Biochemical Pharmacology 87 (2014) 467–476

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Biochemical Pharmacology
journal homepage: www.elsevier.com/locate/biochempharm

Angiotensin (1–7) protects against stress-induced gastric lesions in

rats
Donglin Zhu a,1, Qiang Tong b,1, Wei Liu c, Minjie Tian a, Wei Xie a, Li Ji a, Jingping Shi a,*
a
Department of Neurology, Nanjing Brain Hospital, Nanjing Medical University, P.O. Box 210029, No. 264 Guangzhou Road, Nanjing, PR China
b
Department of Geriatrics, Huai’an First People’s Hospital, Nanjing Medical University, P.O. Box 223300, No. 6 Beijing Road West, Huai’an, PR China
c
Department of Neurology, Suzhou Hospital of Traditional Chinese Medicine, Nanjing University of Chinese Medicine, P.O. Box 215009, No. 889 West
Wuzhong Road, Suzhou, PR China

A R T I C L E I N F O A B S T R A C T

Article history: Stress ulcers can develop with severe physiological stress, and have been proposed as being brain-driven
Received 14 September 2013 events. New findings continue to suggest that stress ulcers can be more effectively managed through
Accepted 31 October 2013 central manipulation rather than by simply altering local gastric factors. Angiotensin (1–7) (Ang (1–7)) is
Available online 11 November 2013
present as an endogenous constituent of the brain and stomach. The beneficial effects of Ang (1–7) have
been confirmed in the vessels, brain, heart, kidney, liver and lungs, but not in the stomach. Given the
Keywords: accumulating evidence suggesting the anti-stress activities of Ang (1–7), its potential gastroprotective
Angiotensin (1–7)
effect in the context of stress requires further investigation. In the present study, rat gastric mucosal
Mas
Cold-restraint stress
lesions were induced by 2 h of cold-restraint stress. We observed that these lesions were significantly
Oxidative stress attenuated after 1 week of intracerebroventricular treatment with Ang (1–7). This gastroprotective
Gastric lesions effect was associated with attenuated oxidative stress and suppressed acid secretion. Brain Ang (1–7)
administration profoundly modified responses to stress, indicated by altered levels of several stress
Chemical compounds studied in this article: hormones, including Ang II, glucocorticoid, norepinephrine, serotonin, and dopamine, in blood or stress-
Angiotensin (1–7) (PubChem CID: 123805) related brain regions. These findings indicate that Ang (1–7) exerts anti-stress activities by restoring the
Angiotensin II (PubChem CID: 172198)
gastric microenvironment and modulating the stress pathways. Ang (1–7) may be a promising agent for
Corticosterone (PubChem CID: 5753)
stress ulcer prophylaxis and therapy, administered through brain-permeable mimics or carriers.
Norepinephrine (PubChem CID: 439260)
Dopamine (PubChem CID: 681) Crown Copyright ß 2013 Published by Elsevier Inc. All rights reserved.
Serotonin (PubChem CID: 5202)
Malondialdehyde (PubChem CID: 10964)

1. Introduction central levels [6,7]. However, responses to stress can also be

Gastric stress ulceration and bleeding are common occurrences
in critically ill patients [1]. Patients with stress ulcers have a longer
hospitalization stay and a higher mortality than patients without
ulcers [1]. Stress ulcerogenesis has been suggested as being a
brain-driven gastric injury [2]. Therefore, therapies targeting both
central stress mechanisms and the gastric microenvironment are
proposed for the development of successful treatment strategies
[3,4].
Stress responses mainly occur through two distinct but
interrelated systems: the hypothalamic–pituitary–adrenocortical
(HPA) axis, and the sympathoadrenal system (SAS) [5]. The SAS can
activate the central noradrenergic system in response to stress
stimuli, and interconnects with the HPA axis at peripheral and
* Corresponding author.
E-mail address: profshijp@163.com (J. Shi).
1
These authors contributed equally to this work.
modulated by higher brain regions than the hypothalamus, such as
the hippocampus, amygdala, and prefrontal cortex, among which
there are complicated interactions [8]. Previous studies have
shown that the activity of the HPA axis is inhibited by the
hippocampus [9–11], but it is activated by the amygdala [8], and
regulated in two directions by the prefrontal cortex [12]. Moreover,
these brain regions influence the hypothalamus through various
neurotransmitters, including norepinephrine, dopamine, 5-hy-
droxytryptamine (5-HT, also called serotonin), and angiotensin II
(Ang II) [8,13]. The balance of these neurotransmitters plays an
important role in the metastasis of the gastric microenvironment
under physical conditions or during responses to mild stress.
Ang II (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe) is an important
vasoconstrictor component of the peripheral and central renin–
angiotensin system, and acts mainly through its type I receptor
(AT1R). Ang II has also been proposed as a fundamental stress
hormone in the body because of its critical role in the activation of
the HPA axis and the SAS, and in the secretion of glucocorticoids
and norepinephrine [13]. The pathogenic mechanisms of Ang II in

