Sigma, USA) and control group used corn oil as vehicle. The mice of control group were 111 fed with basic diet, while mice of other two groups were fed with high fat diet. The mice

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112 from HFD+Ory group were orally administrated with 100 mg/kg of oryzanol 3 days 113 before feeding high fat diet and mice from control group and HFD group were orally 114 administrated with equal volume of corn oil. After HFD treatment for 11 weeks, mice 115 were sacrificed after overnight fasting for 15 h, the blood samples and liver tissues were 116 collected and stored at -80℃. The samples were used for biochemical assay and 117 extractions of RNA and protein. Parts of liver tissues and fat pad tissues were preserved in 118 4% polyoxymethylene and were cut 5 µm thick paraffin sections for H&E staining. 119

Analysis of Blood Samples. All blood samples were collected by venipuncture from

120 an antecubital vein using anticoagulant solution of heparin and ethylenediaminetetraacetic 121 acid (EDTA), then the samples were immediately centrifuged to separate serum of mice. 122 Standard plasma parameters of high-density lipoprotein cholesterol (HDL-C), low-density 123 lipoprotein cholesterol (LDL-C), total cholesterol (TC) and triglycerides (TG) were 124 assayed. 125

Detection of Fat Content in the Liver Tissues. Fat content of liver tissues is 18

126 detected according to the protocol of Peng LV et al. protocol . Briefly, 0.5 g liver tissues 127 were mixed with anhydrous sodium sulphate and were grinded into powder. The mixture 128 was then put into soxhlet extractor, and anhydrous diethyl ether was used to extract the 129 liver fat. 130

Liver and Fat Pad Histology. The liver tissue and fat tissue were cut and fixed in

131 10.0% (100 g/L) formalin solution for 24 h, and the samples were dehydrated with 132 increasing concentrations of ethanol and transparented in xylene. The samples were 133 embedded in melt paraffin and were cut into 5 µm slices on a microtome, and then the

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134 sections were placed on clean glass slides and dried at 37°C overnight. The tissue sections 135 were dewaxed in xylene for 5-10 min twice and dehydrated in different concentrations of 136 ethanol for 2-5 min. These sections were stained with hematoxylin and eosin (H&E) and 137 were observed under the microscope. 138

RNA Isolation and Array Hybridization. Total RNA was extracted from liver

139 tissues according to protocol of Transzol Up kit (Transgen, Beijing, China). RNA quantity 140 and quality were detected by NanoDrop ND-1000. RNA integrity was evaluated by 1.5% 141 agarose gel electrophoresis. About 5 µg of total RNA was reversed transcribed into 142 single-strand cDNA and then synthesized double-strand cDNA according to the protocol 143 of SuperScript ds-cDNA synthesis kit (Invitrogen, Carlsbad, CA). Double-strand cDNA 144 was labelled with fluorescein according to the protocol of NimbleGen Gene Expression 145 Analysis (Nimblegen, Madison, WI, USA). Briefly, double-strand cDNA was added with 146 4.0 µg RNase A and was incubated at 37°C for 10 min, and then double-strand cDNA was 147 purified by mixture solution of phenol, chloroform and isoamyl alcohol. Cy3 was used to 148 label cDNA samples according to the protocol of NimbleGen One-Colour DNA labelling 149 kit (NimbleGen, Madison, WI, USA). Briefly, 1.0 µg double-strand cDNA was added 1.0 150 OD of Cy3-9mer primer and was incubated for about 10 min at 98°C. Then, 100.0 pmol of 151 deoxynucleoside triphosphates and 100.0 U of the Klenow enzyme (New England Biolabs, 152 Beverly, MA, USA) were added and were incubated at 37°C for 2 h. 0.1 volume of 0.5 M 153 EDTA solution was added tubes and stopped reactions. Cy3-labelled cDNA was purified 154 by isopropanol/ethanol precipitation. 4.0 µg of Cy3 labelled cDNA and component A were 155 added to hybridization buffer, and the mixture was added to gene chips for hybridization at

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156 42°C for 18 h. Following hybridization and washing, the slides were scanned using the 157 Axon GenePix 4000B microarray scanner. Differentially expressed genes were screened 158 by more than 2 fold or less than 0.5 fold while student’s t-test was used to further screen 159 altered expression genes between control group and HFD group (p

Oryzanol Modifies High Fat Diet-Induced Obesity, Liver Gene

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