INTRODUCTION

High blood pressure is regarded as cause and consequence ofmany complications associated with vascular endothelium (1).The vascular damage seen in renovascular hypertension (RVH)has been attributed not only to high blood pressure, but also toincreased angiotensin II levels (2). Accordingly, renovascularhypertensive patients are at risk for many cardiovascular andcerebrovascular diseases (3). Treatment of the patients withhypertension or Alzheimer’s disease using inhibitors of reninangiotensin aldosteron system was found to be effective inmaintaining cognitive functions by several mechanisms,including lowering the blood pressure (4).

Two kidney-one clip (2K1C)-induced RVH results in a leakyblood brain barrier (BBB). It has been suggested that high bloodpressure and circulating angiotensin II were responsible forincreased BBB permeability (5). Since angiotensin II inhibitsrelease of acetylcholine in the brain, it can accepted to be partly

responsible for the disrupted cognitive functions (6). Moreover,once the permeability of BBB is enhanced, passage of toxicsubstances and angiotensin II from the systemic to braincirculation is facilitated. Therefore the brain becames vulnerableto injury and impaired memory function.

Hypertensive patients and animals with hypertension havedemonstrated high blood levels of proinflammatory cytokines,such as tumor necrosis factor (TNF)-a, interleukin (IL)-1b andIL-6 (7, 8). In a clinical study; serum levels of anti-inflammatorycytokine IL-10 were found to be lower in hypertensive patientscompared to healthy subjects (9). Inflammation in thebloodstream and the brain can damage cerebral vessels andneurons. Since it has been shown that amyloid beta (Ab)synthesis is increased while its degradation is decreased in theischemic brain, cerebral hypoperfusion due to RVH may causeAb accumulation in the brain. Furthermore, transfer of Ab fromthe systemic to brain circulation could be increased because ofincreased BBB permeability (10).

jOURNAL OF PHYSIOLOGY AND PHARMACOLOGY 2020, 71, 5, 665-677www.jpp.krakow.pl | DOI: 10.26402/jpp.2020.5.07

Z.-A. CEVIKELLI-YAKUT1,2, B. ERTAS1, A. SEN3, T. KOYUNCUOGLU4, B.C. YEGEN4, G. SENER1

MYRTUS COMMUNIS IMPROVES COGNITIVE IMPAIRMENT IN RENOVASCULAR HYPERTENSIVE RATS

1Department of Pharmacology, School of Pharmacy, Marmara University, Istanbul, Turkey; 2Department of Pharmacognosy, School of Pharmacy, Trakya University, Edirne, Turkey; 3Department of Pharmacognosy, School of Pharmacy,

Marmara University, Istanbul, Turkey; 4Department of Physiology, School of Medicine, Marmara University, Istanbul, Turkey

Myrtus communis has anti-inflammatory, neuroprotective and anticholinesterase activities yet there have been limited studiesexamining effects of Myrtus communis on cognitive functions. This study investigated the possible effects of Myrtuscommunis on changes in the cognitive functions of experimental renovascular hypertensive rats. Fifty-six Wistar-Albino ratswere equally divided into 4 groups; sham-operated control, renovascular hypertension (RVH), ramipril (RVH + Ram) andMyrtus communis extract (RVH + MC) treatment groups. Goldblatt’s 2-kidney 1-clip (2K1C) method was used to induceRVH. At the end of 9 weeks of treatment, after blood pressure recording, the animals underwent new object recognition testand Morris water maze (MWM) task. Following these tests, blood brain barrier (BBB) integrity was examined in 6 animalsfrom each group. In the others after decapitation, osteopontin and interleukin (IL)-10 levels were measured in blood samples;while matrix metalloproteinase (MMP)-13, sodium potassium adenosine triphosphatase (Na+,K+-ATPase), cluster ofdifferentiation (CD) 36, amyloid beta (Ab), neprilysin levels, and acetylcholinesterase (AChE) activity were investigated inhippocampal tissues. In RVH group, high systolic blood pressure decreased serum IL-10 levels, increased serum osteopontinlevels and also impaired BBB permeability. Hippocampal MMP-13, CD36, Ab, neprilysin levels and AChE activities wereelevated, while there were decreases in Na+,K+-ATPase levels. In new objet recognition test, discrimination index (DI) wasdetermined as lower in saline-treated RVH group compared to control animals. In MWM training trail, 4th day performancein finding platform was significantly reduced in saline-treated RVH group compared to control group. RVH also decreasedthe time spent in target quadrant in probe test of MWM task compared to control group. In both of the treatment groups, allbiochemical parameters were restored in parallel with improvement in the behavioral test performances. The results of thisstudy suggest that Myrtus communis extract may improve the cognitive dysfunctions in hypertension throughantihypertensive, anti-inflammatory and anticholinesterase activities.

K e y w o r d s : renovascular hypertension, cognitive impairment, blood brain barrier, angiotensin II, ramipril, Myrtus communis,amyloid beta, osteopontin, acetylcholinesterase

Numerous clinical studies have shown positive effects ofcentrally active angiotensin converting enzyme (ACE) inhibitorson the dementia and other cognitive disorders (11). ACEinhibitor therapy was demonstrated to exert protective effectagainst detrimental results of angiotensin II on the cognitivefunctions. Thus, ACE inhibitors crossing the BBB could beinvolved in the repair of inflammatory and oxidative damage inthe cerebral vessels, thereby in maintaining the BBB integrityand enhancing the cerebral blood flow (12).

