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Liver fibrosis can be induced by high salt intake through excess ROS production Guang Wang, Cheung-kwan Yeung, Wing-Yan Wong, Nuan Zhang, Yi-fan Wei, Jing-li Zhang, Yu Yan, Ching-yee Wong, Jun-jie Tang, Manli Chuai, Kenneth Ka-ho Lee, Li-jing Wang, and xuesong Yang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b05897 • Publication Date (Web): 04 Feb 2016 Downloaded from http://pubs.acs.org on February 10, 2016
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.
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Liver fibrosis can be induced by high salt intake through excess ROS production
2 ∥
∥
3
Guang Wang† , Cheung-kwan Yeung † , Wing-Yan Wong †, Nuan Zhang†, Yi-fan Wei†,
4
Jing-li Zhang‡ ,Yu Yan†, Ching-yee Wong†, Jun-jie Tang†,Manli Chuai§, Kenneth Ka
5
Ho Lee#, Li-jing Wang‡, Xuesong Yang*†
6
†
Division of Histology and Embryology, Key Laboratory for Regenerative Medicine of
7
the Ministry of Education, Medical College, Jinan University, Guangzhou 510632,
8
China
9
‡
Institute of Vascular Biological Sciences, Guangdong Pharmaceutical University, Guangzhou 510006, China
10
11
§
Division of Cell and Developmental Biology, University of Dundee, Dundee, DD1 5EH, UK
12
13 14
#
Key Laboratory for Regenerative Medicine of the Ministry of Education, School of Biomedical Sciences, Chinese University of Hong Kong, Shatin, Hong Kong
15
∥
16
contributed to this work equally.
17
18 19
*
The corresponding author：X Yang. E-mail: [email protected] Tel: +86(20)85228316
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Abstract
21
High salt intake has been known to cause hypertension and other side effects.
22
However, it is still unclear whether it also affects fibrosis in mature and the
23
developing liver. In this study, we demonstrated that high salt exposure in mice (4%
24
NaCl in drinking water) and chick embryo (calculated final osmolality of the egg was
25
300 mosm/l) could lead to derangement of the hepatic cords and liver fibrosis using
26
H&E, PAS, Masson and Sirius red staining. Meanwhile, Desmin immunofluorescent
27
staining of mouse and chick embryo liver indicated that hepatic stellate cells were
28
activated after the high salt exposure. pHIS3 and BrdU immunohistological staining
29
of mouse and chick embryo livers indicated that cell proliferation decreased as well as
30
the TUNEL analyses indicated that cell apoptosis increased in the presence of high
31
salt exposure. Next, dihydroethidium staining on the cultured chick hepatocytes
32
indicates the excess ROS was generated following high salt exposure. Furthermore,
33
AAPH (a known inducer of ROS production) treatment also induced the liver fibrosis
34
in chick embryo. Nrf2 and Keap1 positively immunohistological staining on mouse
35
liver suggested the Nrf2/Keap1 signaling was involved in high salt-induced ROS
36
production. Finally, CCK8 assay was used to determine whether or not the growth
37
inhibitory effect induced by high salt exposure can be rescued by anti-oxidant
38
Vitamin C. Meanwhile, the RT-PCR result indicated that the Nrf2/Keap1downsteam
39
genes including HO-1, NQO-1 and SOD2 were involved in this process. In sum, our
40
experiments suggest that high salt intake would lead to high risk of liver damage and
41
fibrosis in both adult and developing embryos. The pathological mechanism may be
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the result from an imbalance between oxidative stress and antioxidant system.
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Key words: high salt, liver fibrosis, mouse, chick embryos, ROS
44 45
Introduction
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A normal and stable osmolarity of the tissue extracellular environment is
47
indispensable for cells to maintain their normal physiological functions. Osmolarity is
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principally determined by ionic compounds, such as sodium chloride (NaCl), which
49
are dissociated into Na+ and Cl-. These ions could affect the osmolarity of blood and
50
extracellular fluids and in this context, a correct amount of salt intake is important for
51
maintaining normal osmolarity in our bodies. The causative relationship between
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dietary salt intake and high osmolarity has long been debated by epidemiologists and
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clinical researchers
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an increase in extracellular osmolarity in epidemiological and clinical studies. In
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animal experiments, monkeys have been placed on a high salt diet for one year
56
developed a significant increase in their extracellular osmolarity 4. Consequently, the
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high osmolarity levels may damage in the arteries, kidneys, eyes and heart, prior to
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hypertension being diagnosed 5-7. However, it is still controversial whether or not high
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salt intake affects the liver morphology and function - especially with regard to the
60
fibrosis in developing liver.
1-3
. Excessive salt consumption has been demonstrated to lead to
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The liver is largest organ in our body and it is crucial to our metabolism since
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many exogenous chemical compounds such as alcohol and drugs are processed and
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degraded there 8. In the embryo, proper development of the liver is dependant of many
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signalling molecules and cell-cell interaction. The mechanism involved is highly
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conserved and involves complex series of developmental signalling pathways and
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Wnt/β-catenin signalling is critical for liver development, differentiation and cellular
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homeostasis 9. Liver fibrosis involves both cellular and extracellular changes which
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are induced by constant insults and damage to the liver, such as chronic inflammation
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and virus infections. These insults activate the normally quiescent hepatic stellate
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cells in the liver to over-produce extra cellular matrix (ECM). Consequently, the
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excess ECM negatively impacts on the hepatocytes altering the liver morphology and
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function over a long and slow pathological process.