0006-2952/$ – see front matter . Crown Copyright ß 2013 Published by Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.bcp.2013.10.026
468 D. Zhu et al. / Biochemical Pharmacology 87 (2014) 467–476
stress ulceration involve reduced gastric blood flow and increased
oxidative stress [13]. Administration of Ang II receptor blockers,
such as candesartan, has been reported to protect against stress-
induced injury [14–17]. However, the metabolite of Ang II through
cleavage of a C-terminal residue, Ang (1–7) (Asp-Arg-Val-Tyr-Ile-
His-Pro), requires further study.
In contrast to the stress-enhancing effects of Ang II, evidence
has emerged recently of the anti-stress activities of Ang (1–7). Ang
(1–7) is mostly a derivation of Ang II, but acts against Ang II in most
conditions through its specific receptor, Mas [18,19]. In the
stomach wall, Ang (1–7) is the predominant Ang peptide [20], and
is speculated to mediate the anti-ulcer effects of AT1R blockers
[20]. In the brain, Ang (1–7) and Mas receptors are abundant in
several stress-related regions, including the hypothalamus,
hippocampus, and amygdala [21]. Many beneficial effects from
brain Ang (1–7) have been confirmed through its brain-permeable
mimics or carriers, including reduced brain Ang II and AT1R
expression [22], increased brain blood flow and thus anti-ischemic
effects [23,24], anti-inflammatory effects [25,26], anxiolytic effects
[27], attenuated oxidative stress [28], abolished sympathetic
activation [29], suppressed norepinephrine release [30–33], and
enhanced synaptic plasticity [34–36], which are all critical to
defense and recovery during stress. These observations suggest an
inhibitory role of Ang (1–7) in stress responses and stress ulcers.
In the present study, stress-related gastric lesions were induced
in a rat model of cold-restraint stress (CRS), and the role of Ang (1–
7) in stress responses and its gastroprotective effects were
investigated by intracerebroventricular infusion of Ang (1–7) or
a MAS receptor antagonist A779.
2. Materials and methods
2.1. Animals
Male Sprague-Dawley rats (from the Center of Laboratory
Animals of Nanjing Medical University) weighing 280–310 g were
housed in a controlled environment at 25  2 8C on a 12-h light/
dark cycle. Rats were provided with food and water ad libitum. Rats
were acclimatized at our animal facility for at least 1 week before
experiments were conducted. Animal care and experiment protocols
were in accordance with the Guide for the Care and Use of Laboratory
Animals of Nanjing Medical University and were approved by the
Biological Research Ethics Committee of Nanjing Medical University.
All efforts were made to minimize the suffering of the animals.
2.2. Implantation of intracranial cannulae
After a 7-day acclimation period, rats were subjected to
intracerebroventricular implantation. Rats were anesthetized with
10% chloral hydrate (0.35 mL/100 g; Amresco, Solon, OH, USA) and
placed in a Kopf stereotaxic frame (David Kopf, Tujunga, CA, USA).
Rats then underwent two intracranial surgeries as previously
described [37]. A stainless steel cannula (Alzet, Cupertino, CA, USA)
was implanted into the left cerebroventricle (1.2 mm posterior and
1.5 mm lateral to the bregma, 4.5 mm below the surface of the
cranium) [26] and coupled to a 1-week osmotic pump (model
2001; Alzet). Osmotic pumps were subcutaneously implanted
between the shoulder blades and were used to infuse low-dose Ang
(1–7) (L-A7, 0.11 nmol/L; Sigma–Aldrich, Germany), high-dose
Ang (1–7) (H-A7, 1.1 nmol/L), 1.1 nmol/L Ang (1–7) + 1.14 nmol/L
A779 (Bachem, Switzerland), 1.14 nmol/L A779, or artificial
cerebrospinal fluid (aCSF) into the left lateral cerebral ventricle.
The wound was closed following surgery. Rats were then returned
to their cages. The pumps were designed to work at a rate of 1 ml/h.
The actual doses of Ang (1–7) delivered into rat brains were 0.1 pg/
h and 1 pg/h. The preparation of aCSF was performed as previously
described (all reagents were from Amresco) [23]. No stress was
induced by this surgical procedure, because there are no significant
alterations in body weight and motor activity in our preliminary
study (data not shown).
After 1 week of continuous infusion, the rats were subjected to
model induction, as described below. To explore the underlying
mechanisms, the experiments were divided into two parts. In part
1, rats were divided into four groups: (1) aCSF + no CRS; (2)
aCSF + CRS; (3) L-A7 + CRS; and (4) H-A7 + CRS. In part 2, rats were
divided into the following four groups: (1) aCSF + CRS; (2) H-
A7 + CRS; (3) H-A7 + A779 + CRS and (4) A779 + CRS. Each group
consisted of 10 rats and no rats or tissues were tested more than
once. Systolic blood pressure was measured by the tail cuff method
at day 0 (pre-surgery), day 3, day 5 and day 7 (after stress), using a
