Naunyn-Schmiedeberg's Archives of Pharmacology https://doi.org/10.1007/s00210-018-1523-3

ORIGINAL ARTICLE

Modulation of brain ACE and ACE2 may be a promising protective strategy against cerebral ischemia/reperfusion injury: an experimental trial in rats Maha Mohammed Abdel-Fattah 1 & Basim Anwar Shehata Messiha 1 & Ahmed Mohamed Mansour 2 Received: 9 March 2018 / Accepted: 1 June 2018 # Springer-Verlag GmbH Germany, part of Springer Nature 2018

Abstract The brain renin-angiotensin system (RAS) is considered a crucial regulator for physiological homeostasis and disease progression. We evaluated the protective effects of the angiotensin receptor blocker (ARB) telmisartan and the angiotensin-converting enzyme 2 (ACE2) activator xanthenone on experimental cerebral ischemia/reperfusion (I/R) injury. Rats were divided into a sham control, a cerebral I/R control, a standard treatment (nimodipine, 10 mg/kg/day, 15 days, p.o.), three telmisartan treatments (1, 3, and 10 mg/kg/day, 15 days, p.o.), and three xanthenone treatments (0.5, 1, and 2 mg/kg/day, 15 days, s.c.) groups. One hour after the last dose, all rats except the sham control group were exposed to 30-min cerebral ischemia followed by 24-h reperfusion. Brain ACE and ACE2 activities and the apoptotic marker caspase-3 levels were assessed. Glutathione (GSH), malondialdehyde (MDA), and nitric oxide end products (NOx) as oxidative markers and tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and IL-10 as immunological markers were assessed. Histopathological examination and immunohistochemical evaluation of glial fibrillary acidic protein (GFAP) were performed in cerebral cortex and hippocampus sections. Telmisartan and xanthenone in the higher doses restored MDA, NOx, TNF-α, IL-6, caspase-3, ACE, and GFAP back to normal levels and significantly increased GSH, IL-10, and ACE2 compared to I/R control values. Histopathologically, both agents showed mild degenerative changes and necrosis of neurons in cerebral cortex and hippocampus compared with I/R control group. Modulation of brain RAS, either through suppression of the classic ACE pathway or stimulation of its antagonist pathway ACE2, may be a promising strategy against cerebral I/R damage. Keywords ACE . ACE2 . Ischemia/reperfusion . Nimodipine . Telmisartan . Xanthenone

Introduction The organ-localized renin-angiotensin system (RAS) is now considered to play an important role in pathological oxidative and inflammatory damage (Paul et al. 2006; Claflin and Grobe 2015). Interference with localized RAS The indicated corresponding author will handle correspondence at all stages of refereeing and publication, as well as post-publication. * Basim Anwar Shehata Messiha [email protected] 1

Pharmacology and Toxicology Department, Faculty of Pharmacy, Beni-Suef University, Beni-Suef, Egypt

2

Pharmacology and Toxicology Department, Faculty of Pharmacy, Al-Azhar University, Cairo, Egypt

was reported to have beneficial outcomes in diverse animal models, namely experimental Alzheimer simulation (Ali et al. 2016; Khallaf et al. 2017), ova-induced bronchial asthma (Abdel-Fattah et al. 2015), chemical hepatotoxicity (Mohammed et al. 2016), and adjuvant-induced rheumatoid arthritis (Wahba et al. 2015). The angiotensin II (Ang-II) produced by the angiotensinconverting enzyme (ACE) in the activated RAS was reported to have pro-oxidant and pro-inflammatory potentials via stimulation of angiotensin II receptor type 1 (AT1 receptor) leading to production of massive amounts of pro-oxidant and proinflammatory mediators including cytokines (da Silveira et al. 2010; Chang and Wei 2015). Activation of NADPH oxidase by angiotensin II leads to the production of massive amounts of reactive oxygen species (ROS) (de Cavanagh et al. 2010; Kossmann et al. 2014; Passaglia et al. 2015). Brain RAS was earlier reported to be implicated in the massive

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progression of ischemic brain injury concerned in the current study (Chen et al. 2009). Interestingly, AT1 receptor blockers (ARBs) have been shown to protect the brain from ischemic injury in animal studies as reported regarding the protective effect of candesartan on cerebral ischemic stroke in spontaneously hypertensive rats (Nishimura et al. 2000) and the protective effect of telmisartan on ischemic brain damage in diabetic mice (Iwanami et al. 2010). Accumulating evidence refers to the presence of a parallel homologous system where the enzyme is ACE2, the product is Ang-(1-7), and the receptor is Mas. Unlike the classic ACE/ Ang-II/AT1 axis, the ACE2/Ang-(1-7)/Mas axis has potent anti-inflammatory effect and is considered the physiological antagonist of the classic system in the brain as well as other organs (Iwanami et al. 2010; Simoes e Silva et al. 2013; Pernomian et al. 2014). Earlier reports showed that central administration of Ang-(1-7) stimulates nitric oxide (NO) release and up-regulates endothelial nitric oxide synthase (eNOS) expression in ischemic tissues following focal cerebral ischemia/reperfusion (I/R) in rats (Chen et al. 2014a). Unlike other ARBs, telmisartan is a unique drug that has a neuroprotective action and acts as an agonistic ligand for peroxisome proliferator-activated receptor-gamma (PPAR-γ) (Khallaf et al. 2017). Many PPAR-γ agonists have been shown to inhibit the activation of signal transducer and activator of transcriptions (STAT), nuclear factor-kappaB (NF-κB), and mitogen-activated protein kinases (MAPK) and down-regulate expression of cytotoxic or proinflammatory gene products (Bernardo and Minghetti 2006). Jung et al. (2007) investigated the therapeutic effect of telmisartan in experimental intracerebral hemorrhage (ICH) in normotensive rats, where the drug was shown to possess anti-oxidant and anti-inflammatory effects coupled with PPAR-γ and eNOS stimulation. Xanthenone, or 9H-Xanthen-9-one, with the molecular formula C13H8O2, is a newly discovered ACE2 activator and previous studies have demonstrated that activation of this enzyme might be a promising strategy to treat cardiovascular and related diseases. Although a number of earlier investigators discussed the role of telmisartan and other ARBs on different experimental ischemic models, too little pharmacological studies were performed on xanthenone as a novel ACE2 activator, including the study performed by Hernández Prada et al. (2008) showing that continuous infusion of xanthenone caused modest decrease in the blood pressure of spontaneously hypertensive rats coupled with reversal of myocardial and perivascular fibrosis and the study performed by Ibrahim et al. (2014) showing that xanthenone could protect experimental pregnant rats against leptininduced hypertension and proteinuria via ACE2 activation. To date, no previous study reported the effect of xanthenone on cerebral ischemic injury. However, Rodrigues Prestes et al. (2017) reported that stimulation of the ACE2/Ang-(1-7)/Mas

