Research Article Open Access
Effect of nifedipine and glibenclamide addition in a model of high fat diet- induced hypertension and hyperlipidemia in rabbits receiving donepezil.
SalwaAbdeltawab Ibrahim1, MahaYehia Kamel1 and Mina Thabet Kelleni1*
1Department of Pharmacology, Faculty of Medicine, Minia University, Minia 61511, Egypt
*Corresponding author: Mina T. Kelleni M. D, Ph. D, Assistant Professor of Pharmacology, Faculty of Medicine, Minia University, Egypt, Tel: (+20)1200382422; E-mail: @
Received: 11 February, 2017; Accepted: 20 February, 2017; Published: 02 March, 2017
Citation: Ibrahim S, Kamel MY, Kelleni MT (2017) Effect of nifedipine and glibenclamide addition in a model of high fat dietinduced hypertension and hyperlipidemia in rabbits receiving donepezil. Int J Sci Res Environ Sci Toxicol 2(1): 4.
Abstract Top
Geriatric individuals suffering from Alzheimer’s disease are frequently co-treated for hypertension and/or diabetes. In the current research, we have explored the addition of nifedipine (1 mg/ kg), glibenclamide (0.45 mg/kg) to donepezil (0.75 mg/kg) in a rabbit model of high fat diet- induced hypertension and hyperlipidemia. Nifedipine was shown to improve hypertension, lipid profile as well as the nitro-oxidative pathway when combined with donepezil. Glibenclamide has shown an improvement regarding hypertension and nitro-oxidative pathway when compared to the high fat diet receiving group.

Keywords: Donepezil; Nifedipine; Glibenclamide; Alzheimer’s disease; hypertension; hyperlipidemia
Introduction
Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by progressive cognitive deterioration and is the most common cause of dementia [1]. Donepezil; an acetylcholine esterase inhibitor, is one of the drugs commonly used worldwide in treatment of AD patients [2]. Patients suffering from AD patients belong mainly to the geriatric age group [3] who are frequently co-suffering from other chronic diseases like diabetes mellitus and hypertension and vice versa. In this research we’ve tested preliminarily the effect of the antihypertensive calcium channel blocker; nifedipine and the anti-diabetic sulfonylurea; glibenclamide in a model of high fat diet- induced hypertension in rabbits receiving donepezil.
Material and Methods
Animals
The present study was conducted on rabbits weighing from 1090–1300g. Rabbits were obtained from an animal market, Minia, Egypt and They were housed for one week as acclimatization period before conduction of the experiment; fed a standard diet of commercial rabbit chow, water was available ad libitum. All animal care and experimental procedures were in accordance with the protocols of the Research Advisory Ethical Committee of Faculty of Medicine, Minia University, Egypt.
Drugs and chemicals
Donepezil powder was obtained from Pfizer pharmaceuticals Company branch in Egypt. Nifedipine and glibenclamide powder was obtained from Egyptian pharmaceutical industries Co. (Eipico), Egypt; copper sulfate (El-Nasr Pharmaceuticals Chemicals Co., Egypt); N-naphthyl ethylenediamine (BDH, England); sulfanilamide (El-Gomhoria, Egypt); thiobarbituric acid (Sigma-Aldrich Chemical Co, USA); trichloroacetic acid (El- Nasr Pharmaceuticals Chemicals Co, Egypt). All other chemicals were of analytical grade and were obtained from commercial sources.
Experimental design
The rabbits(either sex) were divided into five groups (5 rabbits each): (1) control group received distilled water p. o. and normal diet (2) High Fat Diet (HFD) receiving group for 3 weeks [4] (3) HFD plus donepezil (0.75 mg/kg/day, p. o. for the last 5 days) receiving group (4) HFD plus donepezil (as described) plus nifedipine(1 mg/kg/day, p. o. for the last 5 days) receiving group (5) HFD plus donepezil plus glibenclamide (0.45 mg/kg/ day, p. o. for the last 5 days). The doses of the drugs were selected according to previous studies [5-7].
Sampling and methodology
At the end of the experimental period, animal were anesthetized with I. p injection with urethane then systolic and diastolic blood pressure were measured by using (POLYGRAPH, 2006). Animals were scarified and blood samples were collected and prepared for the following biochemical analysis:

Serum levels of Malondialdehyde (MDA) were measured according to the thiobarbituric acid method; it depends on measuring MDA, the breakdown products of lipid peroxides. Trichloroacetic acid was added to the sample for protein precipitation and then thiobarbituric acid was added. The mixture was heated for 10 min in a boiling water bath. One molecule of MDA reacted with two molecules of thiobarbituric acid and the resulting chromogen was centrifuged and the intensity of the color developed in the supernatant was measured spectrophotometrically at 535 nm The absorbance was read at 535 nm and the corresponding concentration was calculated from a standard curve using 1,1,3,3-tetraethoxypropane as a standard [8,9].