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The excess deposits of collagen in the space of tissue also reduce blood flow
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through the liver tissues. Histologically, changes in the hepatic architecture are
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deemed as the “gold standard” for assessing the extent of fibrosis in the liver
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Chronic inflammation and loss of metabolic homeostasis play important roles in the
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pathogenesis of liver fibrosis. In addition, there is accumulating evidence that both of
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these pathological processes are linked with oxidative stress and ROS production
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8-11-12
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ROS production and exacerbate ROS-associated damage in liver tissue. However, it is
81
still controversial whether or not the Nrf2/Keap1 signalling - antioxidant system,
82
which includes key negative transcriptional regulators of ROS production, is involved
83
in this process.
10
.
. Although Uetake et al. (2015) indicated that the salt overload might induce
84
In this study, we investigated the effects of high salt exposure on the liver
85
morphology and the manifestation of fibrosis in the mouse model. We also used the
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chick embryo model to examine how high salt treatment affected the normal
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development of the liver. Lastly, we determined high salt-induced liver fibrosis in
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both adult and developing chick embryo was due to the excess ROS, which may be
89
attributed to reduced inhibitory effect of Nrf2/Keap1 signaling.
90 91 92
Materials and Methods
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Mice
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Five-week-old C57BL/6 mice were obtained from the Guangdong Province
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Animal Centre and maintained at 25°C on a 12 hr light/dark cycle. The mice were
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housed in a pathogen-free animal facility at the Institute of Vascular Biological
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Sciences, Guangdong Pharmaceutical University. As previously described 13, the mice
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were allowed free access to normal sodium food (0.5% NaCl) and water. Female mice
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were divided into control (n=6) and experimental groups (n=8), while the latter
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maintained on 4% NaCl water and control mice on pure water for 4 weeks. The
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animals were experimented on and maintained according to guidelines set by the
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animal experimental ethics committee at Jinan University.
103 104
Chick embryos
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Fertilized chick eggs were obtained from the Avian Farm of the South China
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Agriculture University. The eggs were incubated in a humidified incubator (Yiheng
107
Instrument, Shanghai, China) at 38°C and 70% humidity. After 36 hours of
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pre-incubation, the chick embryos (n=30) were treated with a high salt solution as
109
previously described
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chick eggs and treated with two different concentrations of NaCl (totally added
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volume was 500 µl). For the control group, 0.7% NaCl was injected (final osmolality
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per egg was calculated as 240 mosm/l). In the high salt group, we injected a 16.85%
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NaCl solution (the calculated final osmolality in the egg was 300 mosm/l). The treated
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embryos were incubated for a further 4 or 12.5 days.
14-15
. Briefly, 2 ml of albumen were firstly extracted from the
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The chick embryos were also treated with the 2,2’-azobis(2-amidinopropane)
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Dihydrochloride (AAPH; Sigma-Aldrich, St. Louis, Missouri, USA) as previously
117
described
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AAPH/egg (100 µl AAPH (100mM) injection) were injected into the air egg chamber
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containing 9-day-old chick embryoswhile in the control group, each egg was given
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the same volume of simple saline (0.72% sodium chloride; n=20 in each group). The
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treated embryos were incubated for a further 8 days. Afterwards, the incubated
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embryos were firstly determined whether they were alive or dead using the presence
123
of heartbeats and all dead embryos were excluded from the analysis. The
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experimental embryos were removed from the egg shell and weighed.
16
. 5µmol AAPH/egg (100 µl AAPH (50mM) injection) or 10µmol
125 126
Histology
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Briefly, 5.5-day old chick embryos, mouse livers, 14 and 17 day-old chick
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embryo livers (control, high salt treated and AAPH treated) were fixed in 4%
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paraformaldehyde at 4°C for 24 hours. The specimens were then dehydrated, cleared
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in xylene and embedded in paraffin wax. The embedded specimens were serially
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sectioned at 5 µm using a rotary microtome (Leica, RM2126RT, Wetzlar, Hessen,
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Germany). The sections were stained with for haematoxylin and eosin (H&E),
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Periodic acid Schiff (PAS) reaction 16, Masson's trichrome dyes (Masson staining) 16,
134
Sirius red 17 or immunohistochemically. The Masson and Sirius red staining were used
135
to reveal the presence of fibrosis in liver sections. Photographs were captured of the
136
stained histological sections using by an epi-fluorescence microscope (Olympus IX51,
137
Leica DM 4000B) at 200× magnification.
138 139
Immunohistological staining
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Hydrated sections of chick embryos, chick embryo livers and mouse livers were
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firstly treated with citrate buffer (pH 6.0) and heated in a microwave for antigen
142
retrieval. Immunofluorescent staining was then performed the treated sections. Briefly,
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The primary antibodies were diluted using the PBT-NGS and the sections were
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incubated with p-Histone H3 (pHIS3; 1:400, Santa Cruz Biotechnology, Dallas, Texas,
145
USA), Desmin (1:200, Sigma-Aldrich, St. Louis, Missouri, USA), Nrf2 (1:100, Santa
146
Cruz Biotechnology, Dallas, USA) or Keap1 (1:100, Santa Cruz Biotechnology,
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Dallas, Texas, USA) primary antibody at 4°C overnight. Following 4×30mins washes
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in PBS, the sections were incubated with goat anti-rabbit IgG conjugated Alexa Fluor
149
555 (1:1000, life technologies, Carlsbad, California, USA) for pHIS3, Nrf2 and
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Keap1 or goat anti-mouse IgG conjugated Alexa Fluor 488 (1:1000, life technologies,
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Carlsbad, California, USA) for Desmin for 1 hour. The sections were counterstained
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with DAPI (1:1000, life technologies, Carlsbad, California, USA) at room
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temperature for 30 min before examination. For immunohistochemical staining,
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biotinylated goat anti-rabbit serum IgG (1:1000, Cell signaling technology, Danvers,
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Massachusetts,
156
Daminobenzidine tetrahydrochloride substrate (DAB kit, MXB, Fuzhou, Fujian,
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China) were used to visualize the immunostaining. Photographs were captured of the
158
stained histological sections using by an epi-fluorescence microscope (Olympus IX51,
159
Leica DM 4000B) at 200× magnification.