axis reduced cytokine release and inhibited signaling pathways in experimental models of some human diseases including cerebral ischemia. Experimental cerebral ischemia is a well-established model of brain injury and is considered a simulation to clinical brain ischemic damage in some events like intravascular thrombosis and other forms of stroke (Martínez-Sánchez et al. 2014). Calcium channel blockers are long known to be standard protective agents against experimental I/R injury in different models, as reperfusion injury is mostly related to massive calcium influx in the hypoxic tissue (Cai et al. 2006; Chen et al. 2014b; Shi et al. 2016). Nimodipine is particularly of interest as it is a dihydropyridine agent with a potent action on blood vessels. Additionally, it is a lipophilic agent capable of penetrating the blood–brain barrier (Bielenberg et al. 1990, Tomassoni et al. 2008). Accordingly, nimodipine was applied as a reference standard in a number of investigations concerning the effect of different drugs and extracts on cerebral ischemic injury (Zhu et al. 2017; Zou et al. 2017). Based on the aforementioned data, the present investigation aims to evaluate the possible protective effects of the ARB telmisartan and the ACE2 activator xanthenone, compared with nimodipine as a reference standard, on cerebral I/R injury in experimental rats.

Material and methods Animals Adult male Wistar rats, weighing 200–250 g, were housed in the animal room of the Faculty of Pharmacy, Beni-Suef University in specific pathogen-free conditions at a controlled temperature (25 ± 3 °C) with alternating 12-h light/dark cycles. Animals were allowed standard chow pellets and drinking water ad libitum. Handling of animals and experimental protocols were ethically performed after approval of the Ethical Committee of the Faculty of Pharmacy, Beni-Suef University, which is a member of the Egyptian Network of Research Ethics Committees (ENREC) and which followed the recommendations of the National Institutes of Health (NIH) Guide for Care and Use of Laboratory Animals (NIH Publication No. 8023, revised 1978).

Drugs, chemicals, and reagent kits Nimodipine, telmisartan, xanthenone, and thiopental were obtained from Sigma-Aldrich Chemicals Co., USA. The GSH ELISA kit was purchased from ShangHai BlueGene Biotech Co., LTD, China (Catalog number E02G0367, detection range 50–1000 pg/ml, detection wavelength 450 nm, inter- and intra-assay variabilities are lower than 10%). Caspase-3

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ELISA kit was supplied by CusaBio, USA, with Catalog number CSB-E08857r, detection range 0.312–20 ng/ml, detection wavelength 450 nm, inter-assay variability 10%, and intraassay variability 8%. IL-6 ELISA kit was supplied by CusaBio, USA, with Catalog number CSB-E04640r, detection range 78–5000 pg/ml, detection wavelength 450 nm, and inter- and intra-assay variabilities are lower than 10%. IL-10 ELISA kit was supplied by CusaBio, USA, with Catalog number CSB-E04595r, detection range 3.12– 200 pg/ml, detection wavelength 450 nm, inter-assay variability 10%, and intra-assay variability 8%. TNF-α ELISA kits was obtained from Ray Biotech, USA (Catalog number ELRTNFalpha-001C, detection range 25–20,000 pg/ml, detection wavelength 450 nm, inter- and intra-assay variabilities are lower than 10%). Tissue extraction kits for mRNA of ACE and ACE2 were obtained from Qiagen, USA, while cDNA reverse transcription kit was obtained from Fermentas, USA. Mouse monoclonal GFAP antibody (200 μg/ml, Catalog number MS-280-R7) was obtained from Thermo Fisher Scientific, USA. Secondary HRP-conjugated antibody for GFAP assay (1 μg/ μl, Catalog number bs-0199R-HRP) was obtained from Bioss Inc., USA. All other chemicals and reagents used were of analytical grade.

One hour after the last dose, all rats except those of the sham control group were exposed to 30 min xerebral ischemia followed by 24-h reperfusion (Pulsinelli and Brierley 1979; Ahmed et al. 2014).

Induction of cerebral I/R Cerebral ischemic injury was induced by bilateral carotid artery occlusion according to the modified method described by Ahmed et al. (2014). Briefly, rats were anesthetized with thiopental (50 mg/kg, i.p.) and a midline ventral incision was made in the neck. The bilateral carotid artery was separated from the adjacent tissues and vagus nerve, then occluded using small artery clips to induce global cerebral ischemia for 30 min, followed by a 24-h reperfusion period where the clips were removed to restore circulation. Soon after the start of reperfusion, the abdominal wall and skin were sutured with waxed silk stitches. Rats of the sham control group were exposed to the same procedure except for carotid occlusion. Rectal temperature was regularly checked and maintained at 37 °C throughout the experiment by means of a heating lamp and a heating pad to prevent hypothermia (Fukuoka et al. 2015).

Experimental design Sample preparation for biochemical analyses Rats were randomly divided into nine groups, 13 animals each (Fig. 1). Group 1 was kept as sham control, while group 2 was kept as cerebral I/R control, and both received 1% Tween 80 solution in normal saline by oral gavage (5 ml/kg/day, p.o.) for 15 days. Group 3 was kept as standard treatment and received nimodipine (10 mg/kg/day, p.o.) for 15 days before I/R (Satoh et al. 1996). Groups 4, 5, and 6 received telmisartan (1, 3, and 10 mg/kg/day, respectively, p.o.) for 15 days before I/R (Kobayashi et al. 2009; Abdel-Fattah et al. 2015). Groups 7, 8, and 9 received xanthenone (0.5, 1, and 2 mg/kg/day, respectively, s.c.) for 15 days before I/R (Ibrahim et al. 2014). Nimodipine, telmisartan, and xanthenone were supplied as suspensions in 1% Tween 80 solution in normal saline. Fig. 1 Experimental design showing time schedule of test agent administration, I/R, and sampling

At the end of the experiment, 10 rats in each group were sacrificed by cervical dislocation. The brains were carefully dissected out on ice, and the right hemispheres were washed and homogenized in ice-cold saline to prepare 25% w/v homogenate using a homogenizer (yellow line, DI18 basic, Germany). Brain homogenates were centrifuged at 1000×g for 20 min at 4 °C, then stored at − 80 °C for further biochemical analyses of GSH, MDA, NOx, IL-6, IL-10, TNF-α, and caspase-3. The left hemispheres were used for real-time polymerase chain reaction (RT-PCR) of ACE and ACE2 by homogenization in lysis buffer RLT only for five groups, namely sham, I/R control,

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nimodipine (standard), telmisartan highest dose (10 mg/kg/ day), and xanthenone highest dose (2 mg/kg/day).