Lack of an exact treatment of the vascular dementia and sideeffects of current drugs necessitate development of newalternative treatments. In their in vitro study, Tumen et al. (13)have demonstrated that various extracts of Myrtus communishave acetylcholinesterase (AChE) and butyrylcholinesterase(BChE) enzyme inhibitory activity. It was also reported thatessential oil, water and ethanol extracts of Myrtus communispossess anti-inflammatory activity (8, 14-17). Moreover in anethnobotanical study and in the in vivo studies, Myrtuscommunis was reported to have hypotensive effect (8, 18, 19).

In the light of these findings, the purpose of this study wasto investigate putative beneficial effects of Myrtus communis onthe cognitive impairment of the rats induced with experimentalRVH. In an attempt to guide for the evaluation of its mechanismof action, we also aimed to compare the effects of Myrtuscommunis extract with those of ramipril.

MATERIALS AND METHODS

Animals

Ten week-old Wistar albino rats of both sexes, weighing 200– 300 grams were used. Animals were obtained from MarmaraUniversity (MU) Research Center for Experimental Animals.

Marmara University Animal Care and Use Committeeapproved all experimental protocols (Protocol number:16.2018.mar).

Chemicals

Ramipril was obtained from Santa Cruz Biotechnology, Inc.(California, USA). Evans blue dye, acetylthiocholine iodide and5,5’-dithiobis(2-nitrobenzoic acid) (DTNB) were obtained fromSigma Chemical Co. (St Louis, MO, USA). Rat matrixmetalloproteinase (MMP)-13, sodium potassium adenosinetriphosphatase (Na+,K+-ATPase), cluster of differentiation (CD)36, Ab, neprilysin enzyme-linked immunosorbent assay(ELISA) kits were obtained from YL Biotech Co. (Shanghai,China). IL-10 ELISA kit was obtained from EBIOSCIENCE,Thermo Fisher Scientific (MA, USA). Osteopontin ELISA kitwas obtained from Bioassay Technology Laboratory (Shanghai,China).

Surgery and experimental protocol

Rats were randomly divided into four groups, eachconsisting of fourteen animals: sham-operated control (C) group,saline-treated (RVH), ramipril-treated (RVH + Ram) and Myrtus

communis extract-treated (RVH + MC) RVH groups. To induceanesthesia, the rats were injected intraperitoneally with ketamine(100 mg/kg) and chlorpromazine (0.75 mg/kg). To induce RVH,Goldblatt’s 2K1C method was used (20). Both kidneys were leftin place and a silver clip (internal diameter 0.25 mm) was placedaround the left renal artery of the rats. Three weeks after surgery,blood pressure levels measured above 170 mmHg wereconsidered as hypertensive (21, 22). In the sham-operatedcontrol group, the rats underwent a similar surgical procedurewithout a clip placement. Treatments were started 3 weeks after2K1C surgery (Table 1). The rats were treated perorally witheither saline (control and RVH groups), ramipril (10 mg/kg,RVH + Ram) or Myrtus communis extract (100 mg/kg, RVH +MC) until decapitation (23). The rationale of theantihypertensive dose of ramipril was based on a pilot study(24), while the selected dose of Myrtus communis extract wasthe dose used in the attenuation of scopolamine inducedcognitive impairment in the rats (25).

After 9 weeks treatment period (12 weeks after the surgery),behavioral tests were initiated. Firstly, a new object recognitiontest was performed. After 1 day break between new objectrecognition test and MWM task, training trial of ‘Morris watermaze task (MWM)’ was performed in four consecutive days.Probe test was applied on the 5th day of MWM task. At the endof the behavioral tests, 6 animals from each group were used forthe assessment of BBB permeability. Rest of the animals in eachgroup (n = 8) were decapitated to obtain hippocampal tissues andserum samples. All samples were stored at –80°C forbiochemical analyses.

Body weight measurement

Body weights of all animals were recorded before creatingthe 2K1C model, 3 weeks after the surgery, and 12 weeks afterthe surgery.

Blood pressure measurement

Tail-cuff method was used to measure the blood pressureindirectly. After the rats were placed in a heated chamber (35°C)for 10 min, they were positioned in individual plastic restrainers.Tails of the rats were wrapped with cuff equipped with apneumatic pulse sensor (Biopac MP35 Systems, Inc. COMMATLtd., Ankara, Turkey). At the beginning of the experiment, inorder to prevent stress induced increase in the blood pressurelevels; 7 days before the first blood pressure recording, allanimals were trained daily by blood pressure measurement.Furthermore all animals including control rats were exposedthese stress conditions. Thus it is suggested that stress does notaffect the results of the experiment. Blood pressuremeasurements were recorded in 3 occasions. The blood pressurewas measured before the 2K1C surgery to determine whether theanimals included in the study were normotensive; 3 weeks afterthe surgery to determine the effect of the model on the bloodpresure and at week 12 to evaluate the effect of treatment on theblood pressure levels. During each measurement period, theblood pressure levels were recorded at least 15 times andaverage of 5 of them were used.

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2K1C

surgery

3 weeks waiting

period for hypertension development

9 weeks

treatment period

New object recognition test and Morris water maze task

Blood brain

barrier permeability assessment

Biochemical

analysis Treatments were continued during behavior

tests

Table 1. Experiment timeline summary.