Nitric Oxide (NO) is rapidly oxidized to nitrite and/or nitrate by oxygen and thus the stable oxidation end products of nitric oxide, nitrite and nitrate were used as an index of NO production. The method used to determine NO level in serum depends on reduction of nitrate by copper–cadmium granules, followed by color development with Griess reagent (sulfanilamide and N-naphthylethylenediamine) in acidic medium and then measuring spectrophotometrically the total nitrites at 540 nm [10].

Reduced glutathione (GSH) was measured using colorimetric kit (Biodiagnostic, Egypt) according to kit instructions. The method based on the reduction of 5, 5’dithiobis (2-nitrobenzoic acid) (DTNB) with glutathione to produce a yellow compound. The reduced chromogen is directly proportional to GSH concentration and its absorbance was measured at 405 nm using Beckman-DU-64 spectrophotometer (USA).

Serum triglycerides (TG) and High Density Lipoproteins (HDL) levels were determined using commercially available kits (Biodiagnostics, Giza, Egypt) and expressed as mg/dl according to the kit instructions and quantitated at 500 nm using Beckman- DU-64 spectrophotometer (USA).
Results
Effect on Blood Pressure (table 1, 2)
Table 1: Effect on systolic blood pressure

Group

Systolic blood pressure (mmHg)

Control

86.67±2.11

HFD

125 ± 5.48a

HFD + donepezil

101.7 ± 3.07ab

HFD + donepezil + nifedipine

73.33 ± 3.3bc

HFD + donepezil + glibenclamide

104.2 ±2.7abd

Data represent the mean ± S. E. M. a, b, c, d Significant (P < 0.05) difference from control, HFD, HFD + donepezil, HFD +donepezil + nifedipine, respectively.
Table 2: Effect on diastolic blood pressure

Group

Diastolic  blood pressure (mmHg)

Control

50 ± 2.24

HFD

93.33 ± 5.43a

HFD + donepezil

75.83 ±6.11ab

HFD + donepezil + nifedipine

53.33 ±4.22bc

HFD + donepezil + glibenclamide

74.12 ±4.16abd

Data represent the mean ± S. E. M. a, b, c, d Significant (P < 0.05) difference from control, HFD, HFD + donepezil, HFD + donepezil + nifedipine, respectively.
Effect on nitro oxidative stress biomarkers ( table 3, 4, 5)
Table 3: Effect on serum malondialdehyde

Group

Serum MDA (µmol/l)

Control

87.84 ± 1.08

HFD

237.6±1.54a

HFD + donepezil

175.5±2.75ab

HFD + donepezil + nifedipine

101.1  ±2.23abc

HFD + donepezil + glibenclamide

201.6 ±1.26abcd

Data represent the mean ± S. E. M. a, b, c, d Significant (P < 0.05) difference from control, HFD, HFD + donepezil, HFD + donepezil + nifedipine, respectively.
Table 4: Effect on serum nitric oxide

Group

Serum NO (µmol/l)

Control

90.61±2.54

HFD

30.37 ± 3.14a

HFD + donepezil

70.84 ± 2.14ab

HFD + donepezil + nifedipine

90.27 ± 0.85bc

HFD + donepezil + glibenclamide

57.81 ± 2.17abcd

Data represent the mean ± S.E.M. a, b, c, d Significant (P < 0.05) difference from control, HFD, HFD + donepezil, HFD+ donepezil + nifedipine, respectively.
Table 5: Effect on serum GSH

Group

Serum GSH

Control

6.60± 0.54

HFD

3.20 ± 0.17a

HFD + donepezil

3.56 ± 0.2a

HFD + donepezil + nifedipine

5.34 ± 0.21bc

HFD + donepezil + glibenclamide

3.172 ±0.21acd

Data represent the mean ± S.E.M. a, b, c, d Significant (P < 0.05) difference from control, HFD, HFD + donepezil, HFD + donepezil + nifedipine, respectively.
Effect on lipid profile (Table 6, 7)
Table 6: Effect on serum triglycerides

Group

Serum TG (mg/dl)

