Processed meat protein promoted inflammation and hepatic

Subscriber access provided by UNIV OF SOUTHERN INDIANA

Agricultural and Environmental Chemistry

Processed meat protein promoted inflammation and hepatic lipogenesis by upregulating Nrf2/Keap1 signaling pathway in Glrx-deficient mice Muhammad Ijaz Ahmad, Zou Xiaoyou, Muhammad Umair Ijaz, Muzhair Hussain, Liu Congcong, Xinglian Xu, Guanghong Zhou, and Chunbao Li J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b03136 • Publication Date (Web): 25 Jul 2019 Downloaded from pubs.acs.org on July 26, 2019

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

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

Page 1 of 59

Journal of Agricultural and Food Chemistry

1

Processed Meat Protein Promoted Inflammation and Hepatic Lipogenesis by

2

Upregulating Nrf2/Keap1 Signaling Pathway in Glrx-deficient Mice

3

Muhammad Ijaz Ahmad, Xiaoyou Zou, Muhammad Umair Ijaz, Muzahir Hussain, Congcong

4

Liu, Xinglian Xu, Guanghong Zhou, Chunbao Li*

5

Key Laboratory of Meat Processing and Quality Control, MOE; Key Laboratory of Meat

6

Processing, MARA; Jiangsu Collaborative Innovation Center of Meat Production and Processing,

7

Quality and Safety Control; College of Food Science and Technology, Nanjing Agricultural

8

University, 210095, Nanjing, China

9

Running title: Diets alter gut microbiota and Nrf2/Keap1 signaling

10

Corresponding author:

11

Prof. Dr. Chunbao Li

12

E-mail: [email protected]

13

Tel/Fax: 86 25 84395679

14

1 ACS Paragon Plus Environment


15

ABSTRACT: Oxidative stress may play a critical role in the progression of liver disorders. An

16

increasing interest has been taken in the associations among diet, oxidative stress, gut-liver axis,

17

and non-alcoholic fatty liver disease. Here, we investigated the effects of processed meat

18

proteins on biomarkers of lipid homeostasis, hepatic metabolism, antioxidant functions and gut

19

microbiota composition in glutaredoxin1 deficient (Glrx1-/-) mice. The wild-type (WT) and

20

Glrx1-/- mice were fed soy protein diet (SPD), dry-cured pork protein diet (DPD), braised pork

21

protein diet (BPD) and cooked pork protein diet (CPD) at a dose of 20% of protein for 3 months.

22

Serum and hepatic total cholesterol, serum endotoxin, hepatic liver droplet % and antioxidant

23

capacity were significantly increased by CPD fed WT mice. In addition, CPD fed Glrx1-/- mice

24

significantly increased total cholesterol, triacylglycerol and pro-inflammatory cytokines which

25

are accompanied by higher steatosis scores, intrahepatic lipid accumulation, and altered gene

26

expression associated with lipid metabolism. Furthermore, hepatic gene expression of

27

Nrf2/keap1 signaling pathway and its downstream signaling targets were determined using RT-

28

qPCR. Glrx1 deficiency increased Nrf2 activity and expression of its target genes (GPx, catalase,

29

SOD1, G6pd, and Bbc3), which was exacerbated by intake of CPD. Metagenomic analyses

30

revealed that Glrx1-/- mice fed meat protein diets had higher abundances of Mucispirillum,

31

Oscillibacter, and Mollicutes but lower abundances of Bacteroidales S24-7 group_norank,

32

Blautia, and Anaerotruncus than their wild-type counterparts. In summary, Glrx1 deficiency

33

induced an increase in serum biomarkers for lipid homeostasis, gut microbiota imbalance, and

34

upregulation of Nrf2/Keap1 and antioxidant defense genes, which was aggravated by cooked

35

meat protein diet.

