Tea Catechins Protect Goat Skeletal Muscle against H2O2-Induced

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Tea Catechins Protect Goat Skeletal Muscle against HO-Induced Oxidative Stress by Modulating Expression of Phase 2 Antioxidant Enzymes Rong-Zhen Zhong, Yi Fang, Gui-Xin Qin, Hao-Yang Li, and Dao-Wei Zhou J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b00816 • Publication Date (Web): 28 Jun 2015 Downloaded from http://pubs.acs.org on July 13, 2015

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Journal of Agricultural and Food Chemistry

1

Running head: Tea Catechins as Antioxidants in Goats against Oxidative

2

Stress

3 4

Tea Catechins Protect Goat Skeletal Muscle against H2O2-Induced

5

Oxidative Stress by Modulating Expression of Phase 2 Antioxidant

6

Enzymes

7 8

Rong-Zhen Zhong, †, ‡ Yi Fang, ‡ Gui-Xin Qin,

*†

Hao-Yang Li, ‡ and Dao-Wei Zhou ‡

9 10

†

11

130118, P. R. China

12

‡

13

Changchun, Jilin 130102, P. R. China

College of Animal Science and Technology, Jilin Agricultural University, Changchun, Jilin

Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences,

14 15 16 17 18 19 20 21 *

Corresponding author at College of Animal Science and Technology, Jilin Agricultural

University,

Chanchun,

Jilin

130118

P.R.

China.

Tel.:

+86-431-84531264. Email: [email protected]. (Gui-Xin Qin). 1

ACS Paragon Plus Environment

+86-431-84513789;

Fax:


22

ABSTRACT: To study the mechanisms of tea catechins (TCs) in goat muscles against

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oxidative stress, skeletal muscle cells (SMCs) induced by H2O2 or not were incubated with

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TCs or 3H-1, 2-dithiole-3-thione (D3T) and were defined as H2O2, H2O2D3T, H2O2TC, D3T,

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and TC treatments, respectively. Results showed that, similar to effects of D3T, TCs regulated

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mRNA and protein expression of antioxidant enzymes by suppressing Keap1 protein

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expression in SMCs from 1.58 ± 0.12 to 0.71 ± 0.21 and 1.03 ± 0.11 in H2O2TC and TC

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groups, respectively; however, effects differed in oxidative condition of cells and among

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enzymes. In stressed cells, TCs increased catalase and glutathione S-transferases (GST)

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activities (P < 0.001); whereas, both enzymes’ activities decreased (P < 0.001) to 2.97 ± 0.37

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U/mg protein or 42.1 ± 1.85 mU/mg protein, respectively, in unstressed SMCs. Subsequently,

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an in vivo experiment in goats fed grain supplemented with TCs or D3T following infusion

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with H2O2 was conducted to further verify mechanisms of TC action. As seen in vitro, TCs

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reduced Keap1 protein expression (P < 0.001) from 2.11 ± 0.37 to 1.34 ± 0.13 and 1.43 ± 0.23

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in H2O2TC and TC groups, respectively, in muscle. However, dietary TCs increased plasma

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CuZn superoxide dismutase and GST activities (P < 0.001) regardless of oxidative stress.

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Moreover, feeding TCs to goats under both conditions increased meat color and tenderness (P

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≤ 0.001). In conclusion, TCs protected goat muscles against oxidative stress and subsequent

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improve meat quality by modulating phase 2 antioxidant enzymes and Keap1 expression.

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KEYWORDS: tea catechins, D3T, Keap1-Nrf2 pathway, oxidative stress, goat

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INTRODUCTION

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With the increasing demand for high quality meat from small ruminants by consumers

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worldwide, the number of studies focused on how to improve the health of these animals and

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the quality of their meat has expanded. Stressful conditions, such as those created by early

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weaning, transportation, overcrowding, and poor conditions, create oxidative stress in

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livestock; this stress leads to decreased meat quality and creates significant economic 2


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1

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constraints in small ruminant production.

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stiff lamb disease, joint disease, and nutritional muscular dystrophy, all of which directly

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reduce meat quality.

