Isoorientin Prevents Hyperlipidemia and Liver Injury by Regulating

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Isoorientin prevents hyperlipidaemia and liver injury by regulating lipid metabolism, antioxidant capability and inflammatory cytokine release in high-fructose-fed mice Li Yuan, Xiao Han, Wenfeng Li, Daoyuan Ren, and Xing-Bin Yang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b00290 • Publication Date (Web): 09 Mar 2016 Downloaded from http://pubs.acs.org on March 13, 2016

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Journal of Agricultural and Food Chemistry

Isoorientin prevents hyperlipidaemia and liver injury by regulating

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lipid metabolism, antioxidant capability and inflammatory cytokine release in

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high-fructose-fed mice

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Li Yuan*, Xiao Han, Wenfeng Li, Daoyuan Ren, Xingbin Yang*

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College of Food Engineering and Nutritional Science, Shaanxi Normal University, Xi’an 710119, China

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*Corresponding author: Li Yuan, Ph. D., Xian, Shaanxi, 710119, China.

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Tel./fax: 029-85310517; email: [email protected]

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*Corresponding author: Xingbin Yang, Ph. D., Xian, Shaanxi, 710119, China. Tel./fax: 029-85310517; email: [email protected]

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Abbreviations: ISO, isoorientin; HF, high fructose; TG, total triglyceride; TC, total

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cholesterol; HDL-C, high-density lipoprotein-cholesterol; LDL-C, low-density

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lipoprotein-cholesterol; VLDL, very low-density lipoprotein-cholesterol; ALT,

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alanine

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alkaline phosphatase; T-SOD, total superoxide dismutase; GSH-Px, glutathione

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peroxidase; MDA, malondialdehyde; apoA-Iapolipoprotein A1; apoB, apolipoprotein

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B; IL-6, interleukin 6; IL-1,interleukin 1; FAS, fatty acid synthase.

aminotransferase;

AST,

aspartate

aminotransferase;

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ACS Paragon Plus Environment

ALP,


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ABSTRACT

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Isoorientin (ISO), a natural flavonoid, has been found to have multiple biological

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properties. In the present study, obese mice with high-fructose (HF)-induced liver

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injury were used to investigate the hepatoprotective effects of ISO. The results

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showed that ISO significantly reduced the serum lipid parameters in the mice fed 20%

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HF water. Meanwhile, ISO appeared to alleviate HF-induced lipid metabolic disorders

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by increasing the serum levels of apoA-I and decreasing the serum apoB levels,

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apoB/apoA-I ratio and FAS activity in the liver. ISO also remarkably ameliorated

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HF-induced hepatic oxidative injury and inflammation by decreasing ALT, AST and

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ALP levels; enhancing antioxidant enzyme activities; and inhibiting inflammatory

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cytokine (TNF-α, IL-1, IL-6) release. Histopathology of liver stained by H&E and Oil

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Red O showed the liver steatosis and oxidative injury after HF treatment and the

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protective effect of ISO. Furthermore, aortic pathology observation found that ISO

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had a protective effect on the vascular endothelium. This is the first report that ISO

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efficiently inhibited HF-induced hyperlipidaemia and liver injury by ameliorating

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lipid metabolism, enhancing the antioxidant defensedefence system and regulating the

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secretion of inflammatory cytokines.

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isoorientin,

hyperglycaemia,

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KEYWORDS:

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lipid metabolism, hepatic inflammation

liver

damage,

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oxidative

stress,

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INTRODUCTION

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Fructose is widely used in the food industry as high-fructose syrup which has

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55-90% of fructose, and its consumption has been increasing significantly in recent

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years.1 However, many evidences show that a high-fructose intake can

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ultimately result in metabolic disorders, including obesity, insulin resistance,

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hypertension, hyperlipidaemia and hyperglycaemia.2,3 Moreover, the HF diet also

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results in inflammation, oxidative damage and hepatic steatosis.