non-invasive blood pressure analyzer (BP-2000; Visitech Systems,
Inc., Apex, NC, USA).
2.3. Stress model
After a 24-h fast, one stress session was performed during the
early phase of the light cycle and consisted of a 2-h restraint period
in a container (rat restrainers were metallic cages 20 cm
long  4 cm wide  4 cm high) at 4 8C. Rats were then killed by
decapitation under deep anesthesia.
2.4. Estimation of stress-induced gastric injury
After stress treatment, the area of mucosal injury was
measured. The rat stomach was removed. The mucosa was
exposed by cutting along the greater curvature, and was then
rinsed with cold, sterile saline to remove any gastric content and
blood clots. The severity of mucosal lesions was assessed using a
magnifier and rated for gross pathology according to the ulcer
score described by Dekanski et al. as follows [38]: 0 = no damage;
1 = blood in the lumen; 2 = pin-point erosions; 3 = one to five small
erosions <2 mm; 4 = more than five small erosions <2 mm;
5 = one to three large erosions >2 mm and 6 = more than three
large erosions >2 mm.
The ulcer index (UI) was calculated as the total number of
lesions multiplied by their respective scores by two observers blind
to the experimental protocols. Discussion, which included a
pathology technician, was had and a vote was taken if there were
any differences. The inhibition percentage was calculated by the
following formula [39]: [(UI non-treated UI treated)/UI non-
treated]  100.
2.5. Measurement of H+, K+ ATPase activity, antioxidant enzyme
activity, and malondialdehyde levels in rat stomachs
To prepare tissue homogenates, stomach tissues were homog-
enized with liquid nitrogen using a mortar and pestle. Homoge-
nized tissues were mixed with homogenization Tris-buffer
(10 mM, pH 7.4, Amresco). The mixtures were homogenized on
ice using an Ultra-Turrax homogenizer (IKA Laboratory, Guangz-
hou, China) for 15 min. Homogenates were filtered and centrifuged
at 1000  g at 4 8C for 20 min using a refrigerated centrifuge
(Beckman, CA, USA). The supernatants were used to determine
enzymatic activities.
Superoxide dismutase estimation was based on the generation
of superoxide radicals produced by xanthine and xanthine oxidase,
which react with nitro blue tetrazolium to form a formazan dye
(Sigma–Aldrich). Superoxide dismutase activity was measured at
560 nm by the degree of inhibition of this reaction. Enzyme activity
causing 50% inhibition was considered as 1 unit/mg protein.
Catalase activity was defined as the amount of enzyme required
to decompose 1 mmol of H2O2 (Sigma–Aldrich) per second per mg
 D. Zhu et al. / Biochemical Pharmacology 87 (2014) 467–476
protein at 37 8C. Decomposition of H2O2 in the presence of catalase
was measured at 240 nm (Tecan Trading AG, Männedorf,
Switzerland). Results are expressed as units per mg protein.
The remaining gastric tissues were homogenized using Tris–Cl
buffer and used to measure H+, K+ ATPase activity. The assay
medium contained 70 mM Tris-buffer (pH 6.8), 5 mM MgCl2, and
enzyme solution in 10 mM KCl (all from Amresco) in a total volume
of 1 mL, and was incubated for 1 h. The reaction was initiated by
adding 2 mM ATP (Sigma–Aldrich), and incubated at 37 8C for
20 min. The reaction was stopped by addition of 10% trichlor-
oacetic acid (Amresco). After centrifugation, 2.5 mL of ammonium
molybdate and 0.5 mL of l-amino-2-naphthol-4-sulphonic acid
(Sigma–Aldrich) were added to the supernatant and the absor-
bance was read at 620 nm (Tecan). Results are expressed as mmol
of Pi liberated per min per mg protein.
Tissue malondialdehyde levels were determined using the
method described by Ohkawa et al. [40]. The corpus mucosa was
homogenized in 10 mL of 100 g/L KCl (Amresco). The homogenate
(0.5 mL) was added to a solution containing 0.2 mL of 80 g/L
sodium lauryl sulfate, 1.5 mL of 200 g/L acetic acid, 1.5 mL of 8 g/L
2-thiobarbiturate (all from Amresco), and 0.3 mL of distilled water.
This mixture was heated at 98 8C for 1 h, and 5 mL of n-
butanol:pyridine (15:1, Sigma–Aldrich) was added when the
mixture cooled. The mixture was vortexed for 1 min and
centrifuged for 30 min at 3000  g. Absorbance of the supernatant
was measured at 532 nm (Tecan). A standard curve was obtained
using 1,1,3,3-tetramethoxypropane (Sigma–Aldrich). Recovery
was over 90%. Results are expressed as nmol per mg protein.
2.6. Estimation of Ang II and Ang (1–7) in plasma and brain extracts
Ang II and Ang (1–7) were separated by reversed-phase high-
performance liquid chromatography (HPLC, Waters, Millipore, MA,
USA) using a mBondapak C18 column (300 mm  3.9 mm, 10 mm
particle size, Waters) and a mBondapack C18 precolumn (Waters).
Mobile phase A was 25% methanol, 0.085% H3PO4 containing 0.02%
NaN3 (all from Amresco). Mobile phase B was 75% methanol,
