PBB-71953; No of Pages 9 Pharmacology, Biochemistry and Behavior xxx (2014) xxx–xxx

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Gurpreet Singh a,1, Bhupesh Sharma b,2, Amteshwar Singh Jaggi a,3, Nirmal Singh a,⁎

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Article history: Received 5 February 2013 Received in revised form 1 May 2014 Accepted 8 May 2014 Available online xxxx

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Keywords: Vascular dementia Diabetes Endothelial dysfunction Streptozotocin Endothelin Bosentan Moris water maze Nitrite/nitrate Oxidative stress

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Pharmacology Division, Department of Pharmaceutical Sciences and Drug Research, Faculty of Medicine, Punjabi University, Patiala 147002, Punjab, India Department of Pharmacology, School of Pharmacy, Bharat Institute of Technology, Bypass Road, Partapur, Meerut 250103, Uttar Pradesh, India

The study was designed to investigate the efficacy of bosentan a dual endothelin (ETA and ETB) receptor antagonist in experimental diabetes induced vascular endothelial dysfunction and associated dementia. Diabetes was induced in rats by administration of a single dose (50 mg/kg, i.p.) of streptozotocin (STZ). Drug treatment was started after 1 month of STZ administration and treatment was continued until the end of the study. Morris water maze (MWM) test was employed for testing spatial learning and memory. Endothelial function was measured on isolated aortic rings using student physiograph. Serum glucose, body weight, serum nitrite/nitrate, brain thiobarbituric acid reactive species (TBARS), reduced glutathione (GSH) levels, and brain acetylcholinesterase activity were also tested. STZ treatment resulted in significant development of cognitive and vascular endothelial deficits, manifested in the terms of endothelial dysfunction, impairment of learning and memory, reduction in body weight and serum nitrite/nitrate levels along with increase in serum glucose, brain acetylcholinesterase activity, TBARS, and decreased GSH levels. Treatment of bosentan attenuated diabetes induced impairment of learning, memory, endothelial function, and various biochemical parameters. It may be concluded that bosentan has shown efficacy in STZ induced cognitive and vascular endothelial deficits. Thus, endothelin receptors can be considered as a potential pharmacological target for the management of experimental diabetes induced vascular endothelial dysfunction and associated dementia. © 2014 Published by Elsevier Inc.

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Efficacy of bosentan, a dual ETA and ETB endothelin receptor antagonist, in experimental diabetes induced vascular endothelial dysfunction and associated dementia in rats

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1. Introduction

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Vascular dementia (VaD) is a type of dementia resulting from ischemic or hemorrhagic cerebrovascular disease (CVD) or from cardiovascular or circulatory disturbances that injure brain regions important for memory, cognition, and behavior (Schneck, 2008; Ekonomou et al., 2011). VaD is the second most common type of dementia after Alzheimer's disease (AD). The current prevalence rate of VaD is thought to be 10–30% (Stephan and Brayne, 2008; Peters, 2012). Cardiovascular disorders, diabetes mellitus, hyperhomocysteinemia, hyperlipidemia, obesity, midlife hypertension and smoking are the important risk factors for both VaD and AD (Korczyn et al., 2012). Diabetic people are noted to have a 1.5- to 4-fold risk for AD as well as

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⁎ Corresponding author. Tel.: +91 9815129884. E-mail addresses: [email protected] (G. Singh), [email protected] (B. Sharma), [email protected] (A.S. Jaggi), [email protected] (N. Singh). 1 Tel.: +91 9041749327. 2 Tel.: +91 9958219190. 3 Tel.: +91 9501016036.

VAD (Araki, 2010). Recently we have reported the induction of cognitive and vascular endothelial deficits by utilizing experimental diabetes (Sharma and Singh, 2010, 2011a,b). Endothelial dysfunction is considered to occur early during the development of cardiovascular disease including atherosclerosis and vascular complications associated with diabetes mellitus. A key event in endothelial dysfunction is the reduction in bioavailability and biological activity of nitric oxide (NO). Reduced levels of NO contribute to increased vascular tone, inflammation, platelet aggregation and oxidative stress which all are central features of atherosclerosis and diabetic vasculopathies (Versari et al., 2009). Maintenance of cerebral blood flow across a wide pressure range is critical for brain perfusion; however, alterations to this system may be detrimental and could contribute to cerebrovascular disease. While an acute interruption of cerebral blood flow may cause stroke, chronic impairment of perfusion is associated with neurodegenerative diseases like VaD and AD, all of which are more common in patients with diabetes (Ergul, 2011). Effective treatment of dementia including that of VaD is still a nightmare for the medical fraternity. Only limited drugs are available and these treatment options only provide symptomatic relief. So far not a single agent is available that can provide relief and stop progression of

http://dx.doi.org/10.1016/j.pbb.2014.05.002 0091-3057/© 2014 Published by Elsevier Inc.

