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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
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Journal of Agricultural and Food Chemistry
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Curcumin attenuates N-nitrosodiethylamine induced liver injury in
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mice by utilizing the method of metabonomics Peiyu Qiu,†, & Jiachen Sun,#, & Shuli Man,*, † He Yang,† Long Ma,† Peng Yu,†
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Wenyuan Gao,∗, ‡
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†
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of Industry Microbiology, National and Local United Engineering Lab of Metabolic Control
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Fermentation Technology, College of Biotechnology, Tianjin University of Science &
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Technology, Tianjin, 300457, China.
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‡
Key Laboratory of Industrial Microbiology, Ministry of Education, Tianjin Key Laboratory
Tianjin Key Laboratory for Modern Drug Delivery and High Efficiency, School of
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Pharmaceutical Science and Technology, Tianjin University, Tianjin, 300072, China.
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#
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.
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Abstract
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N-Nitrosodiethylamine (DEN) as one of food additives existed in cheddar cheese,
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processed meats, beer, water and so forth. It possessed a potent hepatocarcinogen in
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animals and humans. Curcumin as a natural dietary compound decreased
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DEN-induced
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histopathological examination of liver tissues, and biomarker detection in serum and
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livers, it demonstrated that curcumin attenuated DEN-induced hepatocarcinogenesis
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through parts of regulating the oxidant stress enzymes (T-SOD and CAT), liver
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function (ALT and AST) and LDHA, AFP level and COX-2/PGE2 pathway. What’s
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more, curcumin attenuated metabolic disorders via increasing concentration of glucose
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and fructose, and decreasing levels of glycine and proline, and mRNA expression of
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GLUT1, PKM and FASN. Docking study indicated that curcumin presented strong
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affinity with key metabolism enzymes such as GLUT1, PKM, FASN and LDHA.
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There were a number of amino acid residues involved in curcumin-targeting enzymes
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of hydrogen bonds and hydrophobic interactions. All in all, curcumin exhibited a
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potent liver protective agent inhibiting chemically induced liver injury through
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suppressing liver cellular metabolism in the prospective application.
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Keywords: Curcumin; diethylnitrosamine; liver cancer; metabolomics; structural
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simulation
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Abbreviation: AFP, alpha fetoprotein; ALT, alanine aminotransferase; AST, aspartate
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transaminase; ATP5b, ATP synthase 5b; CAT, catalase; COX-2, cyclooxygenase-2; DEN,
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diethylnitrosamine; FASN, fatty acid synthetase; GAPDH, glyceraldehyde-3- phosphate
hepatocarcinogenesis
in
this
research.
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According
to
the
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dehydrogenase; GLS, glutaminase; GLU, glucose; GLUT1&4, glucose transporter 1&4; HDL-C,
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high-density lipoprotein cholesterol; HK2, hexokinase 2; LDHA, lactate dehydrogenase A; LDL-C,
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low-density lipoprotein cholesterol; Myc, myelocytomatosis oncogene; PGE2, prostaglandin E2;
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PKM, pyruvate kinase M; Ras, rat sarcoma; RT-PCR, semi-quantitative reverse transcription and
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polymerase chain reaction; T-CHO, total cholesterol; TG, triglyceride; T-SOD, total superoxide
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dismutase.
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Introduction Food additives often induced a risk of cancer such as potassium bromate, nitrite,
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and other chemicals. N-Nitrosodiethylamine, also known as diethylnitrosamine (DEN),
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belonging to nitrite, was found in cheddar cheese, processed meats, beer, water and so
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forth 1 and possessed an underlying toxicity in lives. As previous report, DEN-induced
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liver injury was suffered from the promotion of cell proliferate, redox ratio decrease,
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inflammation, metabolic disturbances and so forth 2 3. Recently, extensive studies have indicated that curcumin (Figure 1) as the main
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4
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bioactive component of the spice turmeric
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pharmacological effects such as hepatoprotective, anti-inflammatory 5, anti-oxidant 6 7,
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anti-carcinogenic
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research data demonstrated that curcumin protected against chronic carbon
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tetrachloride
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tetrachloro-p-benzoquinone 12, aflatoxin B1 13, and heavy metals-induced liver damage
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14
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anti-inflammation
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DEN-induced hepatocarcinogenesis through decreasing the levels of p21, proliferating
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cell nuclear antigen and cell division cycle protein 2 in mice
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oxidative tissue damage 17, and exerted antiangiogenic, antiproliferative, and apoptotic
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effects in rats 18. All these indicated that curcumin would be used in the protection of
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liver cancer. To further reveal the mechanisms of curcumin-treatment in liver injury,
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we adopted metabonomics and network biology approaches. It might help us elucidate
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,
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, possessed different kinds of
and so on. Especially for the hepatoprotective effects, growing
D-galactosamine/lipopolysaccharide
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,
acute
alcoholism
. The protective effects were mainly contributed to the antioxidant activities 15
10
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,
and
of curcumin. Meanwhile, curcumin effectively inhibited
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, protected against
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the pathways involved in curcumin intervening liver injury.