0.085% H3PO4 containing 0.02% NaN3. Concentrated Sep-Pak
extracts were dissolved in 100 mL mobile phase and centrifuged
at 10 000  g for 5 min before injection. The flow rate was 1.5 mL/
min and the working temperature was 45 8C. Elution was
performed as follows: 85% mobile phase A–15% mobile phase B
from 0 to 5 min, followed by a linear gradient to 40% mobile phase
A and 60% mobile phase B until 20 min. Eluates were collected in
0.5 mL fractions in polypropylene tubes containing 20 mL bovine
serum albumin 0.1%. Fractions containing Ang II and Ang (1–7)
were neutralized with 1 N NaOH (Amresco).
The levels of Ang II and Ang (1–7) were assayed in plasma and
brain extracts by commercial enzyme immunoassay kits (Bachem)
according to a previously reported study [41] and the manufac-
turer’s instructions. All samples were analyzed in duplicate.
Concentrations are expressed in pg per mL of plasma or per mg
of brain tissue.
2.7. Western blotting assays for Mas and AT1R
The protein levels of Mas and AT1R were assayed from brain
extracts (cerebellum not included) using western blotting. The
protocols used were as previously described [41]. Briefly, brain
tissue was homogenized in ice-cold lysis buffer using an
ultrasound homogenizer (IKA Laboratory). Tissue homogenates
were centrifuged and collected. Protein concentrations were
determined using the Bradford method. Equal amounts of protein
(60 mg) were electrophoresed using 10% sodium dodecyl sulfate-
polyacrylamide gel electrophoresis and transferred onto
polyvinylidene difluoride membranes (Millipore). After blocking,
469
membranes were incubated overnight at 4 8C with primary
antibodies (anti-Mas antibody; Santa Cruz Biotechnology, Santa
Cruz, CA, USA, 1:100; and anti-AT1R antibody; Abcam, Cambridge,
UK, 1:250) and then incubated with anti-mouse or anti-rabbit
horseradish peroxidase-conjugated secondary antibodies. Blots
were soaked in enhanced chemiluminescent substrate (Pierce,
Rockford, IL, USA) and exposed to film (Kodak, Tokyo, Japan) for
various periods of time (0.05–5 min). Intensities from X-ray film
were quantified using Densitometer Quantity One software (Bio-
Rad, Hercules, CA, USA) after ascertaining linearity.
2.8. Measurement of plasma corticosterone levels
Plasma corticosterone levels were quantified by HPLC coupled
to an ultraviolet detector (Waters). Briefly, 500 mL of plasma
containing a known quantity of dexamethasone was extracted
using 5 mL of dichloromethane (Sigma–Aldrich). The dichloro-
methane extract was evaporated until dry and dissolved in 100 mL
of mobile phase. Twenty mL of extract were injected into the HPLC
system for quantification. Mobile phase consisted of methanol
(Amresco) and water (70:30) at a flow rate of 1.2 mL/min, and
corticosterone was detected at 250 nm using an ultraviolet
detector. The chromatogram was recorded and analyzed with
Breeze software version 3.2 (Waters).
2.9. Measurement of plasma norepinephrine levels, and brain tissue
levels of norepinephrine, serotonin and dopamine
For blood assays, venous blood was sampled from the orbital
vein, transferred into heparinized tubes (Axygen, Union City, CA,
USA) for 10 min of quiescence, and then centrifuged at 1600  g for
10 min at 4 8C (Beckman). A total of 500 mL of plasma sample was
washed with hexane (<Sigma–Aldrich) to remove lipids. Protein
was precipitated with sulfosalicylic acid (10 g/100 mL, Sigma–
Aldrich), and 0.1 mL of internal standard 3,4-dihydroxybenzyla-
mine (Sigma–Aldrich) was added. After centrifugation, the
supernatant was washed with ethyl acetate (Sigma–Aldrich)
saturated with sodium chloride (Amresco). The ethyl acetate
phase containing norepinephrine was evaporated until dry at 37 8C
under a stream of dry nitrogen, and then frozen at 20 8C until
analysis.
Tissues were weighed and homogenized for 15 s in 10 volumes
of 0.1 mol/L perchloric acid (Amresco) using an ultrasound
homogenizer. Homogenates were then centrifuged at 33 000  g
(Beckman) at 4 8C for 1 h, and the supernatants were collected for
analysis.
Levels of norepinephrine, serotonin and dopamine were
measured using HPLC coupled to an electrochemical detector
system (Waters).
2.10. Statistical analysis
Results are expressed as mean  standard deviation (SD).
Statistical significance was determined by one-way analysis of
variance followed by the least significant difference post hoc test. A
P-value <0.05 was considered statistically significant.
3. Results
3.1. Central Ang (1–7) administration reduced systolic blood pressure
and stress-induced systolic blood pressure
As shown in Table 1, when compared with non-stressed rats,
systolic blood pressure measurements in stressed rats showed
significant increases by approximately 30 mmHg (P < 0.01).
Increases were attenuated by brain Ang (1–7) infusion in a
470 D. Zhu et al. / Biochemical Pharmacology 87 (2014) 467–476