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animals is needed.

2

Oxidative stress induces white muscle disease,

Thus, an effective method to reduce oxidative stress levels in these

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The Nrf2-Keap1 (Nuclear factor E2-related factor 2 - Kelch like ECH-associated protein

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1) pathway has been identified as a main regulator of numerous cytoprotective responses to

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oxidative and electrophilic stresses. 3, 4 The core of these signaling pathways is the antioxidant

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response element (ARE, 5´-tga[C/T]NNNGC-3´), which is a cis-acting regulatory element

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found in the promoter region of many genes encoding phase 2 antioxidant enzymes that

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protect against oxidative stress. These protective enzymes include glutathione S-transferases

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(GST), γ-glutamylcysteine ligases, NADPH:quinine oxidoreductase (NQO1), CuZn

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superoxide dismutase (CuZn-SOD), glutathione peroxidase (GPx), and catalase (CAT).

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Under basal conditions, Nrf2 is retained mainly in the cytoplasm through binding to Keap1,

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which in turn facilitates the ubiquitylation and subsequent proteolysis of Nrf2. In response to

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oxidative stress, Nrf2 dissociates from Keap1, translocates into the nucleus and elicits the

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antioxidant response by binding the ARE of target promoters, thus inducing the expression of

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phase 2 antioxidant enzymes. But until now, the Nrf2-Keap1-ARE signaling pathway has not

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been identified in goats and sheep.

65

5

The use of antioxidant enzyme inducers as chemopreventive agents against cancers 6

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associated with oxidative stress in humans has been under clinical investigation.

67

such as 3H-1, 2-dithiole-3-thione (D3T) enhance the detoxification of environmental

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carcinogens and protect against neoplasia through activating Nrf2-Keap1 signaling. D3T

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covalently modifies specific cysteine residues of Keap1 (C257, C273, C288, and C297), reacts

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with Keap1 to format disulfide linkages, and causes a conformational change of Keap1-Nrf2

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complex, thereby disruptiing the Keap1-Nrf2 complex to release Nrf2, which then migrates to 3


Inducers


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the nucleus. 4 As a result of these events, Keap1 has been described as a molecular sensor for

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oxidative stress. 7

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Despite the potential benefit of treating commercially raised animals with antioxidant

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therapies, it is neither economical nor practical to use cancer chemopreventive agents such as

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D3T as feed additives. In addition to these limitations, bans by European countries on the use

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of synthetic antioxidants due to their side effects in livestock feed have made natural

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plant-derived antioxidant compounds increasingly attractive as feed additives. The use of

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abundant plant derived-antioxidants such as tea catechins (TCs) is feasible due to lower costs

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and strong antioxidant properties, provided their effectiveness can be demonstrated in these

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animals. TCs are known to positively regulate the expression of antioxidant enzymes and to

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scavenge reactive oxygen species.

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antioxidant activity through the Nrf2-Keap1 signaling pathway in humans.

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have reported that TCs have potent antioxidant properties against oxidative stress in vivo and

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in vitro, 10 and that TCs improved meat quality by decreasing lipid oxidation, enhancing meat

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color stability, and changing fatty acid composition in goat meat.

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sequence of goat Keap1 has been cloned in our laboratory (GenBank: KF038327.1),

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demonstrating the presence of this antioxidant system. However, functional studies are needed

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to reveal whether TCs can afford protection to goat muscle tissue against oxidative stress

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through modulation of Keap1, and induce phase 2 antioxidant enzyme expression and

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subsequent improvement of meat quality.

8

Similar to D3T, TCs have been reported to exert

11, 12

9

Several studies

The partial gene

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The objectives of this study were (1) to study the effectiveness of TCs against oxidative

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stress in goat skeletal muscle cells (SMCs) through modulation of Keap1-mediated phase 2

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antioxidant enzyme expression compared with a known enzyme inducer in vitro and (2) to

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determine whether the above mechanisms function to improve oxidative health and meat

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quality in live goats when TCs are administered as a dietary supplement in vivo. 4


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MATERIALS AND METHODS

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Chemicals. All chemicals, cell culture reagents, and D3T were obtained from Sigma

99

Chemical Co. (St. Louis, MO, USA) unless otherwise stated. Primary antibodies were

100

purchased from Abcam Co. (San Francisco, CA, USA). Secondary antibodies and all western

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blot reagents were ordered from KenGen Co. (Nanjing, China).