Plant material and preparation of Myrtus communis extract

Leaf samples of Myrtus communis were collected fromTurgutlu region of Manisa in 2010. The leaves were identified byDr. Gizem Bulut, a botanist working at Marmara UniversitySchool of Pharmacy. Voucher specimens were deposited in theHerbarium in School of Pharmacy, Marmara University(Herbarium protocol no: 13006). Myrtus communis leaves (100g) were dried in shade and at room temperature. The driedpowdered leaves were extracted with 96% ethanol using aSoxhlet apparatus and then obtained extract was evaporated todryness in the vacuum at 40°C and stored in a dark container ina refrigerator (+4°C) until its use (26).

Novel object recognition test

The novel object recognition test, which has become awidely used model for investigation of memory alterations, isefficient in evaluating short-term memory. Alterations in the testresults are accepted to be indicative of both hippocampal andcortical lesions (27).

The rats were initially placed in a box (65 × 45 × 65 cm) for 10minutes, which is named as ‘habituation phase’. Then the animalswere first observed for 10 minutes in the same box containing twoidentical objects (F + F), and their behavior was recorded with avideo camera. After this ‘familiarization phase’, rats were allowedto stay in their housing cages for 1 hour. During ‘test phase’, theanimals were put in the box with the familiar object (F) and a novelobject (N), which were different both in color and shape. Thebehavior of the rats was recorded with a video camera for 3minutes. During all experiments, boxes and objects were cleanedwith 70% alcohol solution before placing the next animal (28).

Discrimination index (DI; in seconds (s)) was calculated bydividing the difference in exploration time for familiar object(TF) and novel object (TN) with the total amount of explorationtime for the novel and familiar objects: DI = (TN – TF) / (TN +TF). DI can vary between +1 and –1, where a positive scoreindicates more time spent with the novel object, a negative scoreindicates more time spent with the familiar object, and a zeroscore indicates a null preference (28).

Morris water maze task

MWM task is used frequently for evaluating learning andmemory in rodents (29). MWM task was performed in acircular pool with a diameter of 1.2 m and a height of 0.47 m.The pool was filled (20 cm height) with black-colored waterkept at 24 – 25°C. On the walls around the pool, coloredcardboards were placed as clues for the rats to identify theirenvironment. Two main axes of the pool were determined,splitting the pool into 4 equal quadrants (30).

MWM task consists of a training period and a probe trial.The training period was performed in the first 4 days ofexperiment. During the training period, escape platform (10 cmin diameter, in the same color of water, and hidden 2 cm underthe water) was placed in the middle of one of the quadrants(target quadrant) (31). Each rat was released into the pool fromeach quadrant with a random sequence, and allowed to swim tofind the platform for 75 s, during which video recording wasmade. The animal that could not find the platform within thisperiod was directed to the platform, and its platform finding timewas accepted as 75 s. After finding the platform, the rat wasallowed to stay on the platform for 20 s. (32). Then, the animalwas kept in its homecage for 60 s before it was released to thepool again. On the fifth day of MWM task, probe trial wasperformed. The hidden platform was removed; the rat wasallowed to swim in the pool for 60 s and it was video-taped (33).

During this period, how much time was spent in the targetquadrant was evaluated from the recordings (34, 35).

Evans blue assay for the evaluation of blood brain barrierpermeability

Six animals from each group were used for BBB permeabilityassessment. The BBB integrity was evaluated with Evans blue thatwas used as a marker of albumin extravasation (36). Afterinduction of anesthesia, Evans blue (2% in saline, 4 mL/kg) wasinjected via jugular vein and was allowed to circulate for 30 min.Then, chests were opened and the rats were perfused transcardiallyfor approximately 15 min with a 250 mL of saline at a pressure of110 mm Hg. After decapitation, the brain tissues were removed andweighed for the quantitative measurement of Evans blue-albuminextravasation. Brain samples were homogenized in a 2.5 mLphosphate-buffered saline and mixed by vortexing for 2 min afterthe addition of 2.5 mL of 60% trichloroacetic acid to precipitateproteins. Samples were cooled and then centrifuged for 30 min at1000 g. Absorbance of Evans blue in supernatant was measured at620 nm using a spectrophotometer (Shimadzu UV1208, japan).Evans blue dye was calculated using a standard curve and wasexpressed as mg/g of brain tissue.

Measurement of the serum osteopontin, interleukin-10 andhippocampal matrix metalloproteinase-13, sodium potassiumadenosine triphosphatase, cluster of differentiation 36, amyloidbeta and neprilysin levels

Blood osteopontin (Bioassay Technology LaboratoryShanghai, China), IL-10 (EBIOSCIENCE, Thermo FisherScientific MA, USA) and hippocampal MMP-13, Na+,K+-ATPase, CD36, Ab, neprilysin (YL Biotech Co. Shanghai,China) levels were quantified according to the manufacturer’sinstructions and guidelines of the ELISA kits specific for rats.