Table 1 3.2. Central Ang (1–7) significantly attenuates stress-induced gastric

Central Ang (1–7) administration reduced stress-induced systolic blood pressure
ulcers via Mas
(mmHg).

Treatment 0 3 5 7 The severity of ulcers in the present study was represented by

Part 1 the ulcer index, which was established by Dekanski et al. [38]. As
aCSF + no CRS 129.3  8.0 124.1  8.0 122.1  9.1 124.8  7.6** shown in Fig. 1, many erosions in rat gastric mucosa were induced by
aCSF + CRS 125.9  5.1 120.8  6.0 122.2  7.5 152.8  6.6 2 h of CRS (P < 0.01). After 1 week of pretreatment with 0.11 nmol/L
L-A7 + CRS 126.1  4.2 120.9  8.4 124.6  6.9 147.7  8.9
Ang (1–7), stress-induced gastric injuries were significantly
H-A7 + CRS 127.2  6.3 116.0  7.7 119.8  9.6 144.5  8.3*
attenuated (P < 0.01). These injuries were nearly completely
Part 2 prevented after 1 week of 1.1 nmol/L Ang (1–7) treatment; a few
aCSF + CRS 122.6  7.2 118.8  8.7 124.3  10.5 153.2  5.5#
H-A7 + CRS 126.9  9.1 114.5  8.4 117.3  11.4 146.0  7.3
rats showed blood in the lumen or pin-point erosions (P < 0.01).
H-A7 + A779 + CRS 123.6  7.9 131.7  7.4 126.4  8.9 157.9  8.3## Significant erosions appeared in the gastric mucosa of rats receiving
A779 + CRS 121.1  8.6 130.8  11.6 134.8  7.2 152.9  7.4# 1.1 nmol/L Ang (1–7) and 1.14 nmol/L A779, or A779 alone.
Results are presented as mean  SD.
*P < 0.05 or **P < 0.01 vs. aCSF + CRS. #P < 0.05 or ##
P < 0.01 vs. H-A7 + CRS. L-A: low- 3.3. Central Ang (1–7) significantly altered the activities of H+, K+
dose Ang (1–7), H-A: high-dose Ang (1–7). ATPase and antioxidant enzymes, and the levels of malondialdehyde

dose-dependent manner. Systolic blood pressure before stress After 2 h of CRS, the activities of H+, K+ ATPase in rat gastric
induction was also reduced by 1.1 nmol/L Ang (1–7). When treated mucosa were significantly enhanced (P < 0.01, Fig. 2A). Brain Ang
with its antagonist, A779, systolic blood pressure again increased (1–7) administration attenuated stress-induced H+, K+ ATPase
by CRS, supporting the depressor effect of Ang (1–7). activity (P < 0.01 or <0.05, Fig. 2A). This effect was canceled by its