36

Keywords: Glrx1 deficiency, meat proteins, oxidative stress, gut-liver axis, fecal microbiota

37 2 ACS Paragon Plus Environment

Page 2 of 59

Page 3 of 59


38

INTRODUCTION

39

Nonalcoholic fatty liver disease (NAFLD) is the most common form of liver diseases in western

40

countries.1 Unhealthy Western lifestyle plays a major role in the development and progression of

41

NAFLD, which is characteristic of lack of physical activity but high consumption of fructose,

42

saturated fat and red and processed meats.2-4 Meat does not only contain valuable nutrients for

43

human health including protein, iron, zinc and vitamin B12,5 but also contains saturated fatty

44

acids, cholesterol, heme iron, sodium, and advanced glycation end products that may be harmful

45

for patients with NAFLD.6 Indeed, high red and processed meat consumption is associated with

46

insulin resistance (IR), type 2 diabetes, oxidative stress, chronic liver disease, hepatocellular

47

carcinoma and higher risk of mortality.7,8 Processing of meat may cause protein oxidation that

48

induces fragmentation, aggregation and structural changes of proteins.9,10 The biological

49

consequences of such chemical changes are aging and age-related diseases.11,12 Intake of red

50

meat has been found to increase markers of inﬂammation and systemic oxidative damage.13,14 In

51

a recent study, purified meat protein diets exhibited different impacts from casein and soy

52

protein diets on gut microbiota composition, liver metabolism and muscle anti-oxidation in

53

young rats.15 In contrast, a sustained intake of soy protein has negligible impacts on oxidative

54

stress and blood lipids.16 It remains to be established to what extent intake of red meat proteins

55

affects the composition of gut microbiota and antioxidant function of liver as a result of protein

56

oxidation in processed meat.

57

Oxidative stress, which is cellular redox imbalance, is a main contributing factor for liver injury

58

and NAFLD progression.17,18 Mitochondrial dysfunction is the main source of reactive oxygen

59

species (ROS) in fatty liver and serves as a stimulus for lipotoxicity and hepatic inflammation.19

60

Additionally, ROS levels have been linked to development of hepatic inflammatory disorders 3 ACS Paragon Plus Environment


61

such as NAFLD and non-alcoholic steatohepatitis (NASH).20,21 Intracellular ROS production is

62

controlled by a complex network of antioxidants, including glutathione (GSH), catalase,

63

superoxide dismutase (SOD) and glutathione peroxidases (GPx).22

64

There are two major oxidoreductase protein families: thioredoxin (Trx) and glutaredoxin

65

(Glrx).23 Glrx is a primary redox enzyme and a multi-effect cytokine that takes part in cellular

66

growth, apoptosis, cytoskeletal regulation, angiogenesis, inflammation, and protection of cell

67

against oxidative stress.24,25 Quantitative proteomics and metabolomics analyses revealed that

68

Grx1 knockdown decreased the cellular level of GSH but increased ROS production, resulting in

69

activation of p53 and associated signaling pathways.26 Grx is considered as a potential biomarker

70

and key factor in the pathogenesis of chronic kidney disease and diabetes.27, 28 In physiology,

71

Grx1 regulates Nuclear factor-E2-related factor 2/Kelch-like ECH-associated protein1

72

(Nrf2/Keap1), nuclear factor κB (NF-kB), interleukin 1 beta (IL-1β) glutathionylation and sirtuin

73

1 activity.29-32 Deficiency of Glrx1 in mice would promote the development of obesity,

74

hyperlipidemia and NAFLD under a high-fat diet mode.33 However, the effect of Glrx1 in

75

NAFLD induced by oxidative stress as a result of processed meat proteins remains largely

76

elusive.

77

To assess the importance of Glrx1 in NAFLD, we generated animal model of Glrx1 knock out

78

(KO) by deletion of Glrx1 gene using CRISPR cas9 technology, and studied its consequences on

79

metabolism in physiological conditions, and the effects of processed meat protein diet on the

80

host microbial balance, pro-inflammatory cytokines, and antioxidant function of liver compared

81

with WT mice. We measured hepatic antioxidant capacity, pro-inflammatory cytokines, hepatic

82

genes expression of antioxidant enzymes, and regulator of Nrf2/keap1 signaling pathways. The

83

underlying mechanism was discussed. 4 ACS Paragon Plus Environment

Page 4 of 59

Page 5 of 59


84

MATERIALS AND METHODS

85

Animals and experimental design. Construction of Glrx1

86

deleted using CRISPR cas9 technology according to the protocols of Joung et al.34 Briefly, the

87

sgRNA was designed in the corresponding locations of Glrx1 introns, and the Oligo was ordered.