USA)
for
pHIS3
was
used
as
the
secondary
antibody.
160 161
Cell proliferation analyses
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10 µl of 5-Bromo-2-deoxyUridine (BrdU, 10 mM, Sigma-Aldrich, St. Louis,
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Missouri, USA) solution was administered to high salt-treated or control chick
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embryos for 4 hours before the embryos reached 5.5 day-old. The liver of these
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embryos (n=6) were harvested and transverse sections of the livers were produced.
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These sections were stained with BrdU monoclonal antibody (1:200, BD bioscience,
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Franklin Lakes, New jersey, USA) according to manufacturer’s instructions (Roche,
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Basel, Basel-Stadt, Switzerland). The sections were incubated with goat anti-mouse
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IgG conjugated Alexa Fluor 555 (1:1000, life technologies, Carlsbad, California, USA)
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for BrdU for 1 hour. Cell counts were performed on randomly selected areas of the
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BrdU-stained liver sections (visual field at 200× magnification). We counted the
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number of BrdU positive cells against the total cell number to calculate the
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proliferation index (BrdU+ cells/ total cell numbers). Photographs were captured of
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the stained histological sections using by an epi-fluorescence microscope (Olympus
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IX51, Tokyo, Japan) at 200× magnification.
176 177
TUNEL analyses
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The extent of apoptosis in the mice and chick embryo livers was established using an
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In Situ Cell Death Detection Kit (Roche, Basel, Basel-Stadt, Switzerland). The
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TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end
181
labeling) staining was performed according to the instructions provided by the
182
manufacturer, in which were adapted for tissue labelling on glass slides.
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Daminobenzidine tetrahydrochloride substrate (DAB kit, MXB, Fuzhou, Fujian,
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China) was used to visualize the immunostaining. The presence of TUNEL+ cells was
185
determined using an Olympus microscope attached to an Image Analysis Software
186
(Olympus IX51, Tokyo, Japan). We quantified the percentage of TUNEL+ hepatocytes
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relative to all the hepatocytes in each histological slide. We compared the percentage
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of TUNEL+ hepatocytes at the similar level in the control mice or 5.5-days chick
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embryo and high slat treated mice and 5.5-days chick embryos (n=6 in each group
190
from 6 mouse or chick embryos).
191 192
Dihydroethidium and CCK8 assays
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The livers of 17 day-old chick embryos (N=3) were perfused with Ca2+ free
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Krebs ringer-HEPES buffer containing collagenase (pH 7.4, Wako, Tokyo, Japan) and
195
a trypsin inhibitor (Sigma-Aldrich, St. Louis, Missouri, USA) at 37°C. After breaking
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the eggshell and dissection, 37°C pre-warmed D-Hanks Balanced salt solution was
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injected through hepatic portal vein slowly. When liver began to blanch, made a cut at
198
inferior vena cava to allow erythrocyte efflux till liver became pale. Switch perfusion
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solution to pre-warmed 0.05% collagenase IV (Sigma-Aldrich, St. Louis, Missouri,
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USA). Cut away the liver from embryo when it looked mushy and washed in
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D-Hanks solution. Dissected the liver in DMEM-F12 (Hyclone, Logan City, Utah,
202
USA), and then filtered the cell dispersion through 75µm pore size cell strainer into a
203
EP tube to remove undigested tissue fragments. Centrifuged at 2000rpm for 3 min at
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4 ℃ . Removed the supernatant and gently re-suspended cells with DMEM-12.
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Repeated centrifugation again. Removed the supernatant, and suspended cells with
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DMEM-F12 containing 10%FBS and 10-6 dexamethasone (Sigma-Aldrich, St. Louis,
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Missouri, USA) for a better attachment of hepatocytes. After 48 hours of incubation,
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the culture medium was replaced with fresh medium containing and not containing
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1g/100ml NaCl for 30 mins. Dihydroethidium (DHE) staining were performed at the
210
end of cell culture using DHE fluorescent probe (Beyotime, Shanghai, China) to
211
detecting the presence of superoxide anion (O2-). The cells were incubated with 10
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µM DHE for 30 min, at 37°C and then collected for analyses according to the
213
manufacturer’s instructions.
214
HEK293 cells were seeded into 96-well plates and used in the vitamin C (VC,
215
Sigma-Aldrich, St. Louis, USA) rescue experiment. These cells (1×106 cells/ml) were
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maintained in DMEM + 10% fetal bovine serum at 37°C and 5% CO2. Simple saline
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(control) or 1g NaCl per 100ml culture medium with or without VC (40, 80 or
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160µg/ml)
were added to the HEK293 cultures. The cell viability was assessed
219
using CCK8 assay (Cholecystokinin-8). Briefly, 10 µl of CCK8 reagent (Dojindo,
220
Kumamoto, Japan) was added to the 96-well plates and incubated continually for 4
221
hours at 37°C. The absorbance values were measured at 450 nm using a BIO-RAD
222
Model 450 Microplate Reader (BIO-RAD, Hercules, California, USA). The cell
223
viability was indirectly determined by examining the ratio of the absorbance value of
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high salt-treated cells relative to the control cells, from three independent
225
experiments.