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Figure 1 The chemical formula of curcumin.
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Materials and methods
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Experimental animals
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Thirty-three male Kunming mice weighting 16~18 g were provided by the Laboratory
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Animal Center of academy of Military Medical Sciences (Beijing, China quality certification
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number: SCXK (Jun) 2012-0004). The mice were randomly classified into three groups.
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Normal group: injected with saline solution and oral saline solution. Model group: injected
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with DEN (100 mg/kg body weight, once a week for twenty-five weeks) and oral saline
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solution every other day, drinking water containing 0.05% PB launched in the 3rd week.
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Curcumin group: injected with DEN (100 mg/kg body weight, once a week for twenty-five
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weeks) and oral administration of curcumin (purchased from Zhongda Co. (China)) (10 mg/10
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mL/kg body weight/2 d) started in the 2nd week, drinking water containing 0.05% PB launched
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in the 3rd week. During the process, three mice were sacrificed in the model group.
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Blood samples (0.5 mL) were immediately processed, frozen and maintained at -20°C
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until analyses. Portions of each tissue were fixed in 10% formalin (pH 7.4) for histology. In
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the following experiments, the mice left (more than 9) were classified into three subgroups in
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each group. Using the pooled samples, GC/MS analysis and molecular studies (enzymes,
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western blots and mRNA) were carried out.
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Histopathological examination
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For the histopathological examination, five-µm-thick sections were prepared and stained
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with hematoxylin and eosin. Histopathology examination was evaluated by a pathologist who
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was unaware of whether tissues were treated.
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Biochemical analyses
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Serum levels of aspartate transaminase (AST), alanine transaminase (ALT), lactate
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dehydrogenase (LDH), glucose (GLU), total cholesterol (T-CHO), triglyceride (TG),
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high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C)
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were measured by the detection kits (Nanjing, China).
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Oxidative stress
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The supernatant of the total liver homogenized sample (10% homogenates) was used to
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detect the levels of total superoxide dismutase (T-SOD), and catalase (CAT) activities
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according to the manufacturer’s instructions (Nanjing Jiancheng Bioengineering Institute).
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Semiquantitative reverse transcription PCR
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According to the previous research19, total RNA was isolated from mice liver using
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TRIzol (Sangon Biotech Co., Shanghai, China) according to the manufacturer's instruction.
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Polymerase chain reaction products were electrophorized on 3.0% agarose gel and visualized
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after ethidium bromide staining. The primer sequences and the cycling conditions were
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summarized in Table 1.
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Table 1 Primers sequences and cycling conditions for PCR
Gene
Annealing
Product
temperatures (℃)
(bp)
Nucleotide sequences for primers
TGCAGCCCAAGGATCTCTCT Glut1
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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
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TCGTCTGCTTGAATGGACAG GACGGAAGGAAGGAAAGAGG H-ras
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103
56
411
55
213
AAATTGTCCATGCGAAGGTC GCTCACACCAAAGCGTCAAC
AFP CCTGTGAACTCTGGTATCAG
ATTCAACGGCACAGTCAAGG GAPDH GCAGAAGGGGCGGAGATGA
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Measurements of COX-2 and PGE2 levels in serum
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Serum samples were analyzed for cyclooxygenase-2 (COX-2) (Nanjing SenBeiJia
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Biological Technology Co., Ltd. China) and prostaglandin E2 (PGE2) (Nanjing SenBeiJia
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Biological Technology Co., Ltd. China) with mouse ELISA kits according to the
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manufacturer's instructions 19.
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Western blot assay
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According to previous research19, alpha fetoprotein (AFP) (Boster, China) antibody was
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visualized by Odyssey infrared imaging system (LI-COR Biotechnology, USA) and quantified
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using Image-Pro Plus software.
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GC/MS detection
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Referring to previous methods
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GC/MS detection.
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Molecular docking
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, serum samples were performed on an Agilent 7890A
Docking simulations were carried out with Autodock Vina (version 1.1.2)
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. Three
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dimensional structures of curcumin were downloaded from PubChem Compound
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(http://pubchem.ncbi.nlm.nih.gov).
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MGLTools-1.4.6 was used to prepare protein (protein.pdbqt), write grid parameter file
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(protein.gpf) and dock parameter file (ligand.dpf). Docking was performed sequentially in two
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steps. Initially, a box large enough to cover the whole protein (75 Å×60 Å×60 Å) was used to
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detect potential binding pockets. Then, the second step involved localized docking with a
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smaller box (22.5 Å×22.5 Å×22.5 Å) centered at the potential binding site of interest.
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Result
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Organ weight
All
ligands
were
prepared
to
pdbqt
format.
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As previous report, consequent injection of DEN accompanied with oral
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administration of phenobarbital (PB) in water induced hepatocarcinogen in mice 22. As
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Table 2 indicated, relative organ weight of liver and spleen was significantly elevated
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in the model mice compared with the normal ones (p