Measurement of the hippocampal acetylcholinesterase activity

Activity of AChE, enzyme responsible for degradation ofacetylcholine, was measured in the hippocampal tissues usingspectrophotometer with Ellman method (32, 37). Thehippocampus tissue (20 mg) was homogenized with phosphatebuffer (pH 8, 0.1 M, 1 mL). 0.4 mL of this homogenate was addedto the cuvette containing phosphate buffer (PH 8, 0.1 M, 2.6 mL)and Ellman reactive DTNB (100 µL) was added to the cuvette.The absorbance was read spectrophotometrically at 412 nm.Afterwards, acethyltiocholine iodide (20 µL) was added and theabsorbance was read again. The change in the absorbance wasdetermined. Buffer solution, DTNB and acetylthioline iodide wereused for blind measurement. AchE activity was determined byfollowing formula:

R - rate of hydrolysis per minute in moles of substrate pergram of tissue; DA - change in absorbance per min; C0 - originalconcentration of tissue.

Acetylthiocholine is hydrolyzed to thiocholine and acetateby AChE. Thiocholine hydrolyzes DTNB to nitrobenzoate. Theabsorbance of nitrobenzoate was read spectrophotometrically at412 nm.

Statistical analysis

Statistical analysis was performed using Graphpad Prism 6.0(Graphpad Software, San Diego, CA, USA). All data areexpressed as mean ± standard error (SEM). Biochemical data of

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groups were analyzed with variance analysis (ANOVA)followed by Tukey multiple comparison tests. Novel objectrecognition test and MWM task results were analyzed withMann-Whitney U nonparametric test. For all data, p £ 0.05 wasconsidered statistically significant.

RESULTS

Body weight

In control group, body weight levels increased significantly12 weeks after surgery (p < 0.001) (Fig. 1). In RVH group, there

was no significant change in body weight values at 3 and 12weeks after the surgery. In contrast, body weight increasedsignificantly in both of treatment groups compared to baseline (p< 0.001).

Systolic blood pressure

Basal blood pressure levels were not different betweengroups (Fig. 2). In saline-treated RVH group, mean systolic bloodpressure was found to be significantly higher at 3rd (p < 0.001)and 12th weeks (p < 0.001) than the basal values. Besides, bloodpressure levels measured at 12th week were significantly elevatedas compared to 3rd week values (p < 0.001). Since treatments

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Fig. 1. Body weight levels of groups. Each group consists of 14 animals. Body weight levels were measured at the beginning of study,3 weeks after 2K1C surgery and 12 weeks after 2K1C surgery. Data are expressed as mean ± SEM. Data are analyzed with varianceanalysis (ANOVA) followed by Tukey multiple comparison tests. ***p < 0.001 versus baseline. RVH prevented the increase in bodyweight. Body weight gain in the treatment groups were at the levels of the control group.C, sham operated control; RVH, renovascular hypertension; Ram, ramipril; MC, Myrtus communis extract.

Fig. 2. Systolic blood pressure levels ofgroups. Each group consists of 14 animals.Treatments were started 3 weeks aftersurgery and continued until decapitation.Blood pressure levels were measured at thebeginning of study, 3 weeks after 2K1Csurgery and 12 weeks after 2K1C surgery.Data are expressed as mean ± SEM. Dataare analyzed with variance analysis(ANOVA) followed by Tukey multiplecomparison tests. ***p < 0.001: versusbaseline, +++p < 0.001: versus 3rd week.Since treatments were not started yet at the3rd week, systolic blood pressure levelsincreased in all groups applied 2K1Csurgery. Both of the treatments significantlydecreased systolic blood pressure levels.C, sham operated control; RVH,renovascular hypertension; Ram, ramipril;MC, Myrtus communis extract.

were not started yet at the 3rd week, systolic blood pressure levelswere increased in ramipril and Myrtus communis treated groupsas compared to that of baseline measurements (p < 0.001).However, treatment with either ramipril or Myrtus communis for9 weeks significantly reduced systolic blood pressure of the RVHrats (p < 0.001).

Novel object recognition test

As compared to sham-operated control group,discrimination index (DI) in RVH group was significantly lower(p < 0.001; Fig. 3a). Both ramipril (p < 0.01) and Myrtuscommunis (p < 0.001) treatments caused significant

improvements in novel object recognition test performanceswith respect to saline-treated RVH group. Although memoryperformance of MC-treated RVH was not different than controlgroup, DI of the ramipril-treated RVH group was still lower.

Morris water maze task

In the MWM training trial, 1st day performances of RVHgroups were not significantly different when compared to sham-operated control group (Fig. 3b). When the 4th day performancesof groups were compared with that of sham-operated controlgroup, performance in finding platform was significantly reducedin saline-treated RVH group (p < 0.01), while better learning

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Fig. 3. (a): Performances in novel objectrecognition test of groups. **p < 0.01,***p < 0.001 versus sham-operatedcontrol group, ++p < 0.01, +++p < 0.001versus saline treated RVH group. (b): Performances in Morris water maze(MWM) task training period. *p < 0.05,***p < 0.001 versus first dayperformances of each group, ++p < 0.01versus 4th day performance of sham-operated control group, ap < 0.05: versusfirst day performance of saline treatedRVH group, bbbp < 0.001: versus 4th dayperformance of saline treated RVH group.(c): Probe test performances in MWM taskof groups. *p < 0.05: versus sham-operatedcontrol group, +++p < 0.001 versus salinetreated RVH group.Each group consists of 14 animals.Behavioral tests were initiated 12 weeksafter 2K1C surgery, firstly novel objectrecognition test was performed and in thefollowing days MWM task was performed.Data are expressed as mean ± SEM. MannWhitney U test is used to analyze the data.C, sham operated control; RVH,renovascular hypertension; Ram, ramipril;MC, Myrtus communis extract.

performances were observed in both ramipril- or MC-treated RVHgroup with respect to saline-treated RVH group (p < 0.001). InMWM probe test results, saline-treated RVH group showed asignificantly lower performance (p < 0.05) when compared tosham-operated control group (Fig. 3c), but both ramipril- (p< 0.001) and MC- (p < 0.001) treated RVH groups displayedhigher memory performances than saline-treated RVH group.

