Curcumin Attenuates N-Nitrosodiethylamine-Induced Liver Injury in

Feb 15, 2017 - There were a number of amino acid residues involved in curcumin-targeting enzymes of hydrogen bonds and hydrophobic interactions. All i...
1 downloads 14 Views 2MB Size
Subscriber access provided by University of Newcastle, Australia

Article

Curcumin attenuates N-nitrosodiethylamine induced liver injury in mice by utilizing the method of metabonomics Peiyu Qiu, Jiachen Sun, Shuli Man, He Yang, Long Ma, Peng Yu, and Wenyuan Gao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b04797 • Publication Date (Web): 15 Feb 2017 Downloaded from http://pubs.acs.org on February 17, 2017

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 free 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 accessible to all readers and 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.

Journal of Agricultural and Food Chemistry 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 38

Journal of Agricultural and Food Chemistry

1

Curcumin attenuates N-nitrosodiethylamine induced liver injury in

2

mice by utilizing the method of metabonomics Peiyu Qiu,†, & Jiachen Sun,#, & Shuli Man,*, † He Yang,† Long Ma,† Peng Yu,†

3

Wenyuan Gao,∗, ‡

4 5



6

of Industry Microbiology, National and Local United Engineering Lab of Metabolic Control

7

Fermentation Technology, College of Biotechnology, Tianjin University of Science &

8

Technology, Tianjin, 300457, China.

9



Key Laboratory of Industrial Microbiology, Ministry of Education, Tianjin Key Laboratory

Tianjin Key Laboratory for Modern Drug Delivery and High Efficiency, School of

10

Pharmaceutical Science and Technology, Tianjin University, Tianjin, 300072, China.

11

#

Tianjin University of Traditional Chinese Medicine, Tianjin, 300193, China



Corresponding authors: Shuli Man ([email protected], Tel, 86-022-60601265) and Wenyuan Gao

([email protected], Tel./fax, 86-022-87401895). &

Peiyu Qiu and Jiachen Sun are regarded as co-first authors and contributed equally to this work.

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

12

Page 2 of 38

Abstract

13

N-Nitrosodiethylamine (DEN) as one of food additives existed in cheddar cheese,

14

processed meats, beer, water and so forth. It possessed a potent hepatocarcinogen in

15

animals and humans. Curcumin as a natural dietary compound decreased

16

DEN-induced

17

histopathological examination of liver tissues, and biomarker detection in serum and

18

livers, it demonstrated that curcumin attenuated DEN-induced hepatocarcinogenesis

19

through parts of regulating the oxidant stress enzymes (T-SOD and CAT), liver

20

function (ALT and AST) and LDHA, AFP level and COX-2/PGE2 pathway. What’s

21

more, curcumin attenuated metabolic disorders via increasing concentration of glucose

22

and fructose, and decreasing levels of glycine and proline, and mRNA expression of

23

GLUT1, PKM and FASN. Docking study indicated that curcumin presented strong

24

affinity with key metabolism enzymes such as GLUT1, PKM, FASN and LDHA.

25

There were a number of amino acid residues involved in curcumin-targeting enzymes

26

of hydrogen bonds and hydrophobic interactions. All in all, curcumin exhibited a

27

potent liver protective agent inhibiting chemically induced liver injury through

28

suppressing liver cellular metabolism in the prospective application.

29

Keywords: Curcumin; diethylnitrosamine; liver cancer; metabolomics; structural

30

simulation

31

Abbreviation: AFP, alpha fetoprotein; ALT, alanine aminotransferase; AST, aspartate

32

transaminase; ATP5b, ATP synthase 5b; CAT, catalase; COX-2, cyclooxygenase-2; DEN,

33

diethylnitrosamine; FASN, fatty acid synthetase; GAPDH, glyceraldehyde-3- phosphate

hepatocarcinogenesis

in

this

research.

ACS Paragon Plus Environment

According

to

the

Page 3 of 38

Journal of Agricultural and Food Chemistry

34

dehydrogenase; GLS, glutaminase; GLU, glucose; GLUT1&4, glucose transporter 1&4; HDL-C,

35

high-density lipoprotein cholesterol; HK2, hexokinase 2; LDHA, lactate dehydrogenase A; LDL-C,

36

low-density lipoprotein cholesterol; Myc, myelocytomatosis oncogene; PGE2, prostaglandin E2;

37

PKM, pyruvate kinase M; Ras, rat sarcoma; RT-PCR, semi-quantitative reverse transcription and

38

polymerase chain reaction; T-CHO, total cholesterol; TG, triglyceride; T-SOD, total superoxide

39

dismutase.