226 227
Semi-quantitative RT-PCR
228
The primary culture of embryonic chick hepatocytes was from six 17 day-old chick
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embryos. Simple saline (control) or 1g NaCl per 100ml culture medium with or
230
without VC (80 µg/ml) were added to the cultures and incubated for 30mins. Total
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RNA was isolated from primary cultured hepatocytes using TRIzol (Invitrogen,
232
Carlsbad, California, USA,) according to the manufacturer’s instructions. First-strand
233
cDNA was synthesized to a final volume of 25 µl using the SuperScript RIII
234
first-strand synthesis system (Invitrogen, Carlsbad, California, USA). Following
235
reverse transcription, PCR amplification of the cDNA was performed as described
236
previously
237
(Glyceraldehyde-3-phosphate dehydrogenase): GTCAACGGATTTGGCCGTAT and
238
AATGCCAAAGTTGTCATGGATG;
239
GTCGTTGGCAAGAAGCATCC and ACTCCTTGTGCGAAGCTCTG; NQO-1
19-20
.
The
primers
used
were
as
HO-1(Heme
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oxygenase
GAPDH
1):
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(NAD(P)H dehydrogenase, quinone 1): CGCACCCTGAGAAAACCTCT and
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GTGAAAACGCGGTCAAACCA;
242
AGGAGTGGCAGAAGTAG and CACGGAAGAGCAAGTA; SOD2 (Superoxide
243
dismutase): CTTCCTGACCTGCCTTAC and CGTCCCTGCTCCTTATT. PCR
244
reactions were performed inside a Bio-Rad S1000TM thermal cycler (Bio-Rad,
245
Hercules, California, USA). The final reaction volume was 50 µl, composing of 1 µl
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first-strand cDNA, 25 µM forward primer, 25 µM reverse primer, 10 µl
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PrimeSTARTM Buffer (Mg2+ plus), 4 µl dNTPs mixture (TaKaRa, Tokyo, Japan),
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0.5 µl PrimeSTARTM HS DNA Polymerase (2.5 U/µl, TaKaRa, Tokyo, Japan) and
249
RNase-free water made up to 50 µl. The cDNAs were amplified for 30 cycles. One
250
round of amplification was performed at 94°C for 30s, 58°C for 30s, and 72°C for 30s.
251
The PCR products (20 µl) were resolved in 1% agarose gels (Biowest, Hongkong,
252
China) in 1× TAE buffer (0.04 M Tris acetate and 0.001 M EDTA) and 10,000x
253
GeneGreen Nucleic Acid Dye (TIANGEN, Beijing, China) solution. The resolved
254
products were visualized with a transilluminator (SYNGENE, Cambridge, UK), and
255
photographs were taken using a computer-assisted gel documentation system
256
(SYNGENE, Cambridge, UK). The RT-PCR results were produced from 4
257
independent sets of experiments. The housekeeping gene GAPDH was run in parallel
258
to confirm that equal amounts of RNA were used in each reaction. The ratio intensity
259
for the fluorescently stained bands of gene of interest and GAPDH was calculated and
260
normalized to quantify the level of gene expression.
SOD1
(Superoxide
261
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Data analysis
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Data analyses and construction of statistical charts were performed using a
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GraphPad Prism 5 software (GraphPad Software, La Jolla, CA, USA). The results
265
were presented as the mean value ( x ± SEM). Statistical significance was determined
266
using paired t-test, independent samples t-test and one-way analysis of variance
267
(ANOVA). P < 0.05 was considered to be significant.
268 269
Results
270
High salt exposure disrupts the liver morphology
271
The effects of high salt exposure on liver morphology and functions were
272
investigated by placing 5 week-old C57BL/6 mice on high salt (4% NaCl) or pure
273
(control) waters for 4 weeks
274
determined the liver structure was normal with the hepatocytes arranged as cords and
275
regularly distributed (Fig. 1A). In the high salt exposed livers, the hepatic cords
276
appeared loosely and randomly arranged. Moreover, some of the hepatocytes were
277
balloon-shaped (Fig. 1B) and PAS staining revealed that there were regions in the
278
experimental liver not stained up by the PAS dyes (Fig. 1D) compared to the control
279
(Fig. 1C). This indicates that glycogen synthesis was inhibited in hepatocytes of
280
non-PAS stained regions.
13
. In H&E stained sections of the control liver, we
281
We also stained the liver sections with Masson trichrome stain and established
282
that the control liver was mainly negatively stained (Figs. 1E and E1). In contrast, we
283
could see the high salt-treated liver being more positively stained (Figs. 1F and 1F1).
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We further stained these using Sirius Red stain to reveal the presence of collagen. The
285
result demonstrated that there were more collagen fibres present in the high
286
salt-treated livers (Figs. 1H and H1) than in control livers (Figs. 1G and G1).
287
Similarly, Desmin expression in high salt-treated liver (Figs. 1I and I1) was stronger
288
than in the control (Figs. 1J and J1).