Please cite this article as: Singh G, et al, Efficacy of bosentan, a dual ETA and ETB endothelin receptor antagonist, in experimental diabetes induced vascular endothelial dys..., Pharmacol Biochem Behav (2014), http://dx.doi.org/10.1016/j.pbb.2014.05.002

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Male Wistar rats weighing 200–250 g (3–4 months) (Chaudhary Charan Singh Haryana Agricultural University, Hisar, Haryana, India) were employed in the present study. Animals were provided with standard laboratory feed (Kisan Feeds Ltd., Chandigarh, India) and water ad libitum and were exposed to natural cycle of light and dark. The experimental protocol was approved by the Institutional Animal Ethics Committee (IAEC) and care of the animals was taken as per the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Environment and Forest Government of India, (Reg. No. 107/1999/CPCSEA/2011/07).

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2.2. Drugs and reagents

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Donepezil was obtained as gift sample from Wokhardt Ltd., Baddi, Himachal Pradesh, India. Bosentan was obtained ex-gratia from Cipla

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2.1. Animals

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2.3. Streptozotocin (STZ) diabetes induced vascular endothelial dysfunction 150 and dementia 151

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The rats were injected with a single dose of freshly prepared streptozotocin (50 mg/kg i.p.) in 0.1 M citrate buffer (pH 4.5) to induce experimental diabetes mellitus (Rakieten et al., 1963; Brosky and Logothetopoulos, 1969) and associated dementia (Sharma and Singh, 2010, 2011a,b). Serum glucose levels of the animals were measured every week. The animals were used on the 52nd day for the behavioral and other assessments (Sharma and Singh, 2010, 2011a,b).

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2.4. Bosentan and donepezil administration

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Rats were administered bosentan orally in two doses 50 mg kg−1 and 100 mg kg−1 daily (Said et al., 2005) by suspending in 0.5% w/v of carboxymethyl cellulose (CMC), while donepezil in 1 mg kg−1 (de Bruin et al., 2011) oral dose by dissolving it in saline (0.9% w/v) for 26 days. Furthermore, bosentan (50 mg kg−1; 100 mg kg−1 p.o., daily)/ donepezil (1 mg kg−1 p.o., daily) was administered for 26 days viz. day 30 to day 56 (21 days + 5 days during Morris water maze exposure) in streptozotocin treated rats. The administration of drugs was given 30 min before starting Morris water maze observation.

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2.5. Experimental protocol

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2. Material and methods

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Ltd., Goa, India. Folin–Ciocalteu's phenol and acetylthiocholine were purchased from Merck limited, Mumbai, India. Streptozotocin was purchased from Sigma Aldrich, USA. 5,5,dithiobis (2-nitrobenzoic acid) (DTNB), reduced glutathione (GSH), bovine serum albumin (BSA), sulfanilamide, N-naphthylethylenediamine (NED) and thiobarbituric acid were obtained from Loba Chem, Mumbai, India. Sodium nitroprusside was purchased from SD Fine Chemicals Limited, Mumbai, India. Phenylephrine was obtained as free sample from Aarti industries, Dombivli (East), Maharashtra, India. Bosentan was suspended in 0.5% w/v of carboxymethyl cellulose (CMC) whereas donepezil was dissolved in saline. All the agents were administered orally to the animals. Streptozotocin was dissolved in 0.1 M citrate buffer (pH 4.5) and was administered intraperitoneally.

