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

Biochemical Pharmacology 87 (2014) 467–476

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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 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 b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 14 September 2013 Accepted 31 October 2013 Available online 11 November 2013

Stress ulcers can develop with severe physiological stress, and have been proposed as being brain-driven events. New findings continue to suggest that stress ulcers can be more effectively managed through central manipulation rather than by simply altering local gastric factors. Angiotensin (1–7) (Ang (1–7)) is 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 accumulating evidence suggesting the anti-stress activities of Ang (1–7), its potential gastroprotective effect in the context of stress requires further investigation. In the present study, rat gastric mucosal lesions were induced by 2 h of cold-restraint stress. We observed that these lesions were significantly attenuated after 1 week of intracerebroventricular treatment with Ang (1–7). This gastroprotective 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 hormones, including Ang II, glucocorticoid, norepinephrine, serotonin, and dopamine, in blood or stressrelated brain regions. These findings indicate that Ang (1–7) exerts anti-stress activities by restoring the gastric microenvironment and modulating the stress pathways. Ang (1–7) may be a promising agent for stress ulcer prophylaxis and therapy, administered through brain-permeable mimics or carriers. Crown Copyright ß 2013 Published by Elsevier Inc. All rights reserved.

Keywords: Angiotensin (1–7) Mas Cold-restraint stress Oxidative stress Gastric lesions Chemical compounds studied in this article: Angiotensin (1–7) (PubChem CID: 123805) Angiotensin II (PubChem CID: 172198) Corticosterone (PubChem CID: 5753) Norepinephrine (PubChem CID: 439260) Dopamine (PubChem CID: 681) Serotonin (PubChem CID: 5202) Malondialdehyde (PubChem CID: 10964)

1. Introduction 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: [email protected] (J. Shi). 1 These authors contributed equally to this work.

central levels [6,7]. However, responses to stress can also be 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-hydroxytryptamine (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

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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 stressinduced injury [14–17]. However, the metabolite of Ang II through cleavage of a C-terminal residue, Ang (1–7) (Asp-Arg-Val-Tyr-IleHis-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.

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) HA7 + 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 nontreated]  100.

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

2.5. Measurement of H+, K+ ATPase activity, antioxidant enzyme activity, and malondialdehyde levels in rat stomachs To prepare tissue homogenates, stomach tissues were homogenized with liquid nitrogen using a mortar and pestle. Homogenized 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, Guangzhou, 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

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protein at 37 8C. Decomposition of H2O2 in the presence of catalase was measured at 240 nm (Tecan Trading AG, Ma¨nnedorf, 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% trichloroacetic 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 absorbance 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 nbutanol: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 highperformance 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 manufacturer’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 sulfatepolyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (Millipore). After blocking,

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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 (BioRad, 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 dichloromethane 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 (
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Table 1 Central Ang (1–7) administration reduced stress-induced systolic blood pressure (mmHg). Treatment

0

3

5

7

Part 1 aCSF + no CRS aCSF + CRS L-A7 + CRS H-A7 + CRS

129.3  8.0 125.9  5.1 126.1  4.2 127.2  6.3

124.1  8.0 120.8  6.0 120.9  8.4 116.0  7.7

122.1  9.1 122.2  7.5 124.6  6.9 119.8  9.6

124.8  7.6** 152.8  6.6 147.7  8.9 144.5  8.3*

Part 2 aCSF + CRS H-A7 + CRS H-A7 + A779 + CRS A779 + CRS

122.6  7.2 126.9  9.1 123.6  7.9 121.1  8.6

118.8  8.7 114.5  8.4 131.7  7.4 130.8  11.6

124.3  10.5 117.3  11.4 126.4  8.9 134.8  7.2

153.2  5.5# 146.0  7.3 157.9  8.3## 152.9  7.4#

Results are presented as mean  SD. *P < 0.05 or **P < 0.01 vs. aCSF + CRS. #P < 0.05 or dose Ang (1–7), H-A: high-dose Ang (1–7).