289 290
High salt exposure promotes apoptosis and inhibits proliferation
291
The effects of high salt exposure on hepatocyte survival and proliferation were
292
investigated by TUNEL staining and pHIS3 immunohistochemistry, respectively. It
293
was determined that there were significantly more TUNEL+ hepatocytes in high
294
salt-treated liver sections (Figs. 2B and B1) than control liver sections (Figs. 2A and
295
A1). In the control, 7.41 ± 1.47% hepatocytes were TUNEL+ (n=6) while it was 55.25
296
± 3.79% (n=6, p
Article
Liver fibrosis can be induced by high salt intake through excess ROS production Guang Wang, Cheung-kwan Yeung, Wing-Yan Wong, Nuan Zhang, Yi-fan Wei, Jing-li Zhang, Yu Yan, Ching-yee Wong, Jun-jie Tang, Manli Chuai, Kenneth Ka-ho Lee, Li-jing Wang, and xuesong Yang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b05897 • Publication Date (Web): 04 Feb 2016 Downloaded from http://pubs.acs.org on February 10, 2016
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 37
Journal of Agricultural and Food Chemistry
1
Liver fibrosis can be induced by high salt intake through excess ROS production
2 ∥
∥
3
Guang Wang† , Cheung-kwan Yeung † , Wing-Yan Wong †, Nuan Zhang†, Yi-fan Wei†,
4
Jing-li Zhang‡ ,Yu Yan†, Ching-yee Wong†, Jun-jie Tang†,Manli Chuai§, Kenneth Ka
5
Ho Lee#, Li-jing Wang‡, Xuesong Yang*†
6
†
Division of Histology and Embryology, Key Laboratory for Regenerative Medicine of
7
the Ministry of Education, Medical College, Jinan University, Guangzhou 510632,
8
China
9
‡
Institute of Vascular Biological Sciences, Guangdong Pharmaceutical University, Guangzhou 510006, China
10
11
§
Division of Cell and Developmental Biology, University of Dundee, Dundee, DD1 5EH, UK
12
13 14
#
Key Laboratory for Regenerative Medicine of the Ministry of Education, School of Biomedical Sciences, Chinese University of Hong Kong, Shatin, Hong Kong
15
∥
16
contributed to this work equally.
17
18 19
*
The corresponding author：X Yang. E-mail: [email protected] Tel: +86(20)85228316
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Journal of Agricultural and Food Chemistry
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Abstract
21
High salt intake has been known to cause hypertension and other side effects.
22
However, it is still unclear whether it also affects fibrosis in mature and the
23
developing liver. In this study, we demonstrated that high salt exposure in mice (4%
24
NaCl in drinking water) and chick embryo (calculated final osmolality of the egg was
25
300 mosm/l) could lead to derangement of the hepatic cords and liver fibrosis using
26
H&E, PAS, Masson and Sirius red staining. Meanwhile, Desmin immunofluorescent
27
staining of mouse and chick embryo liver indicated that hepatic stellate cells were
28
activated after the high salt exposure. pHIS3 and BrdU immunohistological staining
29
of mouse and chick embryo livers indicated that cell proliferation decreased as well as
30
the TUNEL analyses indicated that cell apoptosis increased in the presence of high
31
salt exposure. Next, dihydroethidium staining on the cultured chick hepatocytes
32
indicates the excess ROS was generated following high salt exposure. Furthermore,
33
AAPH (a known inducer of ROS production) treatment also induced the liver fibrosis
34
in chick embryo. Nrf2 and Keap1 positively immunohistological staining on mouse
35
liver suggested the Nrf2/Keap1 signaling was involved in high salt-induced ROS
36
production. Finally, CCK8 assay was used to determine whether or not the growth
37
inhibitory effect induced by high salt exposure can be rescued by anti-oxidant
38
Vitamin C. Meanwhile, the RT-PCR result indicated that the Nrf2/Keap1downsteam
39
genes including HO-1, NQO-1 and SOD2 were involved in this process. In sum, our
40
experiments suggest that high salt intake would lead to high risk of liver damage and
41
fibrosis in both adult and developing embryos. The pathological mechanism may be
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the result from an imbalance between oxidative stress and antioxidant system.
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Key words: high salt, liver fibrosis, mouse, chick embryos, ROS
44 45
Introduction
46
A normal and stable osmolarity of the tissue extracellular environment is
47
indispensable for cells to maintain their normal physiological functions. Osmolarity is
48
principally determined by ionic compounds, such as sodium chloride (NaCl), which
49
are dissociated into Na+ and Cl-. These ions could affect the osmolarity of blood and
50
extracellular fluids and in this context, a correct amount of salt intake is important for
51
maintaining normal osmolarity in our bodies. The causative relationship between
52
dietary salt intake and high osmolarity has long been debated by epidemiologists and
53
clinical researchers
54
an increase in extracellular osmolarity in epidemiological and clinical studies. In
55
animal experiments, monkeys have been placed on a high salt diet for one year
56
developed a significant increase in their extracellular osmolarity 4. Consequently, the
57
high osmolarity levels may damage in the arteries, kidneys, eyes and heart, prior to
58
hypertension being diagnosed 5-7. However, it is still controversial whether or not high
59
salt intake affects the liver morphology and function - especially with regard to the
60
fibrosis in developing liver.
1-3
. Excessive salt consumption has been demonstrated to lead to
61
The liver is largest organ in our body and it is crucial to our metabolism since
62
many exogenous chemical compounds such as alcohol and drugs are processed and
63
degraded there 8. In the embryo, proper development of the liver is dependant of many
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signalling molecules and cell-cell interaction. The mechanism involved is highly
65
conserved and involves complex series of developmental signalling pathways and
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Wnt/β-catenin signalling is critical for liver development, differentiation and cellular
67
homeostasis 9. Liver fibrosis involves both cellular and extracellular changes which
68
are induced by constant insults and damage to the liver, such as chronic inflammation
69
and virus infections. These insults activate the normally quiescent hepatic stellate
70
cells in the liver to over-produce extra cellular matrix (ECM). Consequently, the
71
excess ECM negatively impacts on the hepatocytes altering the liver morphology and
72
function over a long and slow pathological process.
