Protective Effects of Functional Chicken Liver Hydrolysates against

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Protective Effects of Functional Chicken Liver Hydrolysates against Liver Fibrogenesis: Antioxidation, Anti-inflammation, and Anti-fibrosis Po-Ju Chen, Jung-Kai Tseng, Yi-Ling Lin, Yi-Hsieng Samuel Wu, Yi-Tse Hsiao, Jr-Wei Chen, and Yi-Chen Chen J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 31 May 2017 Downloaded from http://pubs.acs.org on June 6, 2017

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Journal of Agricultural and Food Chemistry

115x69mm (300 x 300 DPI)

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Protective Effects of Functional Chicken Liver Hydrolysates against Liver Fibrogenesis:

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Antioxidation, Anti-inflammation, and Anti-fibrosis

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Po-Ju Chen¥, #, Jung-Kai Tseng§, &, #, Yi-Ling Lin¥, Yi-Hsieng Samuel Wu¥, Yi-Tse Hsiao¶,

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Jr-Wei Chen¥, £, and Yi-Chen Chen¥, *

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¥

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Taiwan;

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§

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&

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University, Taichung 404, Taiwan;

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¶

School of Veterinary Medicine, National Taiwan University, Taipei City 106, Taiwan;

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£

Poultry Industry Section, Department of Animal Industry, Council of Agriculture, Executive

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Yuan, Taipei 100, Taiwan.

Department of Animal Science and Technology, National Taiwan University, Taipei 106,

Department of Optometry, Asia University, Taichung 413, Taiwan; Department of Medical Research, China Medical University Hospital, China Medical

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# Chen, P. J. and Tseng, J. K. contributed equally as co-first authors.

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* Correspondence to: Tel: 886-2-33664180; Fax: 886-2-27324070; E-mail address:

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[email protected] (YC Chen)

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Running title: Pepsin-digested-chicken liver hydrolysates against liver fibrosis

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Section: Bioactive Constituents, Metabolites, and Functions

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Submitted to Journal of Agricultural and Food Chemistry

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Original research manuscript

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ABSTRACT

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Via an assay of Amino Acid Analyzer, pepsin-digested chicken liver hydrolysates (CLHs)

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contain taurine (365.57+39.04 mg/100g), carnosine (14.03+1.98 mg/100g), and anserine

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(151.58+27.82 mg/100g).

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thioacetamide (TAA)-induced fibrosis.

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increased serum liver damage indices and liver cytokine contents.

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monocytes/macrophages in livers of TAA-treated rats were illustrated by the H&E staining

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and immunohistochemical analysis of cluster of differentiation 68 (CD68, ED1), respectively.

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A significantly increased hepatic collagen accumulation was also observed and quantified

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under TAA treatment.

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and SMAD family member 4 (SMAD4) caused by TAA treatment further enhanced alpha

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smooth muscle actin (α-SMA) gene and protein expressions.

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under TAA treatment were significantly amended by 200 and 600 mg CLHs/kg B.W..

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Hence, the ameliorative effects of CLHs on liver fibrogenesis could be attributed by

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antioxidation and anti-inflmmation.

This study aimed to evaluate whether CLHs could alleviate A dose of 100 mg TAA/kg B.W. significantly Cell infiltration and

A significant upregulation of transforming growth factor-beta (TGF-β)

The liver antioxidant effects

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Keywords: anti-fibrosis; anti-inflammation; antioxidant; chicken liver hydrolysates;

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thioacetamide

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INTRODUCTION

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According to a statistical report from World Health Organization,1 liver cancer ranked second

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among cancer categories globally, and meanwhile, the similar occurrence is also observed in

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Taiwan2.

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ameliorate liver inflammation or fibrogenesis has been concerned by researchers within

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decades.

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model provides advantages including similar clinical symptoms as human’s, a specific target

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in livers, and accessible to obtain.3

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related to oxidative stress attributed to metabolites of TAA (thioacetamide-s-oxide, TASO;

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thioacetamide-S,S-dioxide, TASO2) in livers.4

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Because of the potentially reversible characteristic of liver fibrosis, how to

There are several models for an induction of liver fibrosis.

A TAA-induced rat

The mechanism of TAA-induced liver injury is highly

Food-derived protein hydrolysates own several functional properties, such as antioxidant5

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and antihypertensive6 etc.

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can provide diverse functional effects because of their amino acid composition and peptides

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with specific sequences and structures.7, 8

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of animal by-products, such as feathers, blood, viscera, and skin.