88

The sgRNA vector was constructed by bsal enzyme, and sgRNA and Cas9 mRNA were

89

transcribed using commercial kits (AM1354, AM1908, Ambion Life Technologies, Beijing,

90

China). After that, preparation of single cell fertilized eggs was accomplished by intraperitoneal

91

injection of horse chorionic gonadotropin 5IU. The fertilized eggs were transferred into the

92

prepared M2 strip and arranged in a row (about 30-50). After the injection, the embryos were

93

transferred to the culture dish containing M16 medium. F0+/- generation mice were born about

94

19-21 days after transplantation. At 1 week after birth, F0+/- mice were identified by cutting tail,

95

and positive F0+/- mice were retained. The F0+/- generation mice were backcrossed with

96

C57BL/6J and the six positive F1+/- heterozygotes were obtained. The F1+/- generation is also

97

called the founder lines. The lack of mutation in off-target was verified by PCR amplification

98

and sequencing in founder lines. After creation of a CRISPR edited mice, the founder male mice

99

(F1+/-) were mated to an inbred mice strain having same background used for mutation to

100

eliminate mosaics and off-target effects. Pups carrying the mutation were received through the

101

founder germ line. These mice were denoted “F2-/-”. The F2-/- generation was heterozygous for

102

mutation. Intercross of heterozygous mice yielded healthy off-spring at Mendelian ratios and a

103

sizable experimental cohort was generated. The six positive F1+/- heterozygotes, specific primers

104

for PCR amplification, flow diagram of Glrx1-/- creation and Mendelian genetics to generate

105

cohort are listed in Supporting Information file S1.


-/-

mouse model. Glrx1 gene was


106

Processing of pork products. Pork longissimus dorsi muscles were obtained from a local meat

107

company (Sushi, Jiangsu, China). CPD, BPD and DPD diets were prepared. Briefly, dry-cured

108

pork was prepared by salting pork cuts (size: 10 ×10 ×5cm) with 5% salt, sun-drying for one

109

month and steam-cooking the cuts at 80℃ for 30 min to a core temperature of 72°C. Braised

110

pork was prepared by cutting pork into smaller pieces (size: 1×5 ×5 cm), which were cooked in

111

boiling water for 150 min. Cooked pork was prepared by steam-cooking pork blocks (size: 10

112

×10 ×5cm) at 80°C for 15 min to a core temperature of 72°C.

113

Diet preparation. Cooked meat products were chilled and ground. Methylene chloride and

114

methanol (V/V = 2:1) were used to remove fat. Soy protein was obtained from a commercial

115

company (Shansong Biological Products Inc., Linyi, China) and isoflavones were removed by 80%

116

methanol. The levels of total isoflavones in soy protein were 0.94 mg/g before methanol

117

extraction and 0.03 mg/g after extraction. The diets were prepared according to AIN-93G

118

formulation35 and contained 19.7% protein powder, 39.75% corn starch, 13.2% corn dextrin, 5%

119

fiber, 1.53% mineral premix, 7.0% soybean oil, 1.0% vitamin premix, 10.0% sucrose, 0.25%

120

choline acid salt, 3.0% L-cysteine and 2.27% water. Crude composition, amino acid composition

121

and diet formulation were listed in Supporting Information file S2 (Table S1 to S3).

122

Acclimatization and feeding. All animal studies were carried out in compliance with the

123

relevant guidelines and regulations of the Ethical Committee of Experimental Animal Center of

124

Nanjing Agricultural University, and approved by Experimental Animal Center of Nanjing

125

Agricultural University. Twenty Glrx1-/- and 20 wild-type C57BL/6J male mice (6 weeks old)

126

were kept in a controlled specific-pathogen-free animal center (SYXK < Jiangsu > 2011-0037).

127

After one week of acclimatization, wild-type and Glrx1-/- mice were randomly assigned to one of

128

four diet groups, which contain proteins extracted from soy, dry–cured pork, braised pork or 6 ACS Paragon Plus Environment

Page 6 of 59

Page 7 of 59


129

cooked pork (5 mice per diet group). All mice were housed into separate cages. Mice were given

130

ad libitum access to diets and water for 12 weeks. Body weight and diet intake were recorded

131

weekly.