3.2. Central Ang (1–7) significantly attenuates stress-induced gastric ulcers via Mas The severity of ulcers in the present study was represented by the ulcer index, which was established by Dekanski et al. [38]. As shown in Fig. 1, many erosions in rat gastric mucosa were induced by 2 h of CRS (P < 0.01). After 1 week of pretreatment with 0.11 nmol/L Ang (1–7), stress-induced gastric injuries were significantly attenuated (P < 0.01). These injuries were nearly completely prevented after 1 week of 1.1 nmol/L Ang (1–7) treatment; a few rats showed blood in the lumen or pin-point erosions (P < 0.01). Significant erosions appeared in the gastric mucosa of rats receiving 1.1 nmol/L Ang (1–7) and 1.14 nmol/L A779, or A779 alone.

P < 0.01 vs. H-A7 + CRS. L-A: low-

3.3. Central Ang (1–7) significantly altered the activities of H+, K+ ATPase and antioxidant enzymes, and the levels of malondialdehyde

dose-dependent manner. Systolic blood pressure before stress induction was also reduced by 1.1 nmol/L Ang (1–7). When treated with its antagonist, A779, systolic blood pressure again increased by CRS, supporting the depressor effect of Ang (1–7).

After 2 h of CRS, the activities of H+, K+ ATPase in rat gastric mucosa were significantly enhanced (P < 0.01, Fig. 2A). Brain Ang (1–7) administration attenuated stress-induced H+, K+ ATPase 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).

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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

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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 measured 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

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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

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 norepinephrine 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

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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 gastroprotective 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 glucocorticoid 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.