73
The excess deposits of collagen in the space of tissue also reduce blood flow
74
through the liver tissues. Histologically, changes in the hepatic architecture are
75
deemed as the “gold standard” for assessing the extent of fibrosis in the liver
76
Chronic inflammation and loss of metabolic homeostasis play important roles in the
77
pathogenesis of liver fibrosis. In addition, there is accumulating evidence that both of
78
these pathological processes are linked with oxidative stress and ROS production
79
8-11-12
80
ROS production and exacerbate ROS-associated damage in liver tissue. However, it is
81
still controversial whether or not the Nrf2/Keap1 signalling - antioxidant system,
82
which includes key negative transcriptional regulators of ROS production, is involved
83
in this process.
10
.
. Although Uetake et al. (2015) indicated that the salt overload might induce
84
In this study, we investigated the effects of high salt exposure on the liver
85
morphology and the manifestation of fibrosis in the mouse model. We also used the
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chick embryo model to examine how high salt treatment affected the normal
87
development of the liver. Lastly, we determined high salt-induced liver fibrosis in
88
both adult and developing chick embryo was due to the excess ROS, which may be
89
attributed to reduced inhibitory effect of Nrf2/Keap1 signaling.
90 91 92
Materials and Methods
93
Mice
94
Five-week-old C57BL/6 mice were obtained from the Guangdong Province
95
Animal Centre and maintained at 25°C on a 12 hr light/dark cycle. The mice were
96
housed in a pathogen-free animal facility at the Institute of Vascular Biological
97
Sciences, Guangdong Pharmaceutical University. As previously described 13, the mice
98
were allowed free access to normal sodium food (0.5% NaCl) and water. Female mice
99
were divided into control (n=6) and experimental groups (n=8), while the latter
100
maintained on 4% NaCl water and control mice on pure water for 4 weeks. The
101
animals were experimented on and maintained according to guidelines set by the
102
animal experimental ethics committee at Jinan University.
103 104
Chick embryos
105
Fertilized chick eggs were obtained from the Avian Farm of the South China
106
Agriculture University. The eggs were incubated in a humidified incubator (Yiheng
107
Instrument, Shanghai, China) at 38°C and 70% humidity. After 36 hours of
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pre-incubation, the chick embryos (n=30) were treated with a high salt solution as
109
previously described
110
chick eggs and treated with two different concentrations of NaCl (totally added
111
volume was 500 µl). For the control group, 0.7% NaCl was injected (final osmolality
112
per egg was calculated as 240 mosm/l). In the high salt group, we injected a 16.85%
113
NaCl solution (the calculated final osmolality in the egg was 300 mosm/l). The treated
114
embryos were incubated for a further 4 or 12.5 days.
14-15
. Briefly, 2 ml of albumen were firstly extracted from the
115
The chick embryos were also treated with the 2,2’-azobis(2-amidinopropane)
116
Dihydrochloride (AAPH; Sigma-Aldrich, St. Louis, Missouri, USA) as previously
117
described
118
AAPH/egg (100 µl AAPH (100mM) injection) were injected into the air egg chamber
119
containing 9-day-old chick embryoswhile in the control group, each egg was given
120
the same volume of simple saline (0.72% sodium chloride; n=20 in each group). The
121
treated embryos were incubated for a further 8 days. Afterwards, the incubated
122
embryos were firstly determined whether they were alive or dead using the presence
123
of heartbeats and all dead embryos were excluded from the analysis. The
124
experimental embryos were removed from the egg shell and weighed.
16
. 5µmol AAPH/egg (100 µl AAPH (50mM) injection) or 10µmol
125 126
Histology
127
Briefly, 5.5-day old chick embryos, mouse livers, 14 and 17 day-old chick
128
embryo livers (control, high salt treated and AAPH treated) were fixed in 4%
129
paraformaldehyde at 4°C for 24 hours. The specimens were then dehydrated, cleared
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in xylene and embedded in paraffin wax. The embedded specimens were serially
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sectioned at 5 µm using a rotary microtome (Leica, RM2126RT, Wetzlar, Hessen,
132
Germany). The sections were stained with for haematoxylin and eosin (H&E),
133
Periodic acid Schiff (PAS) reaction 16, Masson's trichrome dyes (Masson staining) 16,
134
Sirius red 17 or immunohistochemically. The Masson and Sirius red staining were used
135
to reveal the presence of fibrosis in liver sections. Photographs were captured of the
136
stained histological sections using by an epi-fluorescence microscope (Olympus IX51,
137
Leica DM 4000B) at 200× magnification.
138 139
Immunohistological staining
140
Hydrated sections of chick embryos, chick embryo livers and mouse livers were
141
firstly treated with citrate buffer (pH 6.0) and heated in a microwave for antigen
142
retrieval. Immunofluorescent staining was then performed the treated sections. Briefly,
143
The primary antibodies were diluted using the PBT-NGS and the sections were
144
incubated with p-Histone H3 (pHIS3; 1:400, Santa Cruz Biotechnology, Dallas, Texas,
145
USA), Desmin (1:200, Sigma-Aldrich, St. Louis, Missouri, USA), Nrf2 (1:100, Santa
146
Cruz Biotechnology, Dallas, USA) or Keap1 (1:100, Santa Cruz Biotechnology,
147
Dallas, Texas, USA) primary antibody at 4°C overnight. Following 4×30mins washes
148
in PBS, the sections were incubated with goat anti-rabbit IgG conjugated Alexa Fluor
149
555 (1:1000, life technologies, Carlsbad, California, USA) for pHIS3, Nrf2 and
150
Keap1 or goat anti-mouse IgG conjugated Alexa Fluor 488 (1:1000, life technologies,
151
Carlsbad, California, USA) for Desmin for 1 hour. The sections were counterstained
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with DAPI (1:1000, life technologies, Carlsbad, California, USA) at room
153
temperature for 30 min before examination. For immunohistochemical staining,
154
biotinylated goat anti-rabbit serum IgG (1:1000, Cell signaling technology, Danvers,
155
Massachusetts,
156
Daminobenzidine tetrahydrochloride substrate (DAB kit, MXB, Fuzhou, Fujian,
157
China) were used to visualize the immunostaining. Photographs were captured of the
158
stained histological sections using by an epi-fluorescence microscope (Olympus IX51,
159
Leica DM 4000B) at 200× magnification.