Serum osteopontin and interleukin-10 levels

Serum osteopontin levels were higher in saline-treated RVHgroup when compared with those of sham-operated control group(p < 0.001) (Fig. 4a), indicating systemic inflammatory responseto RVH. Ramipril and Myrtus communis treatments given to RVHrats decreased the osteopontin levels (p < 0.01 – 0.001).

Serum IL-10 levels decreased significantly in saline treatedRVH group (p < 0.001) (Fig. 4b), indicating damaged anti-inflammatory activity with RVH. Ramipril treatment increasedserum IL-10 levels compared to saline-treated RVH group (p< 0.05), however it was not sufficient to reach the control grouplevels (p < 0.05). Myrtus communis extract increased IL-10levels significantly with respect to saline treated RVH group (p< 0.001).

Blood brain barrier permeability

Evans blue content measured in the brain tissues of saline-treated RVH group was higher with respect to sham-operatedcontrol group (p < 0.001), showing an increased BBBpermeability. On the other hand, Evans blue content in RVHgroups treated with ramipril and Myrtus communis weresignificantly reduced (p < 0.001), and were similar to that ofcontrol group (Fig. 5a).

Hippocampal levels of matrix metalloproteinase-13, cluster ofdifferentiation 36 and sodium potassium adenosinetriphosphatase

MMP-13 and CD36 levels in the hippocampal tissue wereincreased in saline-treated RVH group compared to sham-operatedcontrol group (p < 0.001 and p < 0.05, Fig. 5b and 5c). Treatmentwith ramipril reduced both MMP-13 and CD36 levels in thehippocampi of RVH rats (p < 0.01 and p < 0.05). Similarly, Myrtuscommunis treatment given to RVH rats abolished elevations in bothMMP-13 and CD36 levels in the hippocampal tissues (p < 0.001and p < 0.05). In parallel with these changes, Na+,K+-ATPase inhippocampus tissue was significantly decreased in saline-treatedRVH group as compared to sham-operated control group (p < 0.01,Fig. 5d). However, in both RVH + Ram (p < 0.05) and RVH + MC(p < 0.01) groups, hippocampal Na+,K+-ATPase activity was notdifferent than that of control group.

Hippocampal levels of amyloid beta, neprilysin andacetylcholinesterase activity

Ab, neprilysin and AChE activity levels in the hippocampaltissues of saline-treated RVH group were increased (p < 0.05 –0.001; Fig. 6). When the rats with RVH were treated with eitherramipril or Myrtus communis, these elevations were abolished (p< 0.01 – 0.001), and were measured at control levels.

DISCUSSION

Many epidemiological studies have reported thathypertension, especially during middle age, is a strong riskfactor for late-life cognitive impairment. According to previous

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Fig. 4. (a): Osteopontin serum levels in rats(b): Interleukin 10 (IL-10) serum levels inrats. Each group consists of 8 animals.Biochemical analysis was performed afterdecapitation. Data are expressed as mean ±SEM. DATA are analyzed with varianceanalysis (ANOVA) followed by Tukeymultiple comparison tests. *p < 0.05, ***p< 0.001 versus sham-operated controlgroup; +p < 0.05, ++p < 0.01, +++p < 0.001versus saline treated RVH group. Highlevels of inflammatory biomarkerosteopontin and reduced levels of anti-inflammatory cytokine IL-10 demonstratedinflammation in RVH. Although both of thetreatments alleviated inflammation, Myrtuscommunis was more effective thanramipril.C, sham operated control; RVH,renovascular hypertension; Ram, ramipril;MC, Myrtus communis extract.

experimental studies, hypertension-induced mechanical stressgenerates adaptive changes on the cerebral vessels, such as cellgrowth, remodelling and extracellular matrix proteinaccumulation, all of which narrow the cerebrovascular lumenand eventually result in insufficient cerebral blood supply (38).Additionally, elevated systemic and local cerebral angiotensin IIlevels in RVH exacerbate the endothelial damage (39, 40).Observational studies have verified that antihypertensivetherapy could abolish the dementia risk in the hypertension.