Sample preparation for histopathological and immunohistochemical studies Intracardiac perfusion was performed for histopathological and immunohistopathological assessments. Three rats in the aforementioned five groups, namely sham control, I/R control, nimodipine, telmisartan (10 mg/kg/day), and xanthenone (2 mg/kg/day) groups, were anesthetized with thiopental (50 mg/kg, i.p.). An incision was made along the thorax in order to expose the heart, and the left cardiac ventricle was cannulated. Finally, an incision was made to the rat’s right atrium using scissors to create as large an outlet as possible without damaging the descending aorta. Animals were perfused with 20 ml saline followed by 40 ml of 4% paraformaldehyde in phosphate-buffered saline (PBS) for 5 min. Heads were dissected to remove the brains and placed in a vial of fixative-containing fluid (4% paraformaldehyde in PBS) at least 10× the volume of the brain itself (Pinteaux et al. 2006). Brains were kept in the fixative for 24 h at 4 °C for further manipulations.

Determination of brain tissue cytokines TNF-α, IL-6, and IL-10 level Brain TNF-α, IL-6, and IL-10 were determined in tissue homogenates using ELISA kits according to manufacturer’s instructions based on the ELISA sandwich technique described earlier using rat-specific TNF-α (Bonavida 1991), IL-6 (Engvall et al. 1971; Van Weemen and Schuurs 1971) and IL-10 (Braun et al. 1999) ELISA kits. TNF-α, IL-6, and IL-10 levels were expressed as picogram per gram wet tissue. Determination of apoptotic marker caspase-3 level Brain caspase-3 level was assayed in tissue homogenates using ELISA kit according to the manufacturer’s instructions based on the principle described earlier in a manner typical with cytokines assay just mentioned (Fernandes-Alnemri et al. 1994). The level of caspase-3 was expressed as picogram per gram wet tissue.

Quantitative real-time PCR assessment of ACE and ACE2 activities Isolation of Total RNA

Determination of tissue biomarkers Determination of brain tissue GSH level Brain tissue GSH was measured using GSH ELISA kit according to the manufacturer’s instructions based on the principle described earlier (Van Weemen and Schuurs 1971). The color change was measured at a wavelength of 450 nm and GSH level was expressed as picogram per gram wet tissue.

Determination of brain tissue MDA level Production of MDA was measured colorimetrically using a spectrophotometer (dual wavelength, Beckman, USA) according to the method described by Ohkawa et al. (1979), where the formed colored complex could be extracted in nbutanol and measured spectrophotometrically at 532 nm. The MDA level was expressed as nanomole per gram wet tissue.

Determination of brain tissue NOx level Assay of NOx level was based on the Griess reaction described earlier (Miranda et al. 2001), where the produced nitrite was measured spectrophotometrically at 545 nm as a colored azo dye. Total NOx level was expressed as micromole per gram wet tissue.

Total RNA was isolated using the Qiagen tissue extraction kit according to instructions of manufacturer. The purity (A260/ A280 ratio) and the concentration of RNA were obtained using spectrophotometry. cDNA synthesis The total RNA (0.5–2 μg) was used for cDNA conversion using high capacity cDNA Fermentas reverse transcription kit, where 3 μl of random primers was added to the 10 μl of RNA which was denatured for 5 min at 65 °C in the thermal cycler. The RNA-primer mixture was cooled to 4 °C. The cDNA master mix was prepared according to the kit instructions and was added for each sample. Total volume of the master mix was 19 μl for each sample. This was added to the 31-μl RNA-primer mixture resulting in 50 μl of cDNA. The last mixture was incubated in the programmed thermal cycler 1 h at 37 °C followed by inactivation of enzymes at 95 °C for 10 min and finally cooled at 4 °C. RNA was then changed into cDNA and the converted cDNA was stored at − 20 °C. Amplification and analysis Real-time qPCR amplification and analysis were performed using an applied biosystem with software version 3.1

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(StepOne™, USA). The qPCR assay with the primer sets was optimized at the annealing temperature. The primer sequence of ACE was shown to be as follows: & &

Forward primer: 5′-TTCCCCCAAAGGCCAAGTCC CA-3′ Reverse primer: 5′-GAGGCTGCCCTGGCTTCTGTC-3′

analysis using Bonferroni test to compare between group means pairwise. A significant difference was considered when p value was less than 0.05.

Results Effect of test agents on brain tissue biomarkers

On the other side, the primer sequence of ACE2 was shown to be as follows: & &

Forward primer: 5′-ACCCGTGACCAAGTCTTGAA-3′ Reverse primer: 5′-AAGGAAGTGCCAGGTCAATG-3′

While the primer sequence of the reference beta actin was shown to be as follows: & &

Forward primer: 5′-GGTCGGTGTGAACGGATTTGG-3′ Reverse primer: 5′-ATGTAGGCCATGAGGTCCACC-3′

Histopathological study Brain samples stored in the fixative solution were trimmed and processed by dehydration in alcohol, clearing in Xylene, infiltration with synthetic wax, and blocking out into Paraplast tissue embedding media. Sections that are 3–5-μm in thickness were cut by rotatory microtome. The sections were stained with Harris Hematoxylin and Eosin (H&E) stain as described earlier (Bancroft and Gamble 2008) and microscopically examined at ×100 magnification. Immunohistochemical evaluation of GFAP Brain sections prepared as described above were exposed to immunostaining using mouse monoclonal GFAP antibody and appropriately labeled antiglobulin and chromogen according to the method described by Amenta et al. (1998) and Miguel-Hidalgo et al. (2000). Fields were analyzed for GFAP area percentage by using full HD microscopic camera attached to Leica Application Suite software (Leica Microsystems GmbH, Wetzlar, Germany) and examined at ×400 magnification. Calculating GFAP area is more accurate than absolute cell counting. The more induction of reacting astrocytes, the more branching of cell process is revealed. Statistical analysis Results of the biochemical analyses, RT-PCR estimations, and area percentage of GFAP were expressed as mean ± SEM, and statistical analysis was done using one-way analysis of variance (ANOVA) test using Statistical Package for Social Sciences (SPSS) program (version 16) followed by post hoc