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TCs Preparement. The TCs (purity of 80.11%) were extracted from green tea leaves

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(C.sinensis L.) by high pressure liquid chromatography (Waster 600, Waters Corp., Milford,

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MA) and contained 57.67% (-)-epigallocatechin gallate, 1.61% (+)-catechin and (-)-catechin,

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5.77% (-)-epicatechin, 0.47% (-)-epigallocatechin, 13.03% (-)-epicatechin gallate and 1.56%

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(-)-gallocatechin gallate. In addition, the extracted mixture contained caffeine.

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In Vitro Experimental Procedures

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The use of the animals and the experimental procedures were approved by the Animal Care

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Committee, College of Animal Science and Technology, Jilin Agricultural University,

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Changchun, Jilin, China.

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Isolation, Cell Culture, and Treatment. One male early weaned Boer goat at age of 4

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weeks was electrically stunned and slaughtered by exsanguination using commercial

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procedures conducted according to the recommendations of the animal ethics committee at

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the Jilin Agricultural University. The longissimus dorsi (LD) muscle was collected for cell

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culture. The procedures of LD muscle collection and the primary SMC isolation and culture

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are same as described in Zhong et al. 2011a

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flasks (25 cm2) at a density of 3 × 106 cells/mL and in two 96-well plates at a density of 2.5 ×

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105 cells/mL in Dulbecco’s modified Eagle’s medium (DMEM, Gibco BRL, Gaithersburg,

119

MD)

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penicillin-streptomycin/mL, and 5 µg/mL gentamicin. The cells were cultured at 37°C in a

121

humidified atmosphere of 5% CO2.

supplemented

with

10%

8

Briefly, the isolated SMCs were seeded in 60

heat-inactivated

fetal

5


calf

serum,

100

U

of


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After 24 h of culture, cells in flasks and plates were assigned to 1 of 5 treatments and

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each treatment was replicated six times. The 5 treatments were defined as H2O2-induced as

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control (H2O2), H2O2-induced+D3T incubation (H2O2D3T), H2O2-induced+TC incubation

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(H2O2TC), D3T incubation (D3T), and TCs incubation (TC) groups, respectively. Briefly,

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cells in all H2O2-induced groups were pre-incubated with 1 mM H2O2 for 3 h. Then, cells in

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H2O2D3T, H2O2TC, D3T and TC groups were subsequently treated with 50 µmol/L D3T, 2.5

128

µg/mL TC, 50 µmol/L D3T and 2.5 µg/mL TCs, respectively, according to the concentrations

129

recommended by Zhong et al. 2011a and Cao et al. 2004. 8,

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post-incubated with D3T or TCs. Media from each treatment in two 96-well plates were

131

collected to analyze cell membrane integrity. Cells in 30 flasks (6 flasks per treatment) were

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disrupted in media, centrifuged, and then the supernatant was used to analyze enzyme

133

activities. 15 Cells in the other 30 flasks (6 flasks per treatment) were collected by centrifuging

134

to analyze enzyme mRNA and protein expression levels.

135

13

Cells were harvested at 45 h

Cell membrane integrity was determined by the presence of lactate dehydrogenase (LDH)

136

in the growth media, and was measured using an in vitro LDH activity assay kit (Jiancheng

137

Biology Co., Nanjing, China). Catalase (CAT) activity in the SMC homogenates was assayed

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using spectrophotometric method of Aebi. 1984,

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phosphate buffer is detected at 240 nm. The activity of CuZn-SOD in the SMC homogenates

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was assayed by monitoring inhibition of the rate of xanthine oxidase-mediated reduction of

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cytochrome c described by Spitz and Oberley, 1989.

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homogenates was measured spectrophotometrically by the method of Levander et al. 1983.