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that the hydrolysates from animal by-products provide antioxidant8 and lipid-lowering

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effects9.

Different types of protein after specific hydrolyzing conditions

Protein hydrolyzation offers a feasible application Hence, it has been cited

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The candidates as liver protectants should be not only effective but also low cost.

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Regarding the poultry industry in Taiwan, nearly 7,000 metric tons of chicken livers are

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produced annually,10 and most of them are only used in the fertilizer or incineration. Based

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on the evaluation of antioxidant effects and economic concern, functional chicken liver

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hydrolysates (CLHs) can be produced under a pepsin digestion for 2h and at 37oC.8

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addition, CLHs not only offered antioxidant capacities in D-galactose-induced mice8 but also

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provided lipid-lowering effects in high-fat diet-induced hamsters9.

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attributed to the free amino acid contents and bioactive dipeptides (carnosine and anserine), 2 ACS Paragon Plus Environment

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Those benefits are

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in CLHs.

Carnosine (β-alanyl-L-histidine, CNS) and anserine (β-alanyl-N-histidine) are

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natural histidine-containing and meat-sourced dipeptides which own antioxidant and

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lipid-lowering abilities.11

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damages induced by acetaminophen in mice.12 Besides, it was indicated that anserine is

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able to protect the γ-irradiation induced hepatotoxicity due to the decrease of oxidative

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stress.13

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aimed to evaluate whether CLHs could alleviate TAA-induced liver fibrosis in rats.

A pre-treatment with carnosine can ameliorate acute hepatic

In the view of the economic value and functional properties of CLHs, this study

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MATERIALS AND METHODS

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Chemicals.

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was used to produce chicken liver hydrolysates (CLHs).

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pentobarbital purchased from Sigma-Aldrich Co. (St. Louis, MO, USA) were used in the

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animal treatment.

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solution (30-31%), 2,2’-azino-bis-(3-ethylbenthiazoline sulfonic acid), superoxide dismutase,

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5,5’-dithiobis (2-nitrobenzoic acid), pyrogallol, reduced glutathione, acetic acid, citric acid,

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and Tween-20 purchased from Sigma-Aldrich Co. as well as substrate peroxide solution,

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substrate luminal reagent, sodium hydroxide, methanol, formaldehyde solution, xylene,

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transfer membrane, polyvinylidene difluoride (PVDF), and isopropanol purchased from

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Merck Millipore Co. (Darmstadt, Hesse, Germany) were applied to determine antioxidant

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capacity and lipid peroxidation, and analyze the immunohistochemical observation and

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western blotting in livers.

Pepsin purchased from Calzyme Laboratories Inc. (San Luis Obispo, CA, USA) Carnosine, thioacetamide, and

Thiobarbituric acid, trichloroacetic acid, ethanol, hydrogen peroxide

All chemicals used in this study were in an analytical grade.

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Preparation of CLHs, and Analyses of Taurine and Carnosine/Anserine Contents in CLHs.

Fresh broiler (Arbor acres) livers were collected from Ding-Yao Food Co., Ltd. 3 ACS Paragon Plus Environment


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(New Taipei city, Taiwan) and transported to our lab under a -20oC transportation.

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broilers were obtained from the slaughterhouse which had passed the certification of Certified

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Agricultural Standards (CAS) in Taiwan.

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concerns of heavy metal contents, as well as antibiotic and sulfonamide residues.

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safety concern, the antibiotic and sulfonamide residues in the raw materials, broilers’ livers,

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used in this study were examined again in Inspection Center of National Animal Industry

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Foundation (Pingtung County, Taiwan), and they were all safe.

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manufactured according to the method from our previous report.8

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lyophilized and stored at -20oC before further usages.

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anserine) contents in the CLHs used in this study were assayed in triplicate by using an

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Amino Acid Analyzer (Hitachi L8800 amino acid analyzer, Hitachi High-Technologies Co.,

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Tokyo, Japan) in Food Industry Research and Development Institute (HsinChu City, Taiwan)

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and expressed as mg per 100g lyophilized powders.

The

The slaughtered broilers should be safe from the For a

The CLHs were The CLHs were

Taurine and dipeptide (carnosine and

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Animals and Treatments.

The animal use and protocol were reviewed and approved by

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National Taiwan University Animal Care Committee (IACUU No.: 100-15).

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Wistar rats at the age of 5 weeks old were purchased from BioLASCO Taiwan Co., Ltd.