Oxidative and inflammatory biomarkers The results are given in Fig. 2. Cerebral I/R induced significant decrease in brain GSH content to 23% as compared to sham control. Administration of the standard treatment nimodipine (10 mg/kg/day) or telmisartan (1 mg/kg/day) prior to I/R did not affect GSH level, while administration of telmisartan in doses of 3 and 10 mg/kg/day prior to I/R caused significant increase in brain GSH content by about 68 and 91%, respectively, of the I/R control value. Additionally, administration of xanthenone in doses of 0.5, 1, and 2 mg/kg/day prior to I/R significantly increased GSH content by about 116, 154, and 208%, respectively, of the respective I/R control value. Telmisartan (10 mg/kg/day) and xanthenone (0.5, 1, and 2 mg/kg/day) showed better effects regarding brain tissue GSH content compared with the reference standard nimodipine. Additionally, cerebral I/R caused significant increase in MDA content to reach 322% as compared to sham control. Administration of the standard treatment nimodipine in a dose of 10 mg/kg/day prior to I/R significantly decreased MDA content by 56% of the I/R control value, while administration of telmisartan in doses of 1, 3, and 10 mg/kg/day prior to I/R significantly decreased MDA content by about 38, 60, and 74%, respectively, of the respective I/R control value. Additionally, administration of xanthenone in doses of 0.5, 1, and 2 mg/kg/day prior to I/R showed percentage reductions of MDA content of about 46, 63, and 69%, respectively, of the respective I/R control value. Moreover, cerebral I/R induced significant increase in brain NOx production to 201% as compared to sham control. Administration of the standard treatment nimodipine in a dose of 10 mg/kg/day prior to I/R significantly decreased NOx content by 49% of the I/R control value, while administration of telmisartan in doses of 3 and 10 mg/kg/day prior to I/R caused significant decrease in NOx content by 54 and 66%, respectively, of the I/R control value. Additionally, administration of xanthenone in doses of 0.5, 1, and 2 mg/kg/day prior to I/R caused percent reductions in NOx production by about 58, 45, and 57%, respectively, of the respective I/R control value.

Immunological markers The results are given in Fig. 3. Regarding cytokine levels, cerebral I/R induced significant increase in brain TNF-α achieving

Brain Tissue GSH (Pg/g ssue)

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I/R

Nimodipine

Telmisartan

(10 mg/kg/day, p.o.)

(1 mg/kg/day, p.o.)

Telmisartan

Xanthenone Xanthenone

Xanthenone

(3 mg/kg/day, p.o.) (10 mg/kg/day, p.o.)

Telmisartan

(0.5 mg/kg/day, p.o.) (1 mg/kg/day, p.o.)

(2 mg/kg/day, p.o.)

Brain Tissue MDA (nmol/g ssue)

Sham

I/R

Xanthenone Xanthenone

Xanthenone

(10 mg/kg/day, p.o.) (1 mg/kg/day, p.o.) (3 mg/kg/day, p.o.) (10 mg/kg/day, p.o.) (0.5 mg/kg/day, p.o.) (1 mg/kg/day, p.o.)

Nimodipine

Telmisartan Telmisartan

Telmisartan

(2 mg/kg/day, p.o.)

Brain Tissue NOx (μmol/g ssue)

Sham

Sham

I/R

Xanthenone Xanthenone

Xanthenone

(10 mg/kg/day, p.o.) (1 mg/kg/day, p.o.) (3 mg/kg/day, p.o.) (10 mg/kg/day, p.o.) (0.5 mg/kg/day, p.o.) (1 mg/kg/day, p.o.)

Nimodipine Telmisartan Telmisartan

Telmisartan

(2 mg/kg/day, p.o.)

Fig. 2 Effect of different doses of telmisartan and xanthenone, as compared with nimodipine, on brain tissue GSH, MDA, and NOx in rats subjected to cerebral I/R injury. Data were expressed as mean ± SEM (n = 8–10). Statistical analysis was carried out using one-way analysis of variance (ANOVA) followed by Bonferroni post hoc test to

compare mean values pairwise, where significance was considered at P < 0.05. (a) means significantly different compared with the corresponding sham control group, (b) means significantly different compared with the corresponding I/R control group, and (c) means significantly different compared with the corresponding standard (nimodipine) treatment group

1093% as compared to sham control. Administration of the standard treatment nimodipine in a dose of 10 mg/kg/day prior to I/R caused significant decrease in TNF-α level by 39% as compared to I/R control, while administration of telmisartan in doses of 1, 3, and 10 mg/kg/day prior to I/R caused significant decreases in TNF-α level by about 53, 64, and 79%, respectively, of the respective I/R control value. Additionally, administration of xanthenone in doses of 0.5, 1, and 2 mg/kg/day prior to I/

R caused significant decrease in TNF-α level by 81, 85, and 87%, respectively, of the respective I/R control value. Pretreatment of rats with telmisartan or xanthenone in all dose levels showed more pronounced reduction of brain TNF-α level as compared with nimodipine-treated rats. Side by side, cerebral I/R induced significant increase in IL-6 level to reach 1032% as compared to sham control. Administration of the standard treatment nimodipine in a dose

Brain Tissue TNF-α (Pg/g ssue)

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I/R

Nimodipine Telmisartan Telmisartan

Telmisartan

Xanthenone Xanthenone

Xanthenone

(10 mg/kg/day, p.o.) (1 mg/kg/day, p.o.) (3 mg/kg/day, p.o.)

(10 mg/kg/day, p.o.)

(0.5 mg/kg/day, p.o.) (1 mg/kg/day, p.o.)

(2 mg/kg/day, p.o.)

Brain Tissue IL-6 (Pg/g ssue)

Sham

Sham

I/R

Nimodipine Telmisartan Telmisartan

Telmisartan

Xanthenone Xanthenone

(10 mg/kg/day, p.o.) (0.5 mg/kg/day, p.o.) (1 mg/kg/day, p.o.)

Xanthenone (2 mg/kg/day, p.o.)

Brain Tissue IL-10 (Pg/g ssue)

(10 mg/kg/day, p.o.) (1 mg/kg/day, p.o.) (3 mg/kg/day, p.o.)

Sham

I/R

Nimodipine

Telmisartan Telmisartan

(10 mg/kg/day, p.o.) (1 mg/kg/day, p.o.)

(3 mg/kg/day, p.o.)

Telmisartan (10 mg/kg/day, p.o.)

Xanthenone

Xanthenone

(0.5 mg/kg/day, p.o.) (1 mg/kg/day, p.o.)

Xanthenone (2 mg/kg/day, p.o.)