143

The activity of NQO1 in cells was measured according to method of Prochaska and

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Santamaria, 1988

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tetrazolium bromide (MTT) reduced per minute per mg of protein. The activity of total GST

146

was measured using 1-chloro-2, 4-dinitrobenzene as the substrate and expressed as mU

17

14

in which the disappearance of H2O2 in a

15

The GPx activity in the SMC 16

and expressed as nmol 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl

6


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conjugate formed/mg protein (1 mU = 1 nmol conjujate formed/min). 18 RNA

Isolation,

cDNA

Synthesis,

and

Real-time

Quantitative

RT-PCR.

149

Oligonucleotide primers of CAT, CuZn-SOD, GPx, GST, NQO1, Keap1, and β-actin genes

150

were designed using Premier 5 software (Premier Co., Canada). The details of the

151

oligonucleotide primer sequences, primer lengths, and predicted amplified product lengths are

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listed in Table 1. The procedures of total RNA isolation, cDNA synthesis, and real-time

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qRT-PCR for CAT, CuZn-SOD, GPx, NQO1, GST, and Keap1 expressions are performed

154

according to methods of Zhong et al. 2011a.

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determined by real-time qRT-PCR were calculated according to the 2-△△CT method. 19

8

Changes in mRNA expression levels

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Western Blot Analysis. Total proteins in SMCs were extracted using a commercial kit

157

(KEP210, KeyGEN Biotech, Nanjing, China) and total protein content was measured by the

158

method of Lowry et al. 1951. 20 The extracted proteins were denatured, separated by reducing

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10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel (Bio-Rad, Hercules, CA),

160

transferred onto Polyvinylidene Fluoride membranes (Millipore, USA), and incubated with

161

CAT, CuZn-SOD, GPX, NQO1, GST, Keap1, and β-actin primary antibodies (Abcam Com.,

162

ab50434, ab62800, ab50434, ab166778, ab150000, ab166958, and ab15263) at a

163

concentration of 3.0, 0.02, 2.0, 0.5, 1.0, 1.0, and 2 μg/ml. Subsequently, the protein affinity

164

with primary antibodies was tested by horseradish peroxidase conjugated second antibodies

165

for 2.5 h at 4°C. The intensity of each protein was determined by enhanced

166

chemiluminescence reagent (GE Healthcare, Waukesha, WI) and visualized by exposure to

167

Hyperfilm ECL (GE Healthcare). β-actin served as an internal control. The relative

168

expressions of proteins were expressed as the ratio of band intensities of proteins to β-actin.

169

In Vivo Experimental Procedures

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Animals and Treatment. Thirty male early weaned Boer goats, aged 4 weeks and with

171

initial body weights (BW) averaging 15.8 ± 0.7 kg, were randomly assigned to 1 of 5 groups 7



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(n = 6 goats/group) according to their BWs for a feeding period of 21 days. The 5 treatments

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consisted of (1) H2O2 infused group (H2O2), (2) H2O2 infused and fed 0.5 mmol/kg of BW

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D3T (H2O2D3T), (3) H2O2 infused and fed 0.5% of TCs (dry matter (DM) basis of feed)

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(H2O2TC), (4) fed 0.5 mmol/kg of BW D3T (D3T) without H2O2 infusion and (5) fed 0.5% of

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TCs (TC) without H2O2 infusion. Goats in the stressed control were fed with the basal diet (30%

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Aneurolepidium chinense, 47.5% ground corn grain, 15.0 % solid soybean meal, 5.0%

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molasses, 1.0% dicalcium phosphate, 0.5% limestone, and 1.0% trace mineral salt and

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vitamins for ruminants) formulated according to the NRC, 2007.

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experimental groups were fed with the same basal diet and supplemented with D3T or TCs.