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(Taipei City, Taiwan), and acclimatized for one week before commencement of the

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experiment.

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h light-dark cycle, and randomly assigned to one of the following groups (n=8 per group): 1)

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CON: saline (intraperitoneal injection, i.p.) + normal distilled water (oral gavage); 2) TAA:

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100 mg TAA /kg B.W. (i.p.) + normal distilled water (oral gavage); 3) CLH-L: 100 mg TAA

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/kg B.W. + 200 mg CLHs/kg B.W. (oral gavage); 4) CLH-H: 100 mg TAA/kg B.W. + 600 mg

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CLHs/kg B.W. (oral gavage); 5) CNS: 100 mg TAA /kg B.W. + 200 mg carnosine/kg B.W.

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(oral gavage).

Forty male

Two rats were housed in each cage in an animal room at 22±2 oC with a 12/12

Animal experimental period lasted for 10 weeks with injections of TAA three 4 ACS Paragon Plus Environment

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times weekly (Monday, Wednesday, and Friday) and oral gavage daily. Chow diets

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(Laboratory Rodent Diet 5001, PMI Nutrition International/Purina Mills LLC, Richmond, IN,

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USA) and distilled water were provided ad libitum.

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per cage were calculated as volumes of food and water intakes on per rat daily basis,

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respectively.

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injection in the final day of experiment.

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removed, weighed, and then stored at -80oC for further analyses.

Average daily food and water intakes

All rats were fasted overnight before euthanized by the sodium pentobarbital Liver, heart, kidney, and spleen from each rat were

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Determination of Serum Biochemical Values.

Blood samples were collected via

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abdominal aorta and placed at room temperature for clotting, and then centrifuged (3,000 x g,

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4oC, 15 min; Model 3740; KUBOTA, Tokyo, Japan) to obtain sera, and stored at -80oC.

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Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea

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nitrogen (BUN), total triglyceride (TG), total protein (TP), and total cholesterol (TC) were

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analyzed by using commercial enzymatic kits with an ESPOTCHEMTMEZ SP4430

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biochemistry analyzer (ARKRAY Inc., Kyoto, Japan).

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Preparation of Liver Homogenate.

A 0.5 g sample of liver was homogenized on ice with

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4.5 mL phosphate buffer saline (PBS, pH 7.0).

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supernatant was collected and stored at -80oC for further analyses.

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supernatant were measured by a Bio-Rad protein assay kit (Cat. #: 500-0006, Bio-Rad

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Laboratories, Inc., Hercules, CA, USA).

After a centrifugation (12000×g), Protein levels in

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Determination of Antioxidant Capacity and Lipid Peroxidation in Livers.

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Thiobarbituric acid reactive substances (TBARS), trolox equivalent antioxidant capacity

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(TEAC), and reduced glutathione (GSH) levels, as well as activities of superoxide dismutase 5 ACS Paragon Plus Environment


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(SOD) and catalase (CAT) in livers were assayed according to the previous methods.14

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Malondialdehyde (MDA) is a by-product produced during lipid oxidation.

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acid reactive substances (TBARS) values were applied to assay the MAD level in livers.

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The principle of TEAC assay was based on the measurement of ABTS•+ scavenging ability.

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The reduced GSH level was detected the unique thiol compounds in GSH.

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TBARS, TEAC, and reduced GSH levels were expressed as nmole MDA equivalent (eq.)/mg

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protein, µmole/mg, and nmole/mg protein, respectively.

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determined by the inhibitory effect of SOD on pyrogallol autoxidation and ability to clean

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H2O2, respectively.

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calculated to obtain inhibition (%) by following formula: 1-[(sample or standard∆A/min)/

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(max speed∆A/min)], which max speed was set to reaction of ddH2O loading, indicating

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nearly no inhibition of pyrogallol autoxidation. A standard curve was plotted for SOD,

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which was used for calculation of activities of SOD in liver tissues.

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taking the extinction coefficient (39.4 M−1 cm−1 at 240 nm at 25oC) of H2O2.

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CAT was expressed as the amount of enzyme that decomposes one µmol of H2O2 per min,

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and the specific activity of CAT in liver tissues was expressed in terms of unit/mg protein.

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The glutathione peroxidase (GPx) activity in liver tissues was measured by using a

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commercial kit (Ransel Glutathione Peroxidase, Randox Laboratories Ltd., Antrim, UK) and

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GPx activity in live tissues was expressed as unit/mg protein.