USA)
for
pHIS3
was
used
as
the
secondary
antibody.
160 161
Cell proliferation analyses
162
10 µl of 5-Bromo-2-deoxyUridine (BrdU, 10 mM, Sigma-Aldrich, St. Louis,
163
Missouri, USA) solution was administered to high salt-treated or control chick
164
embryos for 4 hours before the embryos reached 5.5 day-old. The liver of these
165
embryos (n=6) were harvested and transverse sections of the livers were produced.
166
These sections were stained with BrdU monoclonal antibody (1:200, BD bioscience,
167
Franklin Lakes, New jersey, USA) according to manufacturer’s instructions (Roche,
168
Basel, Basel-Stadt, Switzerland). The sections were incubated with goat anti-mouse
169
IgG conjugated Alexa Fluor 555 (1:1000, life technologies, Carlsbad, California, USA)
170
for BrdU for 1 hour. Cell counts were performed on randomly selected areas of the
171
BrdU-stained liver sections (visual field at 200× magnification). We counted the
172
number of BrdU positive cells against the total cell number to calculate the
173
proliferation index (BrdU+ cells/ total cell numbers). Photographs were captured of
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the stained histological sections using by an epi-fluorescence microscope (Olympus
175
IX51, Tokyo, Japan) at 200× magnification.
176 177
TUNEL analyses
178
The extent of apoptosis in the mice and chick embryo livers was established using an
179
In Situ Cell Death Detection Kit (Roche, Basel, Basel-Stadt, Switzerland). The
180
TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end
181
labeling) staining was performed according to the instructions provided by the
182
manufacturer, in which were adapted for tissue labelling on glass slides.
183
Daminobenzidine tetrahydrochloride substrate (DAB kit, MXB, Fuzhou, Fujian,
184
China) was used to visualize the immunostaining. The presence of TUNEL+ cells was
185
determined using an Olympus microscope attached to an Image Analysis Software
186
(Olympus IX51, Tokyo, Japan). We quantified the percentage of TUNEL+ hepatocytes
187
relative to all the hepatocytes in each histological slide. We compared the percentage
188
of TUNEL+ hepatocytes at the similar level in the control mice or 5.5-days chick
189
embryo and high slat treated mice and 5.5-days chick embryos (n=6 in each group
190
from 6 mouse or chick embryos).
191 192
Dihydroethidium and CCK8 assays
193
The livers of 17 day-old chick embryos (N=3) were perfused with Ca2+ free
194
Krebs ringer-HEPES buffer containing collagenase (pH 7.4, Wako, Tokyo, Japan) and
195
a trypsin inhibitor (Sigma-Aldrich, St. Louis, Missouri, USA) at 37°C. After breaking
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the eggshell and dissection, 37°C pre-warmed D-Hanks Balanced salt solution was
197
injected through hepatic portal vein slowly. When liver began to blanch, made a cut at
198
inferior vena cava to allow erythrocyte efflux till liver became pale. Switch perfusion
199
solution to pre-warmed 0.05% collagenase IV (Sigma-Aldrich, St. Louis, Missouri,
200
USA). Cut away the liver from embryo when it looked mushy and washed in
201
D-Hanks solution. Dissected the liver in DMEM-F12 (Hyclone, Logan City, Utah,
202
USA), and then filtered the cell dispersion through 75µm pore size cell strainer into a
203
EP tube to remove undigested tissue fragments. Centrifuged at 2000rpm for 3 min at
204
4 ℃ . Removed the supernatant and gently re-suspended cells with DMEM-12.
205
Repeated centrifugation again. Removed the supernatant, and suspended cells with
206
DMEM-F12 containing 10%FBS and 10-6 dexamethasone (Sigma-Aldrich, St. Louis,
207
Missouri, USA) for a better attachment of hepatocytes. After 48 hours of incubation,
208
the culture medium was replaced with fresh medium containing and not containing
209
1g/100ml NaCl for 30 mins. Dihydroethidium (DHE) staining were performed at the
210
end of cell culture using DHE fluorescent probe (Beyotime, Shanghai, China) to
211
detecting the presence of superoxide anion (O2-). The cells were incubated with 10
212
µM DHE for 30 min, at 37°C and then collected for analyses according to the
213
manufacturer’s instructions.
214
HEK293 cells were seeded into 96-well plates and used in the vitamin C (VC,
215
Sigma-Aldrich, St. Louis, USA) rescue experiment. These cells (1×106 cells/ml) were
216
maintained in DMEM + 10% fetal bovine serum at 37°C and 5% CO2. Simple saline
217
(control) or 1g NaCl per 100ml culture medium with or without VC (40, 80 or
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160µg/ml)
were added to the HEK293 cultures. The cell viability was assessed
219
using CCK8 assay (Cholecystokinin-8). Briefly, 10 µl of CCK8 reagent (Dojindo,
220
Kumamoto, Japan) was added to the 96-well plates and incubated continually for 4
221
hours at 37°C. The absorbance values were measured at 450 nm using a BIO-RAD
222
Model 450 Microplate Reader (BIO-RAD, Hercules, California, USA). The cell
223
viability was indirectly determined by examining the ratio of the absorbance value of
224
high salt-treated cells relative to the control cells, from three independent
225
experiments.