Drugs which improve the endothelial function such as centrallyactive ACE inhibitors warrant more efficient treatment (41).Supportively in a recent study investigating stasis-inducedvenous thrombosis in 2K1C hypertensive rats; over-activatedrenin angiotensin aldosteron system augmented the thrombosisthrough disrupting hemostatic balance and exacerbatingoxidative stress. Moreover supression of renin angiotensinaldosterone system with combined treatment of quinapril andspironolactone alleviated the thrombosis by favour of

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Fig. 5. (a): Blood brain barrierpermeability of groups. Each groupconsists of 6 animals. Blood brainpermeability was investigated 13 weeksafter 2K1C surgery. (b): Matrixmetalloproteinase (MMP)-13 levels inhippocampal tissues of rats. Each groupconsists of 8 animals. (c): Cluster ofdifferentiation 36 levels in hippocampaltissues of rats. Each group consists of 8 animals. (d): Sodium potassiumadenosine triphosphatase (Na+,K+-ATPase)levels in hippocampal tissues of rats. Eachgroup consists of 8 animals. Biochemicalanalysis was performed after decapitation.Data are expressed as mean ± SEM. Dataare analyzed with variance analysis(ANOVA) followed by Tukey multiplecomparison tests. *p < 0.05, **p < 0.01,***p < 0.001 versus sham-operatedcontrol group, +p < 0.05, ++p < 0.01, +++p < 0.001 versus saline treated RVHgroup.Blood brain barrier (BBB) permeability,MMP-13 and CD36 levels increased,Na+,K+-ATPase levels decreased inhippocampi of RVH group. Both of thetreatments protected BBB permeabilityand levels of the biochemical parameters.C, sham operated control; CD, cluster ofdifferentiation; EB, Evans blue; MC,Myrtus communis extract; Ram, ramipril;RVH, renovascular hypertension.

anticoagulant, antiplatelet, profibrinolytic and antioxidantactivities (42). In a more recent study, through antithromboticeffect mechanisms, losartan active metabolite EXP3174 hasalleviated stasis-induced venous thrombosis in 2K1Chypertensive rats (43). Since in RVH both of high blood pressureand over-activated renin angiotensin system play important rolein the cognitive damage (44); in the present study while Myrtuscommunis extract was being evaluated for its effects on thecognitive functions and parameters in RVH rats, centrally activeACE inhibitor ramipril (6) was used as reference compound.

Results of our study demonstrated impaired BBB integrityand cognitive function deficits in 2K1C-induced hypertensiverats, which were supported by biochemical parameters measuredin the serum and hippocampus. Myrtus communis extracttreatment improved alterations in the biochemical parameters,and maintained the BBB integrity and the cognitive functions ina similar manner as ramipril treatment. The studies on thecerebral effects of RVH are restricted (32, 44). Mostly, cerebraleffects of high blood pressure (34, 45) and angiotensin II (35, 46,

47) were investigated in separate studies. Most of these studieshave focused generally on new antihypertensive drugalternatives (12, 44, 46, 47, 48), but effects of these novelantihypertensive drugs on the cerebral damage were found to belimited (38, 49). Thus, there is a need to develop new strategieswith multiple targets for the treatment of RVH-related vasculardementia.

Anti-inflammatory and antioxidant actions of Myrtuscommunis and its inhibitory effect on AChE activity have beenpreviously demonstrated (8, 13, 15, 25). In addition to thesebeneficial effects, recently reported hypotensive effect of Myrtuscommunis in an ethnobotanical study and in vivo experimentssuggests that it could prevent the cognitive deficits in thehypertension (8, 18, 19). In our study, animals that underwent2K1C surgery for induction of RVH have become hypertensive.Treatments with either ramipril or Myrtus communis extractwere efficient in either of antihypertensive activity andpreventing RVH-induced cognitive deficits. It was alreadydemonstrated that novel object recognition test (22, 50) and

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Fig. 6. (a): Amyloid beta levels inhippocampal tissues of rats. (b): Neprilysinlevels in hippocampal tissues of rats. (c): Acetylcholinesterase activity inhippocampal tissues of rats. Each groupconsists of 8 animals. Biochemicalanalysis was performed after decapitation.Data are expressed as mean ± SEM. Dataare analyzed with variance analysis(ANOVA) followed by Tukey multiplecomparison tests. *p < 0.05, **p < 0.01,***p < 0.001 versus sham-operatedcontrol group, ++p < 0.01, +++p < 0.001versus saline treated RVH group. Inhippocampal tissue of RVH group, Ab andneprilysin levels and acetylcholinesteraseactivity increased. Ramipril and Myrtuscommunis extract administrationprevented the increases in levels of thesebiochemical parameters.C, sham operated control; MC, Myrtuscommunis extract; Ram, ramipril; RVH,renovascular hypertension.

MWM task (32, 50) performances were reduced in the animalswith 2K1C, indicating the cognitive deficits induced by RVH.Since novel object recognition test is known to assess short-termmemory function (27), our results revealed that Myrtuscommunis treatment increased the short-term memoryperformance even more efficiently than ramipril treatment.Moreover, the performance of hypertensive rats in MWM taskwas also significantly lower than the control group, while eitherof the treatments have improved their learning and memoryperformances.

In the present study, BBB permeability was significantlyincreased in the RVH group as compared to the control group. Indifferent hypertensive rat models, renin angiotensine aldosteronesystem inhibitors were found to be effective in maintaining theBBB integrity, because they can lower both the blood pressureand angiotensin II levels, and thereby abolish the oxidativestress, inflammation and destruction of BBB tight junctionproteins and collagens (51, 52). In the present study, ramipriltreatment protected the BBB integrity. This is the first study toshow that RVH-induced impairment in the BBB was diminishedby Myrtus communis, implicating its beneficial effect inmaintaining the BBB integrity against the destructiveconsequences of RVH.