Fig. 1. Central Ang (1–7) attenuated stress-induced gastric lesions. Gastric lesions of rats were induced by 2 h of cold-restraint stress (A and B). The ulcer index (C and D) was
assessed according to a well-established score. Results are presented as mean  SD. **P < 0.01 vs. aCSF + CRS. ##P < 0.01 vs. H-A7 + CRS. L-A: low-dose Ang (1–7), H-A: high-dose
Ang (1–7).
 D. Zhu et al. / Biochemical Pharmacology 87 (2014) 467–476 471

Fig. 2. Central Ang (1–7) infusion altered the gastric microenvironment. The activities of H+, K+ ATPase (A and B), catalase (CAT, E and F), and superoxide dismutase (SOD, G and
H), and malondialdehyde levels (MDA, C and D) were assayed in rat gastric mucosa. Results are presented as mean  SD. *P < 0.05 or **P < 0.01 vs. aCSF + CRS. #P < 0.05 or
##
P < 0.01 vs. H-A7 + CRS. L-A: low-dose Ang (1–7), H-A: high-dose Ang (1–7).

antagonist, A779 (Fig. 2B). Similar changes were also observed in
malondialdehyde levels (Fig. 2C and D).
In contrast, the activities of mucosal antioxidant enzymes,
catalase and superoxide dismutase, were remarkably impaired
during CRS (P < 0.01, Fig. 2E and G). The activities recovered with
brain Ang (1–7) infusion (Fig. 2E and G). Activities of catalase and
superoxide dismutase decreased to a similar extent using
combined or single A779 treatment (Fig. 2F and H).
3.4. Intracerebroventricular Ang (1–7) infusion increases Ang (1–7)
levels and its receptor, Mas
Plasma and brain Ang (1–7) levels in stressed rats significantly
decreased compared with levels in non-stressed rats (P < 0.01, Fig.
3A and C). These decreases were partly corrected with a central
low-dose Ang (1–7) infusion. Decreases in Ang (1–7) levels induced
by stress were completely prevented by 1 week of treatment with
an intracerebroventricular supplement of 1.1 nmol/L Ang (1–7)
(P < 0.01, Fig. 3A and C). The largest increase in Ang (1–7) levels
appeared in rat hippocampi. Plasma Ang (1–7) levels also
significantly increased (P < 0.01, Fig. 3A); this effect was blocked
by A779 (Fig. 3B). Ang (1–7) levels did not change with A779
infusion (Fig. 3D), suggesting that the distribution of Ang (1–7) was
not affected by its receptor blocker. Interestingly, similar changes
were observed in the levels of Mas and AT1R, as shown by western
blotting (Fig. 3E and F).
3.5. Intracerebroventricular Ang (1–7) infusion inhibits the genesis of
plasma stress hormones
The effects of Ang (1–7) pretreatment on plasma levels of stress
hormones, including Ang II, corticosterone and norepinephrine, are
472 D. Zhu et al. / Biochemical Pharmacology 87 (2014) 467–476

Fig. 3. Central Ang (1–7) infusion increased Ang (1–7) levels and its receptor, Mas. Ang (1–7) levels were assayed in plasma (A and B) and several stress-related brain regions
(C and D), including the hypothalamus (HYPO), hippocampus (HIP), prefrontal cortex (PFC), and amygdala (AMY). Protein expression of Mas and AT1R (E and F) in the brain
was assayed by western blotting. Results are presented as mean  SD. *P < 0.05 or **P < 0.01 vs. aCSF + CRS. #P < 0.05 or ##P < 0.01 vs. H-A7 + CRS. L-A: low-dose Ang (1–7), H-A:
high-dose Ang (1–7).