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number of studies have suggested that the natural oligosaccharides, flavonoids and

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alkaloids from plants, such as (-)-epocatechin, 6 stachyose and tea polyphenols7 and

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betaine 8, play a promising role in preventing the metabolic syndromes induced by an

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HF diet.

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A large

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Isoorientin (3',4',5,7-tetrahydroxy-6-C-glucopyranosyl flavone; ISO; Fig. 1)

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exists in the human diet and can be isolated from several edible plants, including

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buckwheat, 9 Patrinia10 and corn silks11. It has been reported that ISO exhibits various

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physiological activities,12 for instance, antioxidant activity, anti-nociceptive and

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anti-inﬂammatory activities in mice.

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hepatoprotective effect against CCL4-induced oxidative damage in rats15 and

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t-BOOH-induced oxidative damage in HepG2 cells.16 Our previous studies also found

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that ISO inhibits the proliferation of human hepatoblastoma cancer cells by inducing

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apoptosis and autophagy.17,18 ISO is able to protect the liver cells against hydrogen

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peroxide-induced oxidation stress.19 Additionally, ISO reduces TNF-induced insulin

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resistance in murine 3T3-F442A cells and human sc adipocytes.20 Therefore, we

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speculate that ISO might play an important role in preventing liver injury and

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Furthermore, ISO exhibits a significant



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hyperlipidaemia. However, the hepatoprotective and hypolipidaemic effects of ISO

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have yet to be explored in vivo.

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The objectives of the present study are to test the protective effects of ISO on

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hyperglycaemia, oxidative injury and hepatic steatosis in mice fed an HF diet. In

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addition, its effects on lipid metabolism and vascular endothelium are also

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investigated. This study should provide a clue for substantiating dietary and

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preventing hyperglycaemia and hepatic injury.

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MATERIAL AND METHODS

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Chemicals and reagents

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Isoorientin (purity ≥ 98 %, extracted from bamboo) was purchased from Forever

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Biotechnology, Ltd. (Shanghai, China). Food-grade fructose was from Senbo Biology

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Co., Ltd. (Xi’an, China). Haematoxylin and eosin (H&E) and Oil red O were obtained

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from Shanghai Lanji Technological Development Co., Ltd. (Shanghai, China). Assay

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kits of glucose, total triglyceride (TG), total cholesterol (TC), high-density

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

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low-density lipoprotein-cholesterol (VLDL), alanine aminotransferase (ALT),

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aspartate aminotransferase (AST) and alkaline phosphatase (ALP) were from

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Changchun Huili Biotechnology Co., Ltd. (Changchun, China). The total superoxide

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dismutase (T-SOD), glutathione peroxidase (GSH-Px) and malondialdehyde (MDA)

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assay kits were from the Nanjing Jiancheng Bioengineering Institute (Nanjing,

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Jiangsu, China). ELISA kits of institute, apolipoprotein A1 (apo-A1), apolipoprotein

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B (apo-B), TNF-α, interleukin 6 (IL-6), interleukin 1 (IL-1) and fatty acid synthase

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(FAS) were also purchased from the Nanjing Jiancheng Bioengineering Institute


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(Nanjing, Jiangsu, China). The BCA protein assay kit was from Thermo Scientific

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(Rockford, IL, USA). All other chemicals were analytical grade and were produced in

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China.

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Animals, diets and and experimental design

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Healthy male Kunming mice (weight 18-22 g) were from the Experimental

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Animal Center of Fourth Military Medical University (Xi’an, China), and were

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housed in standard temperature (22 ± 2℃) and humidity (60 ± 5 %) room with 12/12h

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light-dark cycle. All of the animals were allowed free access to tap water and the

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rodent chow (Qianmin Feed Factory, Experimental Animal Center of Fourth Military

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Medical University, Xi’an, China), which contained 40 % corn flour, 26 % wheat

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flour, 10 % bran, 10 % fish meal, 10 % bean cake, 2 % mineral, 1 % coarse meal, and

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1 % vitamin mix.