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

40

Page 4 of 38

Introduction Food additives often induced a risk of cancer such as potassium bromate, nitrite,

41 42

and other chemicals. N-Nitrosodiethylamine, also known as diethylnitrosamine (DEN),

43

belonging to nitrite, was found in cheddar cheese, processed meats, beer, water and so

44

forth 1 and possessed an underlying toxicity in lives. As previous report, DEN-induced

45

liver injury was suffered from the promotion of cell proliferate, redox ratio decrease,

46

inflammation, metabolic disturbances and so forth 2 3. Recently, extensive studies have indicated that curcumin (Figure 1) as the main

47

4

48

bioactive component of the spice turmeric

49

pharmacological effects such as hepatoprotective, anti-inflammatory 5, anti-oxidant 6 7,

50

anti-carcinogenic

51

research data demonstrated that curcumin protected against chronic carbon

52

tetrachloride

53

tetrachloro-p-benzoquinone 12, aflatoxin B1 13, and heavy metals-induced liver damage

54

14

55

anti-inflammation

56

DEN-induced hepatocarcinogenesis through decreasing the levels of p21, proliferating

57

cell nuclear antigen and cell division cycle protein 2 in mice

58

oxidative tissue damage 17, and exerted antiangiogenic, antiproliferative, and apoptotic

59

effects in rats 18. All these indicated that curcumin would be used in the protection of

60

liver cancer. To further reveal the mechanisms of curcumin-treatment in liver injury,

61

we adopted metabonomics and network biology approaches. It might help us elucidate

9

,

8

, possessed different kinds of

and so on. Especially for the hepatoprotective effects, growing

D-galactosamine/lipopolysaccharide

10

,

acute

alcoholism

. The protective effects were mainly contributed to the antioxidant activities 15

10

11

,

and

of curcumin. Meanwhile, curcumin effectively inhibited

ACS Paragon Plus Environment

16

, protected against

Page 5 of 38

Journal of Agricultural and Food Chemistry

62

the pathways involved in curcumin intervening liver injury.

63 64

Figure 1 The chemical formula of curcumin.

65

Materials and methods

66

Experimental animals

67

Thirty-three male Kunming mice weighting 16~18 g were provided by the Laboratory

68

Animal Center of academy of Military Medical Sciences (Beijing, China quality certification

69

number: SCXK (Jun) 2012-0004). The mice were randomly classified into three groups.

70

Normal group: injected with saline solution and oral saline solution. Model group: injected

71

with DEN (100 mg/kg body weight, once a week for twenty-five weeks) and oral saline

72

solution every other day, drinking water containing 0.05% PB launched in the 3rd week.

73

Curcumin group: injected with DEN (100 mg/kg body weight, once a week for twenty-five

74

weeks) and oral administration of curcumin (purchased from Zhongda Co. (China)) (10 mg/10

75

mL/kg body weight/2 d) started in the 2nd week, drinking water containing 0.05% PB launched

76

in the 3rd week. During the process, three mice were sacrificed in the model group.

77

Blood samples (0.5 mL) were immediately processed, frozen and maintained at -20°C

78

until analyses. Portions of each tissue were fixed in 10% formalin (pH 7.4) for histology. In

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

79

the following experiments, the mice left (more than 9) were classified into three subgroups in

80

each group. Using the pooled samples, GC/MS analysis and molecular studies (enzymes,

81

western blots and mRNA) were carried out.

82

Histopathological examination

83

For the histopathological examination, five-µm-thick sections were prepared and stained

84

with hematoxylin and eosin. Histopathology examination was evaluated by a pathologist who

85

was unaware of whether tissues were treated.

86

Biochemical analyses

87

Serum levels of aspartate transaminase (AST), alanine transaminase (ALT), lactate

88

dehydrogenase (LDH), glucose (GLU), total cholesterol (T-CHO), triglyceride (TG),

89

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

90

were measured by the detection kits (Nanjing, China).

91

Oxidative stress

92

The supernatant of the total liver homogenized sample (10% homogenates) was used to

93

detect the levels of total superoxide dismutase (T-SOD), and catalase (CAT) activities

94

according to the manufacturer’s instructions (Nanjing Jiancheng Bioengineering Institute).