Fig. 3 Effect of different doses of telmisartan and xanthenone, as compared with nimodipine, on brain tissue TNF-α and IL-10 in rats subjected to cerebral I/R injury. Data were expressed as mean ± SEM (n = 8–10). Statistical analysis was carried out using one-way analysis of variance (ANOVA) followed by Bonferroni post hoc test to compare

mean values pairwise, where significance was considered at P < 0.05. (a) means significantly different compared with the corresponding sham control group, (b) means significantly different compared with the corresponding I/R control group, and (c) means significantly different compared with the corresponding standard (nimodipine) treatment group

of 10 mg/kg/day prior to I/R significantly reduced IL-6 level by 30% of the I/R control level, while administration of telmisartan in doses of 1, 3, and 10 mg/kg/day prior to I/R induced significant decreases in IL-6 level by about 49, 65, and 75%, respectively, of the I/R control value. Additionally, administration of xanthenone in doses of 0.5, 1, and 2 mg/kg/ day prior to I/R significantly decreased IL-6 level by 77, 80,

and 87%, respectively, of the respective I/R control value. Telmisartan (3 and 10 mg/kg/day) and xanthenone (0.5, 1, and 2 mg/kg/day) pretreatment resulted in significantly lower levels of brain IL-6 compared with nimodipine pretreatment. On the other side, cerebral I/R induced significant decrease in brain IL-10 level to reach 15% as compared to sham control. Administration of the standard treatment nimodipine in a dose

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of 10 mg/kg/day prior to I/R did not cause significant change in IL-10 level as compared to I/R control, while administration of telmisartan in doses of 1, 3, and 10 mg/kg/day prior to I/R caused significant increase in IL-10 level by about 98, 166, and 231%, respectively, of the I/R control level. Additionally, administration of xanthenone in doses of 0.5, 1, and 2 mg/kg/ day prior to I/R significantly increased IL-10 level by about 204, 254, and 345%, respectively, of the respective I/R control level. As with TNF-α and IL-6, telmisartan and xanthenone pretreatments caused more profound modifications of brain IL-10 levels compared with the reference standard nimodipine.

Apoptotic marker The results are given in Fig. 4. Cerebral I/R induced significant increase in the apoptotic marker caspase-3 level to reach 1003% as compared to sham control. Administration of the standard treatment nimodipine in a dose of 10 mg/kg/day prior to I/R significantly decreased caspase-3 by about 33% of the I/ R control, while administration of telmisartan in doses of 1, 3, and 10 mg/kg/day prior to I/R induced significant decreases in caspase-3 level by about 50, 65, and 73%, respectively, of the I/R control level. Additionally, administration of xanthenone in doses of 0.5, 1, and 2 mg/kg/day prior to I/R significantly decreased caspase-3 level by 81, 85, and 88%, respectively, of the I/R control value. Telmisartan and xanthenone in all dose levels showed better caspase-3 improvements compared with the reference standard nimodipine.

compared to sham control. Administration of the standard treatment nimodipine in a dose of 10 mg/kg/day prior to I/R caused significant decrease in ACE gene expression to reach 50% as compared to I/R control, while administration of telmisartan in a dose of 10 mg/kg/day prior to I/R caused significant decrease in ACE gene expression to 34% as compared to I/R control group. Additionally, administration of xanthenone in a dose of 2 mg/kg/day prior to I/R caused significant decrease in ACE gene expression to reach 21% as compared to I/R control group. Xanthenone (2 mg/kg/day) treatment group showed significantly lower ACE mRNA relative expression compared with the nimodipine treatment group. Conversely, cerebral I/R induced significant decrease in ACE2 mRNA gene expression to reach 21% as compared to sham control. Administration of the standard treatment nimodipine in a dose of 10 mg/kg/day prior to I/R caused significant increase in ACE2 gene expression to achieve 275% as compared to I/R control, while administration of telmisartan in a dose of 10 mg/kg/day prior to I/R caused significant increase in ACE2 gene expression to 324% as compared to I/R control group. Additionally, administration of xanthenone in a dose of 2 mg/kg/day prior to I/R caused significant increase in ACE2 gene expression to reach 335% as compared to I/R control group.

Immunohistochemical estimation of GFAP Cortex GFAP immunoreactivity expression

The RT-PCR evaluation of ACE and ACE2 gene expressions

Brain Tissue Caspase-3 (Pg/g ssue)

The results are given in Fig. 5. Cerebral I/R induced significant increase in ACE mRNA gene expression to reach 1591% as

Cerebral I/R induced significant increase in area percentage of GFAP immunoreactivity expression to reach 1359% as compared to sham control (Fig. 6a, b). Administration of the standard treatment nimodipine in a dose of 10 mg/kg/day prior to

Sham

I/R

Nimodipine Telmisartan

Telmisartan

Telmisartan

Xanthenone

Xanthenone

(10 mg/kg/day, p.o.) (1 mg/kg/day, p.o.) (3 mg/kg/day, p.o.) (10 mg/kg/day, p.o.) (0.5 mg/kg/day, p.o.) (1 mg/kg/day, p.o.)

Fig. 4 Effect of different doses of telmisartan and xanthenone, as compared with nimodipine, on brain tissue caspase-3 in rats subjected to cerebral I/R injury. Data were expressed as mean ± SEM (n = 8–10). Statistical analysis was carried out using one-way of variance (ANOVA) followed by Bonferroni post hoc test to compare mean values pairwise,

Xanthenone (2 mg/kg/day, p.o.)

where significance was considered at P < 0.05. (a) means significantly different compared with the corresponding sham control group, (b) means significantly different compared with the corresponding I/R control group, and (c) means significantly different compared with the corresponding standard (nimodipine) treatment group

ACE2 mRNA (Relave expression)

ACE mRNA (Relave expression)

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Fig. 5 Effect of the higher dose levels of telmisartan and xanthenone, as compared with nimodipine, on brain tissue ACE and ACE2 mRNA gene expression in rats subjected to cerebral I/R injury. Date were expressed as mean ± SEM (n = 8–10). Statistical analysis was carried out using oneway analysis of variance (ANOVA) followed by Bonferroni post hoc test to compare mean values pairwise, where significance was considered at

P < 0.05. (a) means significantly different compared with the corresponding sham control group, (b) means significantly different compared with the corresponding I/R control group, and (c) means significantly different compared with the corresponding standard (nimodipine) treatment group

I/R caused significant decrease in area percentage of GFAP immunoreactivity expression to reach 53% as compared to I/R control, while administration of telmisartan in a dose of 10 mg/kg/day prior to I/R caused significant decrease in area percentage of GFAP immunoreactivity expression to 40% as compared to I/R control group. Additionally, administration of xanthenone in a dose of 2 mg/kg/day prior to I/R caused significant decrease in area percentage of GFAP immunoreactivity expression to 11% as compared to I/R control group. The GFAP area percentage in the cerebral cortex of rats receiving xanthenone (2 mg/kg/day) prior to I/R was significantly lower than the respective area in rats pretreated with the reference standard nimodipine.

treatment nimodipine in a dose of 10 mg/kg/day prior to I/R did not cause significant decrease in area percentage of GFAP immunoreactivity expression as compared to I/R control, while administration of telmisartan in a dose of 10 mg/kg/ day prior to I/R caused significant decrease in area percentage of GFAP immunoreactivity expression to 29% as compared to I/R control group. Additionally, administration of xanthenone in a dose of 2 mg/kg/day prior to I/R caused significant decrease in area percentage of GFAP immunoreactivity expression to reach 12% as compared to I/R control group. Both telmisartan (10 mg/kg/day) and xanthenone (2 mg/kg/day) pretreatments resulted in more pronounced suppressions in the hippocampus GFAP area percentage as compared with the reference standard nimodipine pretreatment effect.