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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). References [1] Janicki T, Stewart S. Stress-ulcer prophylaxis for general medical patients: a review of the evidence. J Hosp Med 2007;2(2):86–92. [2] Glavin GB, Murison R, Overmier JB, Pare WP, Bakke HK, Henke PG, et al. The neurobiology of stress ulcers. Brain Res Brain Res Rev 1991;16(3):301–43. [3] Garabadu D, Shah A, Ahmad A, Joshi VB, Saxena B, Palit G, et al. Eugenol as an anti-stress agent: modulation of hypothalamic–pituitary–adrenal axis and brain monoaminergic systems in a rat model of stress. Stress 2011;14(2): 145–55. [4] Ahmad A, Rasheed N, Gupta P, Singh S, Siripurapu KB, Ashraf GM, et al. Novel ocimumoside A and B as anti-stress agents: modulation of brain monoamines and antioxidant systems in chronic unpredictable stress model in rats. Phytomedicine 2012;19(7):639–47. [5] Goldstein DS, McEwen B. Allostasis, homeostats, and the nature of stress. Stress 2002;5(1):55–8. [6] Viljoen M, Panzer A. The central noradrenergic system: an overview. Afr J Psychiatry 2007;10(3):135–41. [7] Gordis EB, Granger DA, Susman EJ, Trickett PK. Salivary alpha amylase-cortisol asymmetry in maltreated youth. Horm Behav 2008;53(1):96–103. [8] Herman JP, Ostrander MM, Mueller NK, Figueiredo H. Limbic system mechanisms of stress regulation: hypothalamo–pituitary–adrenocortical axis. Prog Neuro Psychoph 2005;29(8):1201–13. [9] Sapolsky RM, Krey LC, McEwen BS. The neuroendocrinology of stress and aging: the glucocorticoid cascade hypothesis. Endocr Rev 1986;7(3):284–301. [10] Jacobson L, Sapolsky R. The role of the hippocampus in feedback regulation of the hypothalamic–pituitary–adrenocortical axis. Endocr Rev 1991;12(2): 118–34. [11] Herman JP, Cullinan WE. Neurocircuitry of stress: central control of the hypothalamo–pituitary–adrenocortical axis. Trends Neurosci 1997;20(2): 78–84. [12] Czeh B, Perez-Cruz C, Fuchs E, Flugge G. Chronic stress-induced cellular changes in the medial prefrontal cortex and their potential clinical implications: does hemisphere location matter? Behav Brain Res 2008;190(1):1–13. [13] Saavedra JM, Benicky J. Brain and peripheral angiotensin II play a major role in stress. Stress 2007;10(2):185–93. [14] Saavedra JM, Sanchez-Lemus E, Benicky J. Blockade of brain angiotensin II AT1 receptors ameliorates stress, anxiety, brain inflammation and ischemia: therapeutic implications. Psychoneuroendocrinology 2011;36(1):1–18. [15] Pavel J, Benicky J, Murakami Y, Sanchez-Lemus E, Saavedra JM. Peripherally administered angiotensin II AT1 receptor antagonists are anti-stress compounds in vivo. Ann N Y Acad Sci 2008;1148:360–6. [16] Bregonzio C, Armando I, Ando H, Jezova M, Baiardi G, Saavedra JM. Angiotensin II AT1 receptor blockade prevents gastric ulcers during cold-restraint stress. Ann N Y Acad Sci 2004;1018:351–5. [17] Bregonzio C, Armando I, Ando H, Jezova M, Baiardi G, Saavedra JM. Antiinflammatory effects of angiotensin II AT1 receptor antagonism prevent stress-induced gastric injury. Am J Physiol Gastrointest Liver Physiol 2003;285(2):G414–23. [18] Santos RA, Simoes e Silva AC, Maric C, Silva DM, Machado RP, de Buhr I, et al. Angiotensin-(1–7) is an endogenous ligand for the G protein-coupled receptor Mas. Proc Natl Acad Sci U S A 2003;100(14):8258–63. [19] Gironacci MM, Adamo HP, Corradi G, Santos RA, Ortiz P, Carretero OA. Angiotensin (1–7) induces Mas receptor internalization. Hypertension 2011;58(2):176–81. [20] Olszanecki R, Madej J, Suski M, Gebska A, Bujak-Gizycka B, Korbut R. Angiotensin metabolism in rat stomach wall: prevalence of angiotensin-(1–7) formation. J Physiol Pharmacol 2009;60(1):191–6. [21] Xu P, Sriramula S, Lazartigues E. ACE2/ANG-(1–7)/Mas pathway in the brain: the axis of good. Am J Physiol Regul Integr Comp Physiol 2011;300(4): R804–17. [22] Jiang T, Gao L, Shi J, Lu J, Wang Y, Zhang Y. Angiotensin-(1–7) modulates renin– angiotensin system associated with reducing oxidative stress and attenuating neuronal apoptosis in the brain of hypertensive rats. Pharmacol Res 2013;67(1):84–93. [23] Zhang Y, Lu J, Shi J, Lin X, Dong J, Zhang S, et al. Central administration of angiotensin-(1–7) stimulates nitric oxide release and upregulates the endothelial nitric oxide synthase expression following focal cerebral ischemia/ reperfusion in rats. Neuropeptides 2008;42(5/6):593–600. [24] Passos-Silva DG, Verano-Braga T, Santos RA. Angiotensin-(1–7): beyond the cardio-renal actions. Clin Sci 2013;124(7):443–56. [25] Regenhardt RW, Desland F, Mecca AP, Pioquinto DJ, Afzal A, Mocco J, et al. Antiinflammatory effects of angiotensin-(1–7) in ischemic stroke. Neuropharmacology 2013;71:154–63. [26] Mecca AP, Regenhardt RW, O’Connor TE, Joseph JP, Raizada MK, Katovich MJ, et al. Cerebroprotection by angiotensin (1–7) in endothelin-1 induced ischemic stroke. Exp Physiol 2011;96(10):1084–96.