shown in Fig. 4. Restraint at 4 8C for 2 h led to significant increases in
plasma levels (P < 0.01, Fig. 4A, C and E). These increases were
significantly attenuated by treatment with Ang (1–7) (P < 0.01).
However, when Ang (1–7) was co-infused with its antagonist, A779,
increases were observed in stressed rats (P < 0.01, Fig. 4B, D and F).
3.6. Intracerebroventricular Ang (1–7) infusion alters levels of brain
stress-related hormones or neurotransmitters
Ang II, corticosterone, and norepinephrine levels were mea-
sured in rat hypothalamus, hippocampus, prefrontal cortex, and
amygdala (Fig. 5), and were observed to be remarkably increased
by stress in all or selective brain extracts assayed (P < 0.01 or
<0.05, Fig. 5A, C, E and G). The largest increase of serotonin was
observed in the amygdala; and the largest increase in dopamine
was in the hypothalamus. Dopamine levels significantly increased
in rat prefrontal cortex, amygdala, and hypothalamus, but
decreased in the hippocampus (P < 0.01, Fig. 5G). However, after
1 week of treatment with Ang (1–7), these increases were
significantly prevented (P < 0.01 or <0.05, Fig. 5A, C, E and G).
The effects of Ang (1–7) on these levels were attenuated by A779
infusion (P < 0.01 or <0.05, Fig. 5B, D, F and H).
4. Discussion
Ang (1–7) is becoming a focus of current research. Ang (1–7)
plays a protective role in vessels [42], brain [21], heart [29,43],
kidney [44], liver [45] and lungs [46], but its role has not been
investigated in the stomach. In the present study, we found (for the
first time to the best of the authors’ knowledge) that Ang (1–7)
attenuated stress-induced gastric injury, and this gastroprotective
effect was associated with the modulation of stress pathways and
improved gastric microenvironments.
Gastric ulcer diseases result directly from an imbalance
between defensive factors that protect the mucosa and offensive
factors that disrupt important barriers. Offensive factors include
activated acid secretion and free radical generation, while
defensive factors involve superoxide dismutase and catalase in
tissues. H+, K+ ATPase is the final common pathway of acid
secretion, and blocking its activity is a well-accepted clinical
intervention used in peptic ulcer disease [47]. Free radicals have
been shown to be important etiopathological factors in the genesis
of peptic ulcers, and suppression of oxidative damage is beneficial
for decreasing ulcer progression and promoting healing of gastric
lesions [48]. Malondialdehyde represents an end-product of the
peroxidation of polyunsaturated fatty acids and related esters
within cell membranes, and is currently regarded as a reliable
marker of oxidative damage [49].
In the present study, stress-induced gastric injury was modeled
in rats subjected to 2 h of CRS, a well-studied model [50]. After
exposure to 2 h restraint at 4 8C, systolic blood pressure levels in
stressed rats increased by approximately 30 mmHg. Remarkable
gastric mucosal ulcers were observed in these rats, confirmed by a
mean of 84 on the ulcer index. In the gastric mucosa of stressed
 D. Zhu et al. / Biochemical Pharmacology 87 (2014) 467–476
Fig. 4. Central Ang (1–7) infusion suppressed the genesis of plasma stress hormones. Plasma Ang II (A and B), corticosterone (C and D) and norepinephrine (NE, E and F)
levels were assayed. Results are presented as mean  SD. *P < 0.05 or **P < 0.01 vs. aCSF + CRS. #P < 0.05 or ##P < 0.01 vs. H-A7 + CRS. L-A: low-dose Ang (1–7), H-A: high-dose
Ang (1–7).
rats, the activities of antioxidant enzymes, including catalase and
superoxide dismutase, reduced, while the malondialdehyde levels
and H+, K+ ATPase activities increased, leading to the formation of
stress ulcers. Several blood-borne stress hormones, including
Ang II, glucocorticoid and norepinephrine, were elevated, while
Ang (1–7) decreased. These observations suggest that Ang (1–7)
signaling was impaired during stress.
In recent studies, Ang II, glucocorticoids and norepinephrine
exert ulcer-promoting effects [16,51,52], and other actions against
Ang (1–7) [21,53]. Ang (1–7) signaling may represent an anti-ulcer
system. To explore this hypothesis, stressed rats were treated with
Ang (1–7). Since stress responses and stress ulceration are believed
to be brain-driven events [2], central manipulation has been
proposed as being more effective than simple gastric modulating
agents [3,4]. Although several brain-permeable Ang (1–7) mimics
are available now [21], synthetic Ang (1–7) was delivered directly
into brain ventricles in the present study.
After 1 week of treatment with doses of Ang (1–7), circulating
Ang (1–7) showed dose-dependent elevations. This may be due to
leakage from areas lacking a blood-brain barrier, or stimulated
peripheral angiotensin system by brain-permeable Ang (1–7)
metabolites. Stress-induced systolic blood pressure reduced with
Ang (1–7) administration, which was in accordance with the
vasodilatory action of Ang (1–7) [54]. Interestingly, Ang II levels
decreased after Ang (1–7) treatment. This can be explained by the
observations of a recent study reporting that Ang (1–7) can inhibit
473
the proteolytic function of Ang II-producing enzyme, Ang
converting enzyme, by binding at the COOH-terminal domain of
the enzyme [55]. Plasma glucocorticoids and norepinephrine levels
also reduced with Ang (1–7) treatment. Although it has previously
been reported that Ang (1–7) can inhibit sympathetic tone,
decrease norepinephrine release and Ang II-mediated norepineph-
rine release [30–32], we observed that Ang (1–7) decreased
glucocorticoid release. Accompanied by reduced ulcer-promoting
hormones, stress-induced gastric injury was attenuated by Ang (1–
7) treatment. This gastroprotection was associated with the
attenuation of oxidative damage and gastric acid production,
and enhanced antioxidative activities. The inhibition of H+, K+
ATPase activities and the recovery of antioxidant enzyme activities
by Ang (1–7) may result from it increasing the transcription levels
of these enzymes and/or it directly enhancing the activities of
these enzymes. Ang (1–7) has been well documented as an
effective suppressor of oxidative stress [56]. The beneficial effects
of Ang (1–7) were canceled with the administration of A779, a
blocker of the Ang (1–7) receptor, Mas, suggesting the critical role
of Mas.
Responses to stress are under the control of the brain. The stress
hormones Ang II and norepinephrine, acting as stress-related
neurotransmitters, are abundant in the brain. Several brain areas,
including the hypothalamus, hippocampus, prefrontal cortex, and
amygdala, and other brain neurotransmitters, including 5-HT and
dopamine, are also involved in the regulation of stress [57]. In
474 D. Zhu et al. / Biochemical Pharmacology 87 (2014) 467–476