95

Semiquantitative reverse transcription PCR

96

According to the previous research19, total RNA was isolated from mice liver using

97

TRIzol (Sangon Biotech Co., Shanghai, China) according to the manufacturer's instruction.

98

Polymerase chain reaction products were electrophorized on 3.0% agarose gel and visualized

99

after ethidium bromide staining. The primer sequences and the cycling conditions were

100

summarized in Table 1.

ACS Paragon Plus Environment

Page 6 of 38

Page 7 of 38

Journal of Agricultural and Food Chemistry

101

Table 1 Primers sequences and cycling conditions for PCR

Gene

Annealing

Product

temperatures (℃)

(bp)

Nucleotide sequences for primers

TGCAGCCCAAGGATCTCTCT Glut1

55

76

60

96

53

139

50

409

53

131

54

144

58

94

47

287

55

62

54

139

CGGCTTGCCCGAGATCT GGACAGCCAGCCTACGCCACCATA Glut4 TGTCCGTCGTCCAGCTCGTTCTAC TCAAAGAGAACAAGGGCGAG HK2 AGGAAGCGGACATCACAATC CCATTCTCTACCGTCCTGTTG PKM TCCATGTAAGCGTTGTCCAG GCTCCCCAGAACAAGATTACAG LDHA TCGCCCTTGAGTTTGTCTTC CCGTGAAGGCAATGATTTATAC ATP5b GTCAGCCCAGTCAGAGCTACC AGCACTGCCTTCGGTTCAGTC FASN AAGAGCTGTGGAGGCCACTTG GAGAAAGTGGAGATCGAAATT GLS ACCAACATGGAACGCAAA GCATGAACCGCCGACCTAT P53 CAGAAGGTTCCCACTGGAGTCT Myc

GTCAGAGGAGGAACGAGCTG

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 8 of 38

TCGTCTGCTTGAATGGACAG GACGGAAGGAAGGAAAGAGG H-ras

53

103

56

411

55

213

AAATTGTCCATGCGAAGGTC GCTCACACCAAAGCGTCAAC

AFP CCTGTGAACTCTGGTATCAG

ATTCAACGGCACAGTCAAGG GAPDH GCAGAAGGGGCGGAGATGA

102 103

Measurements of COX-2 and PGE2 levels in serum

104

Serum samples were analyzed for cyclooxygenase-2 (COX-2) (Nanjing SenBeiJia

105

Biological Technology Co., Ltd. China) and prostaglandin E2 (PGE2) (Nanjing SenBeiJia

106

Biological Technology Co., Ltd. China) with mouse ELISA kits according to the

107

manufacturer's instructions 19.

108

Western blot assay

109

According to previous research19, alpha fetoprotein (AFP) (Boster, China) antibody was

110

visualized by Odyssey infrared imaging system (LI-COR Biotechnology, USA) and quantified

111

using Image-Pro Plus software.

112

GC/MS detection

113

Referring to previous methods

114

GC/MS detection.

115

Molecular docking

116

20

, serum samples were performed on an Agilent 7890A

Docking simulations were carried out with Autodock Vina (version 1.1.2)

ACS Paragon Plus Environment

21

. Three

Page 9 of 38

Journal of Agricultural and Food Chemistry

117

dimensional structures of curcumin were downloaded from PubChem Compound

118

(http://pubchem.ncbi.nlm.nih.gov).

119

MGLTools-1.4.6 was used to prepare protein (protein.pdbqt), write grid parameter file

120

(protein.gpf) and dock parameter file (ligand.dpf). Docking was performed sequentially in two

121

steps. Initially, a box large enough to cover the whole protein (75 Å×60 Å×60 Å) was used to

122

detect potential binding pockets. Then, the second step involved localized docking with a

123

smaller box (22.5 Å×22.5 Å×22.5 Å) centered at the potential binding site of interest.

124

Result

125

Organ weight

All

ligands

were

prepared

to

pdbqt

format.

126

As previous report, consequent injection of DEN accompanied with oral

127

administration of phenobarbital (PB) in water induced hepatocarcinogen in mice 22. As

128

Table 2 indicated, relative organ weight of liver and spleen was significantly elevated

129

in the model mice compared with the normal ones (p