475

[27] Bild W, Ciobica A. Angiotensin-(1–7) central administration induces anxiolytic-like effects in elevated plus maze and decreased oxidative stress in the amygdala. J Affect Disorders 2013;145(2):165–71. [28] Moon JY, Tanimoto M, Gohda T, Hagiwara S, Yamazaki T, Ohara I, et al. Attenuating effect of angiotensin-(1–7) on angiotensin II-mediated NAD(P)H oxidase activation in type 2 diabetic nephropathy of KK-Ay/Ta mice. Am J Physiol Renal Physiol 2011;300(6):F1271–82. [29] Zhou LM, Shi Z, Gao J, Han Y, Yuan N, Gao XY, et al. Angiotensin-(1–7) and angiotension II in the rostral ventrolateral medulla modulate the cardiac sympathetic afferent reflex and sympathetic activity in rats. Pflugers Arch 2010;459(5):681–8. [30] Gironacci MM, Yujnovsky I, Gorzalczany S, Taira C, Pena C. Angiotensin-(1–7) inhibits the angiotensin II-enhanced norepinephrine release in coarcted hypertensive rats. Regul Pept 2004;118(1/2):45–9. [31] Gironacci MM, Valera MS, Yujnovsky I, Pena C. Angiotensin-(1–7) inhibitory mechanism of norepinephrine release in hypertensive rats. Hypertension 2004;44(5):783–7. [32] Gironacci MM, Vatta M, Rodriguez-Fermepin M, Fernandez BE, Pena C. Angiotensin-(1–7) reduces norepinephrine release through a nitric oxide mechanism in rat hypothalamus. Hypertension 2000;35(6):1248–52. [33] Rebas E, Zabczynska J, Lachowicz A. The effect of angiotensin 1–7 on tyrosine kinases activity in rat anterior pituitary. Biochem Biophys Res Commun 2006;347(3):581–5. [34] Albrecht D. Angiotensin-(1–7)-induced plasticity changes in the lateral amygdala are mediated by COX-2 and NO. Learn Memory 2007;14(3): 177–84. [35] Hellner K, Walther T, Schubert M, Albrecht D. Angiotensin-(1–7) enhances LTP in the hippocampus through the G-protein-coupled receptor Mas. Mol Cell Neurosci 2005;29(3):427–35. [36] Staschewski J, Kulisch C, Albrecht D. Different isoforms of nitric oxide synthase are involved in angiotensin-(1–7)-mediated plasticity changes in the amygdala in a gender-dependent manner. Neuroendocrinology 2011;94(3): 191–9. [37] Zhu D, Shi J, Zhang Y, Wang B, Liu W, Chen Z, et al. Central angiotensin II stimulation promotes beta amyloid production in Sprague Dawley rats. PLoS ONE 2011;6:e16037. [38] Dekanski JB, Macdonald A, Sacra P. Effects of fasting, stress and drugs on gastric glycoprotein synthesis in the rat. Br J Pharmacol 1975;55(3): 387–92. [39] Demirbilek S, Gurses I, Sezgin N, Karaman A, Gurbuz N. Protective effect of polyunsaturated phosphatidylcholine pretreatment on stress ulcer formation in rats. J Pediatr Surg 2004;39(1):57–62. [40] Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 1979;95(2):351–8. [41] Tian M, Zhu D, Xie W, Shi J. Central angiotensin II-induced Alzheimer-like tau phosphorylation in normal rat brains. FEBS Lett 2012;586(20):3737–45. [42] Akhtar S, Yousif MH, Dhaunsi GS, Chandrasekhar B, Al-Farsi O, Benter IF. Angiotensin-(1–7) inhibits epidermal growth factor receptor transactivation via a Mas receptor-dependent pathway. Br J Pharmacol 2012;165(5): 1390–400. [43] Giani JF, Munoz MC, Mayer MA, Veiras LC, Arranz C, Taira CA, et al. Angiotensin-(1–7) improves cardiac remodeling and inhibits growth-promoting pathways in the heart of fructose-fed rats. Am J Physiol Heart Circ Physiol 2010;298(3):H1003–13. [44] Stegbauer J, Potthoff SA, Quack I, Mergia E, Clasen T, Friedrich S, et al. Chronic treatment with angiotensin-(1–7) improves renal endothelial dysfunction in apolipoproteinE-deficient mice. Br J Pharmacol 2011;163(5):974–83. [45] Lubel JS, Herath CB, Tchongue J, Grace J, Jia Z, Spencer K, et al. Angiotensin-(1– 7), an alternative metabolite of the renin–angiotensin system, is up-regulated in human liver disease and has antifibrotic activity in the bile-duct-ligated rat. Clin Sci 2009;117(11):375–86. [46] Klein N, Gembardt F, Supe S, Kaestle SM, Nickles H, Erfinanda L, et al. Angiotensin-(1–7) protects from experimental acute lung injury. Crit Care Med 2013 [Epub ahead of print]. [47] Shin JM, Cho YM, Sachs G. Chemistry of covalent inhibition of the gastric (H+, K+)-ATPase by proton pump inhibitors. J Am Chem Soc 2004;126(25): 7800–11. [48] Demir S, Yilmaz M, Koseoglu M, Akalin N, Aslan D, Aydin A. Role of free radicals in peptic ulcer and gastritis. Turk J Gastroenterol 2003;14(1):39–43. [49] Dalle-Donne I, Rossi R, Colombo R, Giustarini D, Milzani A. Biomarkers of oxidative damage in human disease. Clin Chem 2006;52(4):601–23. [50] Krishnamurthy S, Garabadu D, Reddy NR, Joy KP. Risperidone in ultra low dose protects against stress in the rodent cold restraint model by modulating stress pathways. Neurochem Res 2011;36(10):1750–8. [51] Filaretova L. The hypothalamic–pituitary–adrenocortical system: hormonal brain-gut interaction and gastroprotection. Auton Neurosci Basic 2006;125(1/ 2):86–93. [52] Morilak DA, Barrera G, Echevarria DJ, Garcia AS, Hernandez A, Ma S, et al. Role of brain norepinephrine in the behavioral response to stress. Prog Neuro Psychoph 2005;29(8):1214–24. [53] Marshall AC, Shaltout HA, Nautiyal M, Rose JC, Chappell MC, Diz DI. Fetal betamethasone exposure attenuates angiotensin-(1–7)-Mas receptor expression in the dorsal medulla of adult sheep. Peptides 2013;44:25–31. [54] Stegbauer J, Oberhauser V, Vonend O, Rump LC. Angiotensin-(1–7) modulates vascular resistance and sympathetic neurotransmission in kidneys of spontaneously hypertensive rats. Cardiovasc Res 2004;61(2):352–9.