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Each

goat

was

assigned

to

a

single

metabolism

21

Goats in the other

cage

in

the

same

182

environmentally-controlled bard barn at 25°C, with free access to fresh water. The

183

experimental diets were prepared every day by mixing D3T or TC powder with the basal diet

184

before morning and afternoon feeding. Goats were fed twice daily at 06:00 and 18:00 h. The

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feed supply and orts for each goat were recorded daily. From days 0 to 21, Goats in stressed

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groups were continuously infused a total amount of 50 µmole/g BW H2O2 into jugular vein

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for 1 h at 05:00 h to induce oxidative stress according to procedure of Lee et al. 2004. 22 Goats

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in unstressed groups were subjected to the same infusion with saline as the goats in stressed

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groups. After 21 days of feeding period, all goats were humanely slaughtered and samples

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collected.

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Sampling and Analytical Procedures. At days 0, 7, 14, and 21, blood samples (7 ml)

192

were taken from jugular vein of each goat before morning feeding and H2O2 infusion, and

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centrifuged at 2500 × g for 15 min at 4°C to harvest plasma for biochemical indices and

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enzyme activity determination. The total protein, albumin, globulin, total cholesterol, high

195

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

196

triglyceride, and urea nitrogen (PUN) concentrations in plasma were measured using an 8


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automated biochemical analyzer (Hitachi, 7020, Beijing, China) and commercial kits

198

(Jiancheng Biology Co., Nanjing, China). The activity of lipoprotein lipase (LPL) was

199

determined according to method of Karpe et al. 1992.

200

GPx, and GST in plasma were determined using the methods described above for cell lysates.

23

The activities of CAT, CuZn-SOD,

201

After 21 days, all goats were electrically stunned and slaughtered by exsanguination

202

using procedures accepted by the animal ethics committee at the Jilin Agricultural University.

203

After slaughter the carcasses were allowed to bleed until they were drained, after which the

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left side of carcass was kept under aseptic conditions at 4°C for sampling. The left LD muscle

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(10 g) from the same region of each animal was removed and snap-frozen at -70°C for

206

determination of Keap1 protein expression according to the method of Kavitha et al. 2013.

207

Another 200 g of the left LD muscle from the same region of each animal was sampled at 24

208

h postmortem for determination of meat quality parameters. Briefly, the pH value of the LD

209

samples was measured using a portable pH-meter (Testo 205, Testo AG, Lenzkirch, Germany)

210

with a plastic body, spear-tipped probe coupled with a temperature probe. Shear force analysis

211

was conducted using a Warner-Bratzler Shear Device (C-LM3B, Beijing, China). The lipid

212

oxidation levels of the LD samples were measured using the 2-thiobarbituric acid reactive

213

substance (TBARS) method modified as described by Olsvik et al. 2005. 25 The intramuscular

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fat content of the LD samples was determined using the methods of ether extraction on

215

Sohxtex.

216

Zhong et al. 2009.

217

Ltd., Osaka, Japan) was used to measure the meat color parameters of lightness (L*), redness

218

(a*), and yellowness (b*).

26

24

The total heme pigment content was determined according to the methods of 11

In addition, a Minolta color-meter (model CR410, Minolta Camera Co.

219

Statistical Analyses. The data from analysis of gene and protein expression levels and

220

enzyme activities in cells and meat quality parameters were analyzed using a General Linear

221

Model followed by Duncan’s multiple range tests.

27

The data from the blood metabolite

9



222

measurements were analyzed with SAS, 2002 27 using the MIXED model procedure described

223

by Littell, et al. 1996, 28 with a model consisting of dietary treatment, sampling time, and

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treatment × time interaction as fixed effects and with the animal as the random effect. The

225

measurements obtained from each goat at different sampling times were treated as repeated

226

measures for each blood metabolite measurement. The means were separated using the least

227

squares mean and presented with the standard error of the mean (SEM). The results were

228

considered statistically significant at P ≤ 0.05.

229

RESULTS

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Treatment of goat SMCs with D3T or TCs affected phase 2 antioxidant enzyme activities

231

(Table 2). However, effects were different between H2O2-induced and non-induced cells and

232

changes in enzyme activity were different among enzymes. Treatment with D3T or TCs

233

increased CuZn-SOD and NQO1 activities regardless of oxidative stress induction (P

Tea Catechins Protect Goat Skeletal Muscle against H2O2-Induced

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