Hippocampus GFAP immunoreactivity expression Histopathological study Cerebral I/R induced significant increase in area percentage of GFAP immunoreactivity expression to 917% as compared to sham control (Fig. 7a, b). Administration of the standard

Light microscopic examination of the cerebral cortex of sham control rats showed more or less normal neurons

Naunyn-Schmiedeberg's Arch Pharmacol

a

I/R

Sham

Telmisartan (10 mg/kg/day, p.o.)

Nimodipine (10 mg/kg/day, p.o.)

Xanthenone (2 mg/kg/day, p.o.)

GFAP area percentage (cortex)

b

Sham

I/R

Nimodipine (10 mg/kg/day, p.o.)

Telmisartan

Xanthenone

(10 mg/kg/day, p.o.)

(2 mg/kg/day, p.o.)

Fig. 6 a Photomicrographs of cerebral cortex sections immunostained with GFAP antibody, showing the effect of the higher dose levels of telmisartan an xanthenone, as compared with nimodipine, on rats subjected to cerebral I/R injury regarding GFAP expression in reacting astrocytes (white arrows). b Effect of the higher dose levels of telmisartan and xanthenone, as compared with nimodipine, on cerebral cortex GFAP area percentage in rats subjected to cerebral I/R injury. Data were expressed as mean ± SEM (n = 3). Statistical analysis was carried out

using one-way analysis of variance (ANOVA) followed by Bonferroni post hoc test to compare mean values pairwise, where significance was considered at P < 0.05. (a) means significantly different compared with the corresponding sham control group, (b) means significantly different compared with the corresponding I/R control group, and (c) means significantly different compared with the corresponding standard (nimodipine) treatment group

with central large vesicular nuclei that contained one or more nucleoli (Fig. 8a). Alternatively, the cerebral cortex sections obtained from the I/R control group showed severe degenerated and necrotized neurocytes, in the form of shrunken, pyknotic, and hyperchromatic nuclei (Fig. 8b). The cerebral cortex sections obtained from the I/R control group pretreated with the standard treatment nimodipine showed moderate degenerated and necrotised neurocytes in the cerebral cortex (Fig. 8c). The cerebral cortex sections obtained from the I/R control group pretreated with telmisartan in both doses 1 and 3 mg/kg/ day showed moderate to severe degenerative changes and necrosis of neurons with mild congestion in the cerebral and meningeal blood vessels (Fig. 8d, e). Meanwhile,

sections obtained from the I/R control group pretreated with telmisartan (10 mg/kg/day) showed mild neuronal degeneration. Minimal necrosis of neurons could be detected (Fig. 8f). The cerebral cortex sections obtained from the I/R control group pretreated with xanthenone in both doses 0.5 and 1 mg/kg/day showed moderate degenerative changes and necrosis of neurons (Fig. 8g, h). Alternatively, sections of the highest dose (2 mg/kg/day) showed mild degenerative changes and necrosis of neurons (Fig. 8i). Light microscopic examination of the hippocampus of sham control rats showed more or less normal histological structure of the pyramidal layer, and most of the neurocytes appeared normal (Fig. 9a). On the other side, the hippocampus

Naunyn-Schmiedeberg's Arch Pharmacol

a

Sham

Nimodipine (10 mg/kg/day, p.o.)

I/R

Xanthenone (2 mg/kg/day, p.o.)

Telmisartan (10 mg/kg/day, p.o.)

GFAP area percentage (hippocampus)

b

Sham

I/R

Nimodipine (10 mg/kg/day, p.o.)

Telmisartan

Xanthenone

(10 mg/kg/day, p.o.)

(2 mg/kg/day, p.o.)

Fig. 7 a Photomicrographs of hippocampus sections immunostained with GFAP antibody, showing the effect of the higher dose levels of telmisartan and xanthenone, as compared with nimodipine, on rats subjected to cerebral I/R injury regarding GFAP expression in reacting astrocytes (white arrows). b Effect of the higher dose levels of telmisartan and xanthenone, as compared with nimodipine, on hippocampus GFAP area percentage in rats subjected to cerebral ischemia/reperfusion injury. Data were expressed as mean ± SEM (n = 3). Statistical analysis was

carried out using one-way analysis of variance (ANOVA) followed by Bonferroni post hoc test to compare mean values pairwise, where significance was considered at P < 0.05. (a) means significantly different compared with the corresponding sham control group, (b) means significantly different compared with the corresponding I/R control group, and (c) means significantly different compared with the corresponding standard (nimodipine) treatment group

sections obtained from the I/R control group showed severely damaged apoptotic neurons of the pyramidal layer in the form of pyknotic and shrunken neurons with hyperchromatic nuclei (Fig. 9b). The hippocampus sections obtained from I/R the I/R control group pretreated with the standard treatment nimodipine showed moderate degeneration in the neurons of the pyramidal layer (Fig. 9c). The hippocampus sections obtained from I/R group pretreated with telmisartan in both doses 1 and 3 mg/kg/day showed moderate thickness of the pyramidal layer and with more or less neurons. Presence of pericellular edema was also evident (Fig. 9d, e). On the other side, sections obtained from the I/R control group

pretreated with telmisartan (10 mg/kg/day) showed mild thickening of the pyramidal layer with more or less neurons and presence of pericellular edema (Fig. 9f). The hippocampus sections obtained from I/R group pretreated with xanthenone (0.5 mg/kg/day) showed moderate to severe necrosis of the pyramidal layer (Fig. 9g). The hippocampus sections obtained from I/R group pretreated with xanthenone (1 mg/kg/day) showed moderate degenerative changes (Fig. 9h). Sections of the highest dose (2 mg/kg/ day) showed more or less normal histological structure of the hippocampus where most of the neurocytes appeared normal (Fig. 9i).