476

D. Zhu et al. / Biochemical Pharmacology 87 (2014) 467–476

[55] Tom B, de Vries R, Saxena PR, Danser AH. Bradykinin potentiation by angiotensin-(1–7) and ACE inhibitors correlates with ACE C- and N-domain blockade. Hypertension 2001;38(1):95–9. [56] Zimmerman MC. Angiotensin II and angiotensin-1–7 redox signaling in the central nervous system. Curr Opin Pharmacol 2011;11(2):138–43. [57] Ranabir S, Reetu K. Stress and hormones. Indian J Endocrinol Metab 2011;15: 18–22. [58] Heisler LK, Pronchuk N, Nonogaki K, Zhou L, Raber J, Tung L, et al. Serotonin activates the hypothalamic–pituitary–adrenal axis via serotonin 2C receptor stimulation. J Neurosci 2007;27(26):6956–64. [59] Graeff FG, Guimaraes FS, De Andrade TG, Deakin JF. Role of 5-HT in stress, anxiety, and depression. Pharmacol Biochem Behav 1996;54(1):129–41.

[60] Landeira-Fernandez J, Grijalva CV. Participation of the substantia nigra dopaminergic neurons in the occurrence of gastric mucosal erosions. Physiol Behav 2004;81(1):91–9. [61] Saad SF, Agha AM, Amrin Ael N. Effect of bromazepam on stress-induced gastric ulcer in rats and its relation to brain neurotransmitters. Pharmacol Res 2001;44(6):495–501. [62] Ozdemir V, Jamal MM, Osapay K, Jadus MR, Sandor Z, Hashemzadeh M, et al. Cosegregation of gastrointestinal ulcers and schizophrenia in a large national inpatient discharge database: revisiting the brain-gut axis hypothesis in ulcer pathogenesis. J Invest Med 2007;55(6):315–20. [63] Pezze MA, Feldon J. Mesolimbic dopaminergic pathways in fear conditioning. Prog Neurobiol 2004;74(5):301–20.