132

Collection of samples. The mice were sacrificed by cervical dislocation. The mouse is firmly

133

grasped and left eye ball is quickly pulled out with a forcep causing it to bleed. About 200µl of

134

blood samples were collected in Eppendorf tubes and the samples were centrifuged at 12000 ×g

135

for 10 min. Serum samples were collected and stored at -80°C until further analyses. The fecal

136

samples were collected before killing the mice and frozen in liquid nitrogen for 16S rRNA gene

137

sequencing. Liver samples were immediately removed and the largest liver lobe was dissected

138

into three parts, and stored at -80°C until further analyses.

139

Biochemical analyses. Liver samples (0.5 g) were homogenized in 1 mL ice-cold physiological

140

saline for 1 min, and the homogenates were centrifuged at 5000 ×g at 4°C for 10 min.

141

Triacylglycerol (TAG), total cholesterol (T-CHO), low-density lipoprotein cholesterol (LDL-C)

142

levels and high-density lipoprotein cholesterol (HDL-C) were measured in the soluble extracts

143

from serum and liver using a Chemray 240 automatic chemistry analyzer (SPOTCHEM EZ SP-

144

4430, ARKARY, Inc., Kyoto, Japan) following the manufacturer’s instructions. Alanine

145

aminotransferase (ALT) and aspartate aminotransferase (AST) in serum were measured using a

146

commercial kit (K752, K753) (Bio Vision, San Francisco, CA) at 525 nm and 542 nm

147

respectively by a SpectraMax ABS Plus microplate reader (Molecular devices, San Jose,

148

California, USA) following the manufacturer’s instructions. The lipopolysaccharide binding

149

protein (LBP) and lipopolysaccharide (LPS) in serum were measured using an ELISA Kit (No.

150

RGB-60178M, Beijing Rigor, Beijing, China) and commercial AOGENE kit (ANG-E21618M,

151

Shanghai, China) respectively according to the manufacture’s protocols respectively. IL-1β, 7 ACS Paragon Plus Environment


152

tumor necrosis factorα (TNF-α), and interleukin 6 (IL-6) in serum were measured using a

153

commercial Bio-plex kit (5827, Bio-Rad Laboratories, Inc., Hercules, California, USA)

154

according to the manufacturer’s instructions.

155

Determination of oxidative stress and antioxidant enzyme activities. Liver samples (0.5 g)

156

were homogenized in 1 mL ice-cold physiological saline for 1 min, and the homogenates were

157

centrifuged at 5000×g at 4°C for 10 min. The protein concentration in the supernatant was

158

measured by a SpectraMax ABS Plus microplate reader (Molecular devices, San Jose,

159

California, USA) using a Bio-Rad protein assay (Hercules, CA). The supernatants were aliquoted

160

and commercial kits were used to measure the antioxidant activities and oxidative status

161

including catalase, GPx, malondialdehyde (MDA), SOD, and ROS using assay kits (Cayman,

162

Chemical, Co., Ann Arbor, MI) according to the manufacturer’s protocols.

163

Histochemical analysis. Liver samples were fixed in 10% formalin for 48 h at 20°C and

164

embedded in paraffin. The paraffined tissues were sectioned to 6 µm thick for H&E staining to

165

analyze hepatic vacuolization. For Oil Red O staining, snap-frozen liver samples were sectioned

166

to 8 µm thick and stained with 0.2% Oil Red O in 60% of isopropanol for 20 min, and then the

167

sections were rinsed three times with PBS for 10 min each. Images were captured by a light

168

microscope and image analysis was done with the Image-Pro Plus (version 7.01 for Windows

169

(Media Cybernetics, Silver Springs, MD, USA) for estimating lipid droplet percent in liver. The

170

liver inflammation, necrosis, denaturation, hepatocyte ballooning and steatosis scores were

171

assessed using Image-Pro Plus (version 7.01) as described previously.36

172

16S rRNA sequencing. Total genomic DNA in fecal samples was extracted using the QIAamp

173

DNA Stool Mini Kit (DP328, Qiagen, Dusseldorf, Nordrhein-Westfalen, Germany). The purity


Page 8 of 59

Page 9 of 59


174

and concentration of DNA were assessed by a Nanodrop® spectrophotometer (Nanodrop2000,

175

Thermo Fisher Scientific, Waltham, MA). The bacterial 16S rRNA gene sequences of the

176

purified DNA samples were used to amplify the V4 region, which is related with the lowest

177

taxonomic assignment error rate.37 The resulting amplicons were purified, quantified, and

178

construction of a paired-end library was performed following the Illumina genomice DNA

179

library procedure, and the DNA library was sequenced with paired-end (2×250) on an Illumina

180

MiSeq platform (San Diego, CA).