Thiobarbituric

The liver

The SOD and CAT activities were

The alteration of absorbance per min under 420 nm wavelength was

The CAT activity was One unit of

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Determination of Collagen and Inflammatory Cytokine Levels in Livers.

The collagen

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content was determined by using commercial kits (Cat#: 6012, Chondrex Inc., Redmond, WA,

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USA).

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binding to helical structure of collagen.

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by using the standard curve plotted by bovine Type I collagen and expressed as µg/mg

Basically, the absorbance alteration under 530 nm was detected after Sirius dye The collagen contents in samples were calculated

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protein.

The hepatic TNF-α, IL6, IL-1β, and TGF-β levels were measured by using

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enzyme-linked immunosorbent assay (ELISA) and conducted according to the commercial

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manufacturer’s instructions (TNF-α, IL6, IL-1β, and TGF-β kits, eBioscience Inc., San Diego,

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CA, USA).

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Histopathological Analyses.

Liver tissues were fixed in neutral buffered formalin, and then

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dehydrated in graded alcohol from 30 to 95%. After impregnating the tissues in xylene,

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they were embedded in paraffin for sectioning using a microtome to cut into 5 µm thick

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sections.

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graded alcohol and stained with hematoxylin and eosin (H&E) as well as Sirius red staining,

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respectively.

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AxioCam ERc 5s camera system with AxioVision Release 4.8.2 (06-2010) 341 software (Carl

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Zeiss Microscopy, LLC, Thornwood, NY, USA).

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collagen accumulation for hepatocirrhosis were given by Metavir score, and samples were

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categorized into 5 levels dependent on the severity from fibrosis to cirrhosis.15

On a completion of de-paraffinization with xylene, tissues were dehydrated in

The microscopic analysis was conducted by a Zeiss Axioskop 340 microscope

Via a double-blind test, the values of

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Determination of Hepatic Messenger Ribonucleic Acid (mRNA) Expressions of TGF-β

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Pathway.

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carried out by using commercial kits (GoScriptTM Reverse Transcriptase, Promega Co.,

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Madison, Fitchburg, WI, USA).

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commercial kits (Fast SYBR Green Master Mix, Applied Biosystems, Molecular Probes Inc.,

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Eugene, OR, USA).

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NM_017008.4; F: 5’-GACCCCTTCATTGACCTCAAC-3’, R:

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5’-GGAGATGATGACCCTTTTGGC-3’); Tgfb1 (GenBank No.: NM_021578.2; F:

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5’-CCTGAAAGGGCTCAACACC-3’, R: 5’-CAGTTCTTCTCTGTGGAGCTGA-3’);

The transcription of RNA to complementary deoxyribonucleic acid (cDNA) was

The real-time PCR was carried out following the

All the primers used were designed: Gapdh (GenBank No.:



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Tgfbr1 (GenBank No.: NM_012775.2; F: 5’-TCCAAACCACAGAGTAGGCAC-3’, R:

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5’-TGGATTCCGCCAATGGAACA-3’); Tgfbr2 (GenBank No.: NM_031132.3; F:

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5’-CCGTGTGGAGGAAGAACGAC-3’, R: 5’-TGAAGCCGTGGTAGGTGAAC-3’);

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SMAD3 (GenBank No.: NM_013095.3; F: 5’-TTATGGGGTGCTGTGGTCTT-3’, R:

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5’-ACATGCGTGACTGCACATAC-3’); SMAD4 (GenBank No.: NM_019275.2; F:

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5’-GATAGCGTCTGTGTGAACC-3’, R: 5’-GTACTGGTGGCATTAGACTC-3’); Col1α

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(GenBank No.: NM_053304.1; F: 5’-GACTGTCCCAACCCCCAAAA-3’, R:

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5’-CTTGGGTCCCTCGACTCCTA-3’); Acta2/αSMA (GenBank No.: NM_031004.2; F:

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5’-TTCGTTACTACTGCTGAGCGTGAGA-3’, R:

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5’-AAAGATGGCTGGAAGAGGGTC-3’); Ctgf (GenBank No.: NM_022266.2; F:

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5’-GTGAGGCTGAAGCCAGCTAT-3’, R: 5’-CGGCAAATTCACTTGCCACA-3’).

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fluorescence signals were detected by StepOne Real time PCR system (Applied Biosystems;

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Thermo Fisher Scientific, Inc., Waltham, MA, USA), and the expression levels of the mRNA

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genes were transformed by cycle of threshold (Ct) value.