Control

54.47±1.02

HFD

193.9 ±1.24a

HFD + donepezil

192.2±2.63a

HFD + donepezil + nifedipine

82.05±1.76abc

HFD + donepezil + glibenclamide

191.9±0.89ad

Data represent the mean ± S.E.M. a, b, c, d Significant (P< 0.05) difference from control, HFD, HFD + donepezil, HFD + donepezil + nifedipine, respectively.
Table 7: Effect on serum high density lipoporteins

Group

Serum HDL (mg/dl)

Control

32.79  ±1.01

HFD

16.90±0.42a

HFD + donepezil

16.73±0.64a

HFD + donepezil + nifedipine

21.02±0.58abc

HFD + donepezil + glibenclamide

17.04 ±0.52ad

Data represent the mean ± S.E.M. a, b, c, d Significant (P < 0.05) difference from control, HFD, HFD + donepezil, HFD + donepezil + nifedipine, respectively.
Discussion
Cerebrovascular disease and ischemic brain injury secondary to cardiovascular disease are common causes of dementia and cognitive decline in the elderly [11]. It’s common to find geriatric individuals treated from multiple diseases like Alzheimer’s, diabetes and hypertension. Further, hypertensive patients with concomitant diabetes must take both antihypertensive and hypoglycaemic medications, for which there is a lack of experimental and clinical guidelines [12]. In this research we’ve explored the possible interactions that may be encountered when donepezil is used together with nifedipine or glibenclamide.

In the current research, the addition of nifedipine to donepezil significantly improved both systolic and diastolic blood pressure compared with HFD, HFD + donepezil as well as HFD + donepezil + glibenclamide groups. This effect was predicted since nifedipine is a well-known antihypertensive drug. However, Donepezil +/- glibeclamide groups have also significantly improved systolic and diastolic blood pressure as compared to HFD.

Donepezil was shown to attenuate the development of hypertension in spontaneously hypertensive rats with a mechanism probably involving anti-inflammatory effects, indicating that acetyl cholinesterase inhibition yields beneficial effects for antihypertensive therapy [13]. Glibenclamide was also suggested to act as a competitive antagonist of thromboxane receptors inhibiting vasoconstriction [14].

Our research has shown that the addition of nifedipine to donepezil has improved the nitro-oxidative stress pathway; decreased serum MDA and increased serum NO and GSH compared with HFD, HFD + donepezil as well as HFD + donepezil + glibenclamide groups. Donepezil +/- glibeclamide groups have also decreased serum MDA and increased serum NO compared with HFD group.

Donepezil was previously shown to decrease lipid per oxidation (MDA); increase the contents of endogenous antioxidant (GSH) and the activities of antioxidant enzymes (catalase and SOD) in stroke rat model [15]. It was also shown that calcium disarrangement and free radical formation play a role in hepatotoxicity and nifedipine was shown to decrease MDA, increase GSH and SOD in diethyl dithiocarbamate-induced hepatic toxicity in rats [16]. Additionally, Nifedipine was shown to increase the activity of superoxide dismutase and catalase; elevate the contents of GSH and nitrites; and decrease the MDA levels [17].

Moreover, glibenclamide was shown to possess antioxidant effect contributing to the protective effect against oxidative stress-induced damage during diabetic complications in alloxaninduced diabetic rats [18]; further glibenclamide decreased MDA and increased GSH in streptozotocin-nicotinamide induced diabetic rat model [19].

The current research has also shown a favorable effect on serum TG and HDL when nifedipine was added to donepezil; an effect that was not shown by donepezil alone or donepezil + glibenclamide. Nifedipine was previously shown to decrease total cholesterol, LDL-cholesterol, TG, atherogenic index and elevated HDL level in rats under ethanol and sucrose feeding (dzeufiet). Nifedipine has also significantly decreased fasting TG level and increased HDL-C in the elderly group and was shown to have favorable metabolic effects that are beyond the known enhancement of insulin sensitivity [20].
Conclusion
Administration of both donepezil and the calcium channel blocker nilvadipine was shown to attenuate hyperhomocysteinemia-induced memory deficits and neuropathology’s in rats and the combination was suggested as a promising therapeutic candidate for AD [21]. In the current research we further confirm the favorable metabolic effect of nifedipine on the lipid profile as well as on the nitro-oxidative pathway when combined with donepezil. Glibenclamide has shown a significant improvement as regard to the nitro-oxidative pathway when compared to HFD. Further studies to test the combination and to explore other potentials are encouraged.
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