High blood pressure, angiotensin II, pro-inflammatorycytokines and hypoxia elevate expression and activation ofMMPs (53). It has been suggested that MMP-13 impairs theBBB integrity by disintegrating basal membrane proteins (54).MMP-13 expression was elevated in experimental cerebralischemia and in spontaneous hypertension, while angiotensin IIhas been reported to stimulate MMP-13 activity inatherosclerotic lesions (55, 56). Liebetrau et al. have reportedthat ramipril decreased cerebral MMP-2 and MMP-9 levels instroke-prone spontaneously hypetensive rats (57). Our resultsshowed that hippocampal MMP-13 levels were lower in theramipril-treated RVH group than those measured in the saline-treated hypertensive rats. Previously, in an in vitro study, Zeidan-Chulia et al. have reported that essential oil obtained from leavesof Myrtus communis inhibited MMP-2 and MMP-9 activity (58).In our in vivo study, we demonstrated that hippocampal MMP-13 levels were also significantly lower in Myrtus communisextract treatment group.

Endothelial damage reduces Na+,K+-ATPase activity, andresultant ionic imbalance triggers vasoconstriction, decreasescerebral blood flow and elevates inflammatory cytokine levels(59, 60). In our study, reduced hippocampal Na+,K+-ATPaseactivity in the saline-treaed RVH group increased when the ratsreceived ramipril treatment. Since ramipril improves theendothelial function through many mechanisms, it is expected toprotect Na+,K+-ATPase activity (61). Similarly, jiang and Yinhave found that ramipril treatment increased Na+,K+-ATPaseactivity and Na+,K+-ATPase a1 subunit expression in thoracicaortic smooth muscle cells of salt-sensitive hypertensive rats(62). Until today, efficacy of Myrtus communis on Na+,K+-ATPase activity has not been reported. In the present study,Myrtus communis extract has also maintained Na+,K+-ATPaselevels in the hippocampal tissue.

It has been reported that inflammation has an important rolein pathogenesis of neurodegenerative dementia (63).Osteopontin acts as a cytokine that elevates proinflammatorycytokine levels and decreases IL-10 levels (64). Levels ofosteopontin, whose roles in kidney diseases are well known,have also been reported to increase in rats with 2K1C model(65). According to the limited number of studies, osteopontinlevels in hypertension increase due to both high angiotensin IIlevels and strain on the vessel wall (66). In recent studies,osteopontin levels were found to be high in both brain andplasma samples of Alzheimer’s patients. In addition, a

correlation was detected between osteopontin levels and Abdensities in the brain (67, 68). Supportively in our study serumlevels of osteopontin were increased in saline-treated RVHgroup, and these elevations were suppressed in both Myrtuscommunis- and ramipril-treated groups. Besides decreased anti-inflammatory cytokine IL-10 levels were elevated with bothMyrtus communis and ramipril treatments. Supportively,ramipril treatment given to patients with acute coronarysyndrome has increased serum IL-10 levels (69). Supporting ourresults, in our pilot study where we examined peripheralinflammation in 2K1C hypertensive rats, Myrtus communisextract significantly decreased serum IL-1b, IL-6 and TNF-alevels (8). In addition, Myrtus communis extract increasedpancreatic IL-10 levels in the rats with acute pancreatitis (70). Inthe present study, Myrtus communis treatment demonstratedhigher anti-inflammatory activity compared to ramipril.

CD36 plays a remarkable role in phagocytosis, angiogenesis,lipid metabolism, inflammation and eventually development ofatherosclerosis (71), which is caused by excessive lipid uptake(72). Ueno et al. have reported that hippocampal CD36expression is elevated in stroke-prone spontaneous hypertensiverats compared to control group (73). In an experimental study inmice, the researchers have demonstrated that angiotensin IIincreases CD36 expression via the stimulation of IL-6production, while losartan treatment before angiotensin IIadministration has eliminated this effect (74). Followingcerebral ischemia, cerebral CD36 expression was increased (75)along with the levels of many CD36 ligands, such as Ab fibrils,oxidized low density lipoprotein (LDL), long chain fatty acidsand membrane components of apoptotic cells (76). In the studythat evaluated the effects of ramipril on the development ofatherosclerosis in apolipoprotein E deficient mice, ramipriltreatment significantly reduced CD36 messenger ribonucleicacid (mRNA) levels in the peritoneal macrophages andattenuated the severity of atherosclerosis (77). Our resultsshowing decreased hippocampal CD36 levels with ramipriltreatment are consistent with this study. Similarly, Myrtuscommunis extract decreased the hippocampal CD36 levels via itsantiinflammatory and antioxidant effects, as well as itshypotensive action.

In the present study, Ab levels in the hippocampal tissues ofsaline-treated RVH rats were significantly elevated, suggestingthe hypertension-induced cerebral ischemia. ACE inhibitorswere shown to reduce Ab levels in the brain and alleviate thecognitive damage due to the dementia (78). Our findingsdemonstrated that both ramipril and Myrtus communis extractlowered the hippocampal Ab levels of rats with impairedcognitive function due to RVH.