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housekeeping gene with the mRNA gene expressions of CON group setting to 1.

The

The Gapdh was used as a

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Immunohistochemical Analysis (IHC).

For an IHC analysis, paraffin-embedded liver

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sections were used.

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with gradient graded alcohol from 100, 90, 70, 50 and 30%, followed by ddH2O and PBS

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incubation at 25oC for 10 min each concentration, respectively.

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conducted by incubating sections in retrieval buffer (2.581 g citric acid and 500 µL Tween-20

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in 1 L deionized water) at 120oC for 2 min.

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sections were incubated in 1% H2O2/methanol solution followed by blocking with

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avidin/biotin kit (SP-2001; VECTOR Laboratories, Inc., Burlingame, CA, USA) for 15 min,

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respectively.

Firstly, liver sections were de-paraffinized with xylene and hydrated

Antigen retrieval was

After cooling with running water, tissue

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(twice, 5 min for each).

After finishing aforementioned steps, blocking buffer containing

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horse serum was used to cover liver sections, reacting at 4oC overnight.

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anti-αSMA mouse monoclonal antibody (Merck Millipore Co.) with 100X dilution or

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anti-ED1 (Santa Cruz, Dallas, Texas, USA) with 50X dilution were added to sections, and

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then incubated at 4oC overnight.

Washing steps were taken 5 times with 5 min for each after

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the primary antibody treatment.

Next, tissue sections were treated diluted biotinylated

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secondary antibody solution for 30 min, followed by washed with PBS 5 times with 5 min for

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each.

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was then added to sections, and reaction was conducted for 30 min.

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taken for 10 min twice, the DAB peroxidase substrate (Cat. No. SK-4100, VECTOR

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Laboratory, Burlingame, CA, USA) was treated on sections for 30 min (depending on the

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color intensity) in dark room at room temperature (25oC), and the reactions were stopped by

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rinsing with PBS.

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nuclei of cells.

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clearing, and mounting, which were ready for microscopic analysis, under a Zeiss Axioskop

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340 microscope AxioCam ERc 5s camera system with AxioVision Release 4.8.2 (06-2010)

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341 software (Carl Zeiss Microscopy, LLC, Thornwood, NY, USA).

Then, an

VECTASTAIN® Elite ABC Reagent (VECTOR Laboratory, Burlingame, CA, USA) After washing steps

In order to obtain better observations, hematoxylin was then used to stain

Finally, the sections were gone through process including dehydration,

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Western Blotting.

Briefly, the radioimmunoprecipitation assay (RIPA) lysis buffer (Merck

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Millipore Co.) was mixed with liver homogenates and 5X western sample dye, and then

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incubated at 100 oC for 10 min.

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(10% running gels and 4% stacking gels) and running in condition at 100V, followed by

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transferring to polyvinylidene fluoride (PVDF) membranes (10V).

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PVDF was blocked by skimmed milk (5%) for 2h at 4oC.

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soaked in PBS-Tween 20 (0.1%, v/v) containing either anti-αSMA monoclonal antibody

After cooling, the samples were loaded into SDS-PAGE gel

The protein loaded

Then, PVDF membranes were



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(Merck Millipore Co.) or anti-GAPDH polyclonal antibody (Thermo Fisher Scientific, Inc.)

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with 1000X dilution and incubated at 4oC overnight.

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PBS-Tween 20 for 15 min each, the anti-mouse secondary antibody (1:4000) was added,

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incubating for one h at room temperature.

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secondary antibody incubation.

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chemiluminescence (ECL) kit (Immobilon™ Western, Millipore Co., Billerica, MA, USA)

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under a MultiGel-21 (TOP BIO CO., New Taipei City, Taiwan).

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(National Institutes of Health, Bethesda, MD, USA) was used to quantify the optical density

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of protein bands with the value of the GAPDH band as a reference.

After washing three times by

The same washing steps were taken after a

Protein bands were visualized with an enhanced

Image J. software

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Statistical Analysis.

The experiment was conducted using a completely randomized design

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(CRD).

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using analysis of variance (ANOVA).

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was detected by the one-way analysis of variance (ANOVA), differences among treatments

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were tested by using the Least Significant Difference (LSD) test.

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data were carried out by using SAS (SAS Institute Inc., 2002).

Data were shown as the mean±standard error of the mean (SEM) and analyzed by When a significant difference (p

Protective Effects of Functional Chicken Liver Hydrolysates against

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