Fig. 5. Central Ang (1–7) infusion altered the levels of brain stress hormones and neurotransmitters. The levels of Ang II (A and B), norepinephrine (NE, C and D), 5-HT (E and F)
and dopamine (DA, G and H) were assayed in rat hypothalamus (HYPO), hippocampus (HIP), prefrontal cortex (PFC) and amygdala (AMY). Results are presented as mean  SD.
*P < 0.05 or **P < 0.01 vs. aCSF + CRS. #P < 0.05 or ##P < 0.01 vs. H-A7 + CRS. L-A: low-dose Ang (1–7), H-A: high-dose Ang (1–7).

recent studies, anti-stress agents were reported to exert gastro-
protective effects through modulation of hormonal systems
[3,4,50]. We therefore assessed the effects of Ang (1–7) on the
central stress pathways. Ang II, norepinephrine, 5-HT, and
dopamine levels were elevated in the above brain regions;
dopamine, however, decreased in the hippocampus. These changes
were inhibited by Ang (1–7) treatment. Ang (1–7) inhibition of
brain Ang II and AT1R expression was observed to be linked to
reduced oxidative stress and attenuated neuronal apoptosis in a
previous study [22].
Norepinephrine has been implicated in the formation of stress
ulcers [52]. Activation of the central noradrenergic system
increases plasma glucocorticoid levels and stimulates glucocorti-
coid secretion, leading to norepinephrine release at different levels
and thus forming a feed-forward cycle between the HPA axis and
the SAS [3]. In response to 2 h of CRS, a vicious cycle is activated,
which concludes with the collapse of stress defenses. Ang (1–7)
may break the cycle by suppressing norepinephrine release. 5-HT
is an important neurotransmitter that promotes glucocorticoid
release [58] and anxiety [59]. CRS-induced 5-HT release provides
further evidence of the link between anxiety and stress. The finding
that Ang (1–7) decreased 5-HT levels suggests an anti-anxiety
effect of Ang (1–7), as reported recently [27].
Dopamine is a precursor of norepinephrine and an important
stress modulator in the brain. Administration of dopamine, or
related agents, attenuates stress ulcerogenesis, while opposite
effects have been observed with dopamine-lytic drugs [60–62].
Stress-induced dopamine release in the hypothalamus, prefrontal
cortex, and amygdala may contribute to elevated norepinephrine
levels, while depletion in the hippocampus is due to the
vulnerability of hippocampus during stress. As noted, excess
dopamine in the prefrontal cortex and amygdala is associated with
the reconsolidation of fear memories after stress disorder, a severe
anxiety disorder [63]. Increases in dopamine levels during stress
were prevented by Ang (1–7) treatment, further suggesting its
anxiolytic effects. Furthermore, the effects of brain Ang (1–7) were
attenuated by blocking its receptor, Mas, suggesting Ang (1–7)
signaling by Mas.
Taken together, the results suggest that Ang (1–7), via its
receptor, Mas, provides significant gastroprotection through the
modulation of peripheral and central stress pathways, and
the restoration of gastric microenvironments. Considering its
beneficial effects in vessels, brain, heart, kidney, liver and lungs,
Ang (1–7) administration by available brain-permeable mimics or
carriers may be a promising strategy for comprehensive anti-stress
prophylaxis and therapy.
Conflicts of interest
The authors declare that there are no conflicts of interest.
 D. Zhu et al. / Biochemical Pharmacology 87 (2014) 467–476
Acknowledgments
This work was supported by the Medical Science and
Technology Development Foundation, Nanjing Department of
Health (Nos. ZKX 08035 and YKK11031) and Nanjing Bureau of
Science and Technology (No. 201104009).
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