Neprilysin is one of the proteases that contribute todegradation of Ab peptides, and neprilysin levels in the brain arereduced when both cerebral endothelium and neurons aredamaged due to the cerebral hypoxia. However, when neuronsare not yet damaged, neprilysin levels can be elevated inresponse to high Ab levels. In postmortem study, high levels ofAb accompanied by increased neprilysin mRNA and proteinlevels have been reported in Alzheimer’s patients (79, 80).Supportively, in our study, hippocampal neprilysin and Ab levelswere significantly higher in non-treated RVH group, while bothlevels were decreased in the RVH groups treated with eitherramipril or Myrtus communis extract, which emphasizes thatchanges in the cerebral Ab and neprilysin levels are incorrelation with the cerebral injury.

Angiotensin II adversely affects cholinergic system byreducing acetylcholine release (6). This action is partlyresponsible for the high blood pressure levels in RVH.Accordingly, it has been reported that ACE inhibitors mayincrease the acetylcholine release (6), and thereby be effective in

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the treatment of the hypertension. On the other hand, perindopriltreatment has decreased AChE activity and increasedacetylcholine levels in the streptozotocin-injected brain areas(81). Based on the previous studies and our results with ramipriltreatment, it can be suggested that inhibition of renin angiotensinsystem in addition to its blood pressure controlling effect, mayhave a positive effect on the memory functions by decreasing thedegradation of acetylcholine and increasing its activity.Recently, in rats with scopolamine-induced Alzheimer model,ethanol extract of Myrtus communis leaves was shown toincrease acetylcholine receptor expression, decrease AChEactivity and prevent increase in AChE mRNA expression in thedifferent brain areas, including the hippocampus (25). Thisinhibitory action of Myrtus communis extracts on the AChEactivity was also shown in the in vitro studies (13, 82). In thepresent study, Myrtus communis extract significantly decreasedRVH-induced elevation in the hippocampal AChE activity inparallel with its beneficial effects on the memory.

The ethanol extract of Myrtus communis leaves containsphenolic compounds such as phenolic acids, tannins andflavonoids (83). In the recent study conducted by our team, ahigh total phenol and flavonoid content of Myrtus communis wasdetected (84). Flavonoids are known to have the neuroprotectiveeffects and cross the BBB (85, 86). Ayaz et al. reported thatflavonoids alleviate the neuronal apoptosis that develops due tothe free radicals and Ab accumulation (87). Quercetin, myricetinand catechin derivatives are among the flavonoids detected inHPLC analysis of Myrtus communis leaves ethanol extract.Especially, myricetin and catechin derivative compounds havebeen found as major compounds in the plant (88). Theseflavonoids have been found to be effective in the treatment ofAlzheimer’s disease in different studies (85, 89, 90).Epicatechin-3-gallate, quercetin and myricetin prevent the Abaccumulation, tau aggregation and reduce the oxidative stress(87). Anticholinesterase activity of quercetin and myricetin isalso known (91, 92). Moreover, it was reported catechinderivative compounds and myricetin can permeate across theBBB effectively (93, 94). In addition, the neuroprotective role ofphenolic acids has previously been reported in previous studies(95). Gallic acid, caffeic acid, ellagic acid were found in theleaves of Myrtus communis as phenolic acids (88). In previousstudies, these compounds have been reported to have theneuroprotective effects (96, 97, 98). In the light of theseinformation, we suggest that the phenolic compounds in theextract, especially the catechin and myricetin derivativecompounds found as major compounds, could play an importantrole in the positive effects of Myrtus communis extract on thecognitive functions in the present study.

Phenolic compounds in Myrtus communis leaf extract couldlower the blood pressure and positively affect the cognitivefunctions with its ACE inhibitory activity. Supportivelyepigallocatechin and epigallocatechin gallate inhibited ACEactivity in the HUVEC cell line (99). Similarly in the in vitrostudy of Chen et al., myricetin and quercetin showed ACEinhibitory activity (100). Furthermore quercetin treatmentreduced systolic blood pressure levels of the rats with 2K1Cmodel (101). Regarding to ACE inhibitory effect mechanism;Persson et al. (99) claimed that polyphenols bind to Zn+2 on theACE active area. Selvaraj et al. (99) also reported that myricetinhas the ability to chelate the metal ions such as Fe+2 and Zn+2. Inthe light of these information, it is suggested that the effect ofMyrtus communis in our study is due to ACE inhibition andreduction of angiotensin II levels.

In conclusion, our results demonstrate that Myrtus communisextract, similar to ramipril, significantly improves the RVH-induced cognitive impairment. In addition to their hypotensiveeffects, both agents appear to exert their supportive effects on

memory and BBB integrity by inhibiting AChE activity.Additionally, the herb decreases the levels of osteopontin, MMP-13, CD36, Ab and NEP, and increases Na+,K+-ATPase and IL-10levels. The effects of Myrtus communis on cognitive functionsare comparable to those of ramipril. Further experimental andclinical studies are needed to establish the use of this herb toprotect the cognitive functions in vascular dementia-relatedpathologies.

Acknowledgements: The authors would like to thank Dr.Gizem Bulut for her valuable assistance in identification of theplant material.

Source of funding: This study was funded by MarmaraUniversity Scientific Research Projects Committee with thegrant number SAG-C-DRP-120418-0159.

Conflict of interests: None declared.

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R e c e i v e d : September 24, 2020A c c e p t e d : October 30, 2020

Author’s address: Prof. Goksel Sener, Marmara University,School of Pharmacy Tibbiye Cad. 34668 Istanbul, Turkey.E-mail: gsener@marmara.edu.tr; gokselsener@hotmail.com

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