Naunyn-Schmiedeberg's Arch Pharmacol

Fig. 8 Photomicrographs of cerebral cortex sections (H&E stain) prepared from rats of different groups, where a is sham control, b is positive control, c is standard (nimodipine) treatment; d–f Telmisartan 1, 3, and 10 mg/kg/day, respectively; g–i Xanthenone 0.5, 1, and 2 mg/kg/day, respectively

Discussion In the current study, the effects of the ARB telmisartan and the ACE2 activator xanthenone were studied against cerebral I/R injury in experimental rats, as compared with the calcium channel blocker nimodipine as a reference standard. Cerebral ischemia is a well-known experimental model of cerebral oxidative and inflammatory damage in which proinflammatory cytokines are involved (Min et al. 2017; Nasoohi et al. 2017). According to Chang et al. (2017), production of cytokines is not just a result of ischemic injury but seems to play a mechanistic role in neuronal ischemic damage. Side by side, oxidative stress may activate the redox sensitive transcription factor NF-κB, which consequently enhances transcription of pro-inflammatory genes and expression of pro-inflammatory cytokines (Reuter et al. 2010). This comes in agreement with our findings where rats exposed to cerebral I/R showed oxidative and inflammatory damage evidenced by GSH depletion coupled with elevated MDA and NOx levels (Fig. 2). In particular, TNF-α contributes to the

pathogenesis of I/R injury through induction of cell adhesion molecules and facilitating leukocyte infiltration, enhancing reactive oxygen species production, and exaggerating the inflammatory response and cerebral damage (Yin et al. 2013). Additionally, blockade of TNF-α receptors was reported to reduce brain infarct volume and cerebral edema caused by transient focal ischemia in rats (Hosomi et al. 2005). Similarly, the pleiotropic cytokine IL-6 is involved in many central nervous system disorders including stroke (Suzuki et al. 2009). On the other side, the anti-inflammatory cytokine IL-10 is a crucial mediator in cerebral I/R recovery (Zhang et al. 1994; Ahmed et al. 2014). In support to such idea, the IL10 knockout mice showed greater brain damage after focal ischemia (Grilli et al. 2000), while the administration of IL10 protected against such injury and inhibited TNF-α production (Manzanero et al. 2013). These findings again come in agreement with our findings regarding elevated proinflammatory cytokines IL-6 and TNF-α levels coupled with suppressed anti-inflammatory cytokine IL-10 level in ischemic rats (Fig. 3).

Naunyn-Schmiedeberg's Arch Pharmacol

Fig. 9 Photomicrographs of hippocampus section (H&E strain) prepared from rats of different groups, where a is sham control, b is positive control, c is standard (nimodipine) treatment; d–f Telmisartan 1, 3, and 10 mg/kg/day, respectively; g–i Xanthenone 0.5, 1, and 2 mg/kg/day, respectively

Apoptosis was earlier reported to be well correlated with cerebral ischemic damage (Li et al. 2017). Caspase-3 in particular is an exceptional apoptotic effector found to increase soon after cerebral ischemia with a persistent increase after reperfusion (Li et al. 2010). This comes in agreement with our findings where apoptosis was enhanced by cerebral I/R as evidenced by increased caspase-3 level (Fig. 4). Activation of caspase-3 leads to cleavage of actin with subsequent loss of its ability to inhibit deoxyribonuclease (DNase) activity and hence DNA fragmentation (Mashima et al. 1995). Caspase-3 also mediates the cleavage of certain proteins vital for DNA repair and cell stability, causing finally cell death primarily by apoptosis (Loetscher et al. 2001). Interestingly, binding of TNF-α to what is known as death receptor tumor necrosis factor-α receptor 1 (TNFR1) initiates activation of caspase pathway (Love 2003). Brain RAS was recently paid attention as a major pathway of oxidative and inflammatory ischemic damage (Orobei and Kulikov 2014; Wang et al. 2016). Interestingly, cerebral ischemic injury in the current investigation was associated with increased expression of ACE and decreased expression of its aforementioned physiological antagonist ACE2 (Fig. 5).

Additionally, GFAP expression was enhanced in rats exposed to cerebral I/R (Fig. 6). This also comes in agreement with previous investigations showing enhanced GFAP expression in rats exposed to cerebral ischemic injury (de laTremblaye et al. 2017; Liao et al. 2017). Based on the well-established role of brain RAS in the progression of oxidative and inflammatory damage, blockade of brain angiotensin II receptors by telmisartan is expected to exert anti-oxidant and anti-inflammatory effect, at least by down-regulation of NADPH oxidase activity (de Cavanagh et al. 2010; Kossmann et al. 2014; Passaglia et al. 2015) and cytokine production by leucocytes (da Silveira et al. 2010; Chang and Wei 2015). This might explain our findings where telmisartan pretreatment significantly ameliorated ischemic oxidative and inflammatory brain damage (Figs. 2 and 3). In agreement too, we earlier reported a neuroprotective effect of telmisartan in a rat simulation of Alzheimer disease (Khallaf et al. 2017). Telmisartan also attenuated serum IL-6 levels in TNF-α-infused mice, and IL-6 production from rat aorta stimulated with TNF-α ex vivo (Takagi et al. 2013). Based on a meta-analysis of nine randomized controlled trials, telmisartan therapy was shown to be effective in reducing IL-6 and

Naunyn-Schmiedeberg's Arch Pharmacol

TNF-α levels (Takagi et al. 2013). IL-10 suppressed the inflammatory response by inhibiting the production of IL-1β, TNF-α, and IL-6. Administration of ARB was shown previously to increase urinary levels of the anti-inflammatory cytokine IL-10 (Jia et al. 2012). Fortunately, most clinically used angiotensin II type 1 receptor blockers readily penetrate blood brain barrier and hence may be of value in managing CNS disorders (Unger 2001; Iwanami et al. 2010; Villapol and Saavedra 2014; Saavedra 2017). A number of other ARBs with varying properties are now clinically available including candesartan, irbesartan, losartan, olmesartan, telmisartan, and valsartan. For instance, losartan was reported to be the best one lowering serum uric acid level (Nishida et al. 2013). Olmesartan and valsartan are the only members that do not affect CYP2C9 liver microsomal subfamily and avoid related drug-drug interactions (Kamiyama et al. 2007). Interestingly, previous investigations showed that telmisartan is the only angiotensin II type 1 antagonist that acts as a selective PPAR-γ receptor modulator (Kurtz 2005, Ernsberger and Koletsky 2007). It has been reported to exert anti-oxidative and anti-inflammatory effects in accordance. Meanwhile, PPAR-γ inhibits the secretion of inducible NO synthase which is involved in tissue damage (Abdel-Fattah et al. 2015; Rabie et al. 2015). Confirming such fact, it was observed that chronic administration of troglitazone, a selective ligand for PPAR-γ, is associated with a greatly attenuated responsiveness toward inducers of hepatic TNF-α and IL-6 production (Sigrist et al. 2000). Side by side, telmisartan was reported to reduce neuronal apoptosis via a PPAR-γ-dependent caspase-3 inhibiting mechanism (Haraguchi et al. 2010). It should be mentioned that a number of investigators concluded that the neuroprotective effects of telmisartan are independent on its antihypertensive effects. Washida et al. (2010) reported that administration of telmisartan in a dose of 1 mg/ kg/day for 30 days attenuated cognition impairment without any significant effect on blood pressure in mice exposed to bilateral common carotid artery stenosis. In an earlier study, Kumai et al. (2008) concluded that chronic treatment of rats with telmisartan in nonhypotensive doses improved cerebral blood flow autoregulation and vascular integrity without lowering blood pressure. More recently, Justin et al. (2014) reported that the neuroprotective effect of telmisartan (5 and 10 mg/kg) in a cerebral ischemic model in rats was performed by the nonhypotensive doses of the drug. Too little pharmacologic research was conducted regarding ACE2 activators, of which the most commonly reported are xanthenone and diminazene aceturate (Singh et al. 2015). Although we reported no previous data regarding xanthenone neuroprotective effect against experimental cerebral I/R injury, a strong relationship between ACE2 and oxidative stress was earlier confirmed in a mouse neuroblastoma cell line