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After a week for adjustable feeding, mice were randomly divided into four

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groups (n = 10). In the normal control (ND) group, the mice were allowed free access

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to tap water and were given 0.4 mL physiological saline by gavage once daily. In the

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high-fructose (HF) group, mice both received 20 % high-fructose water and 0.4mL

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physiological saline. In the ISO-treated groups, the mice were administered with ISO

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at 20 and 40 mg/kg·bw (ig, 0.4 mL) as low- (HF+LISO) and high-dose (HF+HISO)

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treatments, respectively, except 20 % high-fructose water. The mice both in HF and

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ISO group were allowed free access to 20 % high-fructose water, and the 20 % HF

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water was renewed every other day. During 56 consecutive days of treatment, the

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body weights of all of the mince were measured once a week. After two hours of the



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last administration, mice were allowed free access to water for 8 h except food. Then,

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all of the mice were anesthetized by ether, and weighed sacrificed to obtain blood and

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livers. The blood samples were centrifuged at 1200×g for 20 min and stored at 4 ℃,

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and the liver samples were frozen at -80 ℃. All experimental protocols were adhered

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the Guidelines of Experimental Animal Administration.

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Biochemical and enzymatic activities evaluation

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The serum levels of TG, TC, VLDL-C, LDL-C, HDL-C, insulin, glucose, ALT,

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AST and ALP were determined using commercial kits with a Multiskan Go automatic

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analyser (Thermo Electron, America) according to the manufacturer's protocol.

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Lipid metabolism analysis

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The levels of apoA-I and apoB in serum and FAS levels in the liver were tested

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using a commercial RIA kit or an ELISA kit according with the manufacturer's

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instructions.

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Estimation of Biochemical properties and cytokines of the liver

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The liver samples were homogenized using an automatic homogenizer

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(F6/10-10G, FLUKO Equipment Shanghai Co., Ltd., Shanghai, China). After

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centrifuging at 5000 rpm for 10 min at 4 °C the supernatants were collected and

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measured protein concentrations using BCA assay kit. The levels of GSH-Px, T-SOD,

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MDA, TNF-α, IL-1 and IL-6 in the liver supernatant were determined using

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commercial kits or an ELISA kit according to the manufacturer's instructions.

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Histopathological and Thoracic Aortas observation


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Histopathology of the liver was measured using H&E and Oil Red O staining. The

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liver tissue samples were fixed with 10% neutral formalin solution, and then

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embedded in paraffin, cut into slices (5-6µm thickness) and stained only with H&E

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dye. Next, the stained sample tissues were processed using cryostat (CM1950, Leika,

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Germany) and stained by Oil Red O. The slides were examined under a light

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microscope (Olympus Optical Co., Ltd., Tokyo, Japan) for observations and

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photography.

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The thoracic aortas were fixed with 4% paraformaldehyde buffer solutionand

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were processed with the method of paraffin embedding and H&E staining, followed

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by observation under a light microscope.

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Statistical analysis

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All results were presented as the means ± standard errors (SE), and were

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analysed using the one-way factorial analysis of variance (ANOVA) and Duncan’s

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post-hoc test (SPSS 16.0).

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RESULTS

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Effects on food intake, body weight, BMI and liver weight

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Table 1 shows the effects of ISO on the food intake, body weights, BMI, liver and

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fat weights of the experimental mice. As shown in Table 1, food intake of HF-fed

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mice was lower than that of ND group mice (p < 0.05), and compared with HF group,

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there were no significant change of food intake in ISO-treated group. Water intake of

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ND group mice was slight lower than that of other groups, even though there was no

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significant difference (p < 0.05), and all groups had no differences (p < 0.05) at



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fructose intake. Additionally, there was no significant difference in the initial body

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weight among the all groups. However, by the end of 8 weeks trial, the final body

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weights of HF-fed mice were significantly higher than that of ND group mice.

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Comparing with the HF group, 20 and 40 mg/kg·bw of ISO supplementation

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effectively reduced body weight by 11.90 % and 11.07 % (p

Isoorientin Prevents Hyperlipidemia and Liver Injury by Regulating

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