181

The raw data were trimmed and chimeric sequences were removed. The operational

182

taxonomic units (OTUs) were clustered with ≥97% similarity. The community richness

183

estimators (Chao) and abundance-based coverage estimators (ACE), α-diversity indices

184

(Shannon and Simpson indices), and Good’s coverage were calculated.38 Principal coordinate

185

analysis (PCoA) was used on the basis of OTUs to cluster the microbial composition.39

186

Multivariate analysis of variance (MANOVA) was performed to further confirm the observed

187

differences. Linear discriminant analysis effect size (LEfSe) analysis was conducted to identify

188

fecal bacterial biomarkers and to differentiate OTUs among different groups.40

189

In addition, the Spearman correlation was calculated between gut microbiota and serum

190

biomarker, key genes involving hepatic inflammatory and oxidative stress response pathways, or

191

hepatic lipid biomarkers. The Cytoscope (http://www.cytoscope.org) and R (pheatmap package)

192

were applied.

193

mRNA isolation and RT-qPCR. RNA was extracted from liver tissues using a commercial

194

RNeasy mini kit (Takara Bio, Tokyo, Japan) and quantified by a Nanodrop spectrometer

195

(Thermo Scientific, MD, USA) at 260 nm and 280 nm. cDNA was prepared by mixing 4 µL 9 ACS Paragon Plus Environment


196

RNA, 2 µL 5x Prime Script RT Master MIX (Takara) and 4 µL RNase free water. RT-qPCR was

197

performed as described by Nolan, Hands and Bustin.41 SYBER green probe was used for the

198

relative quantification of target gene. Analyses were performed by the comparative (2-ΔΔCT)

199

method,42 and followed by normalization to reference gene β-actin in which SPD group was set

200

as a control. The primers of target genes were listed in Supporting Information file S2 (Table

201

S4).

202

Western blotting. Protein was extracted by homogenizing the liver tissues (50mg) in RIPA

203

buffer that contained protease inhibitor and phosphatase inhibitor (Roche, Penzberg, Germany).

204

The homogenate was centrifuged at 10,000 ×g at 4°C for 5 min and the protein concentration in

205

the supernatant was measured by a SpectraMax ABS Plus microplate reader (Molecular devices,

206

San Jose, CA) using a Bio-Rad protein assay (Hercules, CA). The total protein mass (20 µL) was

207

loaded on 7-10% SDS-PAGE gels. Electrophoresis was run at 100 V for 120 min at 4°C and the

208

gels were soaked in methanol for 30 min. Blotting was performed with primary antibodies Nrf2

209

(1:1000; Cell Signaling Technology, Danvers, MA), Keap1 (1:1000; Cell Signaling Technology,

210

Danvers, Massachusetts, USA), and Glrx1 (1:1000; Abcam; Cambridge, UK), and then changed

211

by goat anti-mouse IgG (H + L) secondary antibody (1:1000; Thermo Pierce, Rockford, lL).

212

Target proteins were normalized against GADPH (1: 1000; Abcam). The protein bands were

213

examined by an Image Quant LAS4000 (GE, Uppsala, Sweden) and the intensities of these

214

proteins were analyzed by the Image J software (Version 1.4.3.67, Broken Symmetry Software,

215

Madison, Wisconsin, USA).

216

Statistical analysis. The main effects of genotype, diet, and their interaction were evaluated by

217

multiple ANOVA and means were compared by Tukey’s multiple comparisons test. Differences


Page 10 of 59

Page 11 of 59


218

were considered significant for P0.05, Table 1). Diet had no significant effect (P>0.05) on diet

223

intake, body weight, liver weight. However, it significantly affected body weight gain and BPD

224

group had the smallest value in both wild-type and Glrx1-/- mice (P

Processed meat protein promoted inflammation and hepatic

Recommend Documents