(Neuro2A cells) exposed to Ang-II and infected with AdhACE2, where ACE2 overexpression reduced ROS formation (Xia et al. 2011). Diminazene aceturate was also reported to protect experimental rats against ischemic heart injury (Qi et al. 2013). However, Baldissera et al. (2016) reported that diminazene aceturate might enhance oxidative liver and kidney damage. Such intrinsic pro-oxidant effect was not reported regarding xanthenone. Being a potent antioxidant and anti-inflammatory pathway, the ACE2/Ang-(1-7)/Mas axis is considered the physiological antagonist of the classic ACE/Ang-II/AT1 axis (Bennion et al. 2018). Previous reports revealed that Ang-(1-7) could modulate the expression profile of various cytokines in various tissues and cell lines. For instance, Ang-(1-7) treatment reduced TGFβ levels in renal tubular cells, as well as both basal and pro-renin induced expression of various cytokines in microglial cells through reduction in the expression of NF-κB subunits (Khajah et al. 2017). Interestingly, a Mas receptor agonist showed an anti-inflammatory outcome in wild-type mice but failed to show such effect in Mas knockout mice (da Silveira et al. 2010). This may explain our findings where xanthenone showed potent antioxidant, antiinflammatory (Fig. 2), immunomodulator (Fig. 3), and antiapoptotic effects (Fig. 4). Side by side, Santos et al. (2017) performed a lipophilicity study on some xanthenone derivatives to help understand the ability of such agents to penetrate blood brain barrier, and the results were highly promising. Although there is very little work regarding xanthenone biological effects, it was earlier reported that several derivatives of the drug can cross blood– brain barrier and show central effects (Chen et al. 1993; Pytka et al. 2015; Waszkielewicz et al. 2017). No sufficient data are available regarding the effect of xanthenone on blood pressure and impact of this effect on its neuroprotective activity, but Hernández Prada et al. (2008) reported that xanthenone administration (1, 5, and 10 mg/kg) to Wistar-Kyoto rats showed significant blood pressure-lowering effect only at the higher doses (5 and 10 mg/kg), which further supports the idea that xanthenone beneficial effects in the current study (0.5, 1, and 2 mg/kg/ day) are almost not related to blood pressure lowering effect as with the above-described telmisartan effects. It is worthy to mention that there seems to be a threshold level of brain ACE activity beyond which oxidative and inflammatory cascades begin. A similar threshold of GSH depletion might be present, below which oxidative damage takes place. This is evident upon observation of the completely normalized brain MAD and NOx levels by telmisartan and xanthenone (Fig. 2), while GSH, ACE, and ACE2 levels were only partially improved (Figs. 2 and 5). In agreement, Pompella and Casini (1984) reported that lipid peroxidation and MDA production in mice brains after exposure to bromobenzene did not take place except when GSH was

Naunyn-Schmiedeberg's Arch Pharmacol

depleted to a threshold level. Meanwhile, Wright and Harding (1992) reported that brain RAS plays important physiologic roles in the brain at physiological levels while overstimulated brain RAS is involved in different pathologies, which may support our conclusion regarding the threshold pro-oxidant and pro-inflammatory ACE level. A number of previous investigations discussed the role of ACE and ACE2 systems on the pathogenesis of cerebrovascular ischemic stroke and post-stroke neuronal damage. Kikuchi et al. (2013) reported that the ARB telmisartan could offer an acute treatment of stroke clinically but with failure to achieve significant neuroprotection after stroke. On the other side, Jiang et al. (2013) and Bennion et al. (2015) reported that activation of the ACE2/Ang-(1-7)/Mas axis could offer significant neuroprotection after cerebral ischemic stroke with notable reduction of the cerebral infarct area. The authors reported that ACE2 activation down-regulates the classic RAS via conversion of Ang-II to Ang-(1-7), and consequently Ang-(1-7) interacts with nitric oxide and bradykinin, thus attenuating the pathologic progress via antithrombotic activity and preventing thrombogenic events contributing to ischemic stroke. These results might explain our findings in the current study where the ACE2 activator xanthenone showed better protection compared with the ARB telmisartan as ACE2 activation causes a dual effect: first, the direct anti-oxidant, anti-inflammatory, and antithrombotic effects of Ang-(1-7) described earlier; second, the indirect effect of the activated ACE2 through inhibition of the classic ACE/Ang-II/AT-1 axis. Conclusively, interference with brain RAS, either through suppression of the classic ACE pathway by telmisartan or through stimulation of its antagonist ACE2 pathway by xanthenone, may be a promising strategy, with ACE2 activation probably being more efficient. It is worthy to mention that telmisartan and xanthenone neuroprotective effects were significantly better than nimodipine effect, which confirms the major role of brain ACE/ACE2 expressions in the pathogenesis of ischemic brain damage. Further controlled clinical trials are claimed. For instance, ACE2 activation by xanthenone or other novel analogues may be performed in patients with hypertension, atherosclerosis, surgery, immobility, or other risk factors of intravascular thrombosis to decrease the risk of ischemic stroke. Acknowledgements The authors are grateful to Dr. El-Shima Nabile Ahmed, Lecturer of Pathology, Faculty of Veterinary Medicine, BeniSuef University for providing help regarding the histopathological study conducted in the current investigation. Author contribution BAS and AM conceived and designed research. AM set and supervised the experimental model. MM conducted experiments. MM and BAS analyzed data. BAS wrote the manuscript. All authors read and approved the manuscript.

Compliance with ethical standards All authors strictly followed the ethical standards of scientific research. Conflict of interest No conflict of interest is evident among all authors.

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