226 227
Semi-quantitative RT-PCR
228
The primary culture of embryonic chick hepatocytes was from six 17 day-old chick
229
embryos. Simple saline (control) or 1g NaCl per 100ml culture medium with or
230
without VC (80 µg/ml) were added to the cultures and incubated for 30mins. Total
231
RNA was isolated from primary cultured hepatocytes using TRIzol (Invitrogen,
232
Carlsbad, California, USA,) according to the manufacturer’s instructions. First-strand
233
cDNA was synthesized to a final volume of 25 µl using the SuperScript RIII
234
first-strand synthesis system (Invitrogen, Carlsbad, California, USA). Following
235
reverse transcription, PCR amplification of the cDNA was performed as described
236
previously
237
(Glyceraldehyde-3-phosphate dehydrogenase): GTCAACGGATTTGGCCGTAT and
238
AATGCCAAAGTTGTCATGGATG;
239
GTCGTTGGCAAGAAGCATCC and ACTCCTTGTGCGAAGCTCTG; NQO-1
19-20
.
The
primers
used
were
as
HO-1(Heme
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oxygenase
GAPDH
1):
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(NAD(P)H dehydrogenase, quinone 1): CGCACCCTGAGAAAACCTCT and
241
GTGAAAACGCGGTCAAACCA;
242
AGGAGTGGCAGAAGTAG and CACGGAAGAGCAAGTA; SOD2 (Superoxide
243
dismutase): CTTCCTGACCTGCCTTAC and CGTCCCTGCTCCTTATT. PCR
244
reactions were performed inside a Bio-Rad S1000TM thermal cycler (Bio-Rad,
245
Hercules, California, USA). The final reaction volume was 50 µl, composing of 1 µl
246
first-strand cDNA, 25 µM forward primer, 25 µM reverse primer, 10 µl
247
PrimeSTARTM Buffer (Mg2+ plus), 4 µl dNTPs mixture (TaKaRa, Tokyo, Japan),
248
0.5 µl PrimeSTARTM HS DNA Polymerase (2.5 U/µl, TaKaRa, Tokyo, Japan) and
249
RNase-free water made up to 50 µl. The cDNAs were amplified for 30 cycles. One
250
round of amplification was performed at 94°C for 30s, 58°C for 30s, and 72°C for 30s.
251
The PCR products (20 µl) were resolved in 1% agarose gels (Biowest, Hongkong,
252
China) in 1× TAE buffer (0.04 M Tris acetate and 0.001 M EDTA) and 10,000x
253
GeneGreen Nucleic Acid Dye (TIANGEN, Beijing, China) solution. The resolved
254
products were visualized with a transilluminator (SYNGENE, Cambridge, UK), and
255
photographs were taken using a computer-assisted gel documentation system
256
(SYNGENE, Cambridge, UK). The RT-PCR results were produced from 4
257
independent sets of experiments. The housekeeping gene GAPDH was run in parallel
258
to confirm that equal amounts of RNA were used in each reaction. The ratio intensity
259
for the fluorescently stained bands of gene of interest and GAPDH was calculated and
260
normalized to quantify the level of gene expression.
SOD1
(Superoxide
261
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Data analysis
263
Data analyses and construction of statistical charts were performed using a
264
GraphPad Prism 5 software (GraphPad Software, La Jolla, CA, USA). The results
265
were presented as the mean value ( x ± SEM). Statistical significance was determined
266
using paired t-test, independent samples t-test and one-way analysis of variance
267
(ANOVA). P < 0.05 was considered to be significant.
268 269
Results
270
High salt exposure disrupts the liver morphology
271
The effects of high salt exposure on liver morphology and functions were
272
investigated by placing 5 week-old C57BL/6 mice on high salt (4% NaCl) or pure
273
(control) waters for 4 weeks
274
determined the liver structure was normal with the hepatocytes arranged as cords and
275
regularly distributed (Fig. 1A). In the high salt exposed livers, the hepatic cords
276
appeared loosely and randomly arranged. Moreover, some of the hepatocytes were
277
balloon-shaped (Fig. 1B) and PAS staining revealed that there were regions in the
278
experimental liver not stained up by the PAS dyes (Fig. 1D) compared to the control
279
(Fig. 1C). This indicates that glycogen synthesis was inhibited in hepatocytes of
280
non-PAS stained regions.
13
. In H&E stained sections of the control liver, we
281
We also stained the liver sections with Masson trichrome stain and established
282
that the control liver was mainly negatively stained (Figs. 1E and E1). In contrast, we
283
could see the high salt-treated liver being more positively stained (Figs. 1F and 1F1).
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We further stained these using Sirius Red stain to reveal the presence of collagen. The
285
result demonstrated that there were more collagen fibres present in the high
286
salt-treated livers (Figs. 1H and H1) than in control livers (Figs. 1G and G1).
287
Similarly, Desmin expression in high salt-treated liver (Figs. 1I and I1) was stronger
288
than in the control (Figs. 1J and J1).
289 290
High salt exposure promotes apoptosis and inhibits proliferation
291
The effects of high salt exposure on hepatocyte survival and proliferation were
292
investigated by TUNEL staining and pHIS3 immunohistochemistry, respectively. It
293
was determined that there were significantly more TUNEL+ hepatocytes in high
294
salt-treated liver sections (Figs. 2B and B1) than control liver sections (Figs. 2A and
295
A1). In the control, 7.41 ± 1.47% hepatocytes were TUNEL+ (n=6) while it was 55.25
296
± 3.79% (n=6, p