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the disease. Hence, there is a need to develop an agent which not only provides symptomatic relief but also halts progression of the disease. Endothelin (ET) and nitric oxide (NO) are well known mediators produced by endothelial cells to maintain hemodynamic responses (Bohm and Pernow, 2007; Wang et al., 2011). ET is the most important endothelium derived vasoconstriction factor. There are three mature endothelin isoforms: ET-1, ET-2, and ET-3. ET-1 represents the most potent and long lasting vasoconstrictor. ET-1 binds to two receptors, endothelin A (ETA) and endothelin B (ETB) and is responsible for a variety of physiological functions, primarily blood flow control (Burnier and Forni, 2012). Cerebrovascular tone plays a key role in controlling cerebral blood flow. Studies have demonstrated that the ET system is upregulated in type 2 diabetes leading to increased sensitivity to ET-1 and decreased relaxation in basilar artery (Li et al., 2011). ET-1 also contributes to endothelial dysfunction observed in subjects with insulin resistance and patients with type-2 diabetes (Pernow et al., 2012). ET receptor antagonists (such as bosentan) are noted to improve endothelium dependent vasodilatation (Pernow et al., 2012); reduce oxidative stress and inflammation process (Chen et al., 2010). ET receptor antagonists have also been shown to provide beneficial effect in various cerebrovascular disorders such as ischemic stroke (Kaundal et al., 2012), subarachnoid hemorrhage (Komotar et al., 2011) and moyamoya disease. Recently, it has been suggested that, ET receptor antagonists may be beneficial in the treatment of AD (Palmer and Love, 2011). ET-1 has been reported to induce neurovascular dysfunction in hypoxia condition associated with stroke and dementia (Capone et al., 2012). Role of ETA and ETB receptors in dementia is not clear and the potential of endothelin receptor antagonists in vascular dementia is unexplored. Therefore, the present study has been undertaken to investigate the potential of bosentan a dual ETA and ETB endothelin receptor antagonist in a rat model of STZ induced vascular endothelial dysfunction and associated dementia. Bosentan, a dual ETA and ETB endothelin receptor antagonist, has shown to be metabolized by the hepatic cytochrome P450. The oral bioavailability is 50% and peak plasma levels are reached within 2–3 h. Hepatic metabolism followed by biliary excretion of the metabolites apparently represents the major pathway of elimination for bosentan (Ono and Matsumori, 2002). Donepezil an AChE inhibitor is a well-established drug for the management of dementia and is clinically used for memory deficits. We have used it earlier for its benefits in experimental AD (Sharma et al., 2008a,b) and vascular endothelial dysfunction associated cognitive deficits (Koladiya et al., 2008, 2009; Sharma and Singh, 2012a,b,c, 2013). Donepezil has been used as a positive control in the present study.

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Eight groups were employed in the present study and each group 170 was comprised of 6 male Wistar rats. 171 2.5.1. Group I — control 172 Rats were exposed to Morris water maze for acquisition trial from day 173 1 to day 4 and retrieval trial on day 5. 174 2.5.2. Group II — vehicle control group (0.5% CMC) Rats were administered CMC (10 ml/kg, p.o. daily) for 21 days followed by exposure to Morris water maze. The treatment was continued during acquisition (from the 22nd to 25th day) and retrieval trials (on the 26th day) on Morris water maze.

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2.5.3. Group III — STZ treatment group Rats were administered a single dose streptozotocin (50 mg/kg, i.p.) and then were exposed to Morris water maze on the 52nd day of STZ administration.

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2.5.4. Group IV — bosentan per se Rats were administered bosentan (100 mg/kg, p.o., daily) for 21 days followed by exposure to Morris water maze. The treatment was continued during acquisition (from the 22nd to 25th day) and retrieval trials (on the 26th day) on Morris water maze.

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2.5.5. Group V — STZ and bosentan low dose 189 Bosentan (50 mg/kg, p.o., daily) was administered to the STZ 190 (50 mg/kg, i.p.) treated rats, starting from the 30th day of STZ treatment 191

Please cite this article as: Singh G, et al, Efficacy of bosentan, a dual ETA and ETB endothelin receptor antagonist, in experimental diabetes induced vascular endothelial dys..., Pharmacol Biochem Behav (2014), http://dx.doi.org/10.1016/j.pbb.2014.05.002

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2.5.6. Group VI — STZ and bosentan high dose Bosentan (100 mg/kg, p.o., daily) was administered to the STZ (50 mg/kg, i.p.) treated rats, starting from the 30th day of STZ treatment followed by exposure to Morris water maze on the 52nd day of STZ administration. The treatment was continued during acquisition (from the 52nd to 55th day) and retrieval trials (on the 56th day) on Morris water maze. 2.5.7. Group VII — donepezil per se Rats were administered donepezil (1 mg/kg, p.o., daily) for 21 days followed by exposure to Morris water maze. The treatment was continued during acquisition (from the 22nd to 25th day) and retrieval trials (on the 26th day) on Morris water maze.

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2.7.2. Estimation of serum glucose Streptozotocin was used to induce diabetes in the animals. To ascertain the diabetes animas, blood glucose levels were estimated (Sharma and Singh, 2010, 2011a,b). The glucose levels were estimated spectrophotometrically (DU 640B Spectrophotometer, Beckman Coulter Inc., CA, USA) at 505 nm by glucose oxidase peroxidase (GOD–POD) method using a commercially available kit (Reckon Diagnostics Pvt. Ltd. Vadodara, India).

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2.7.3. Estimation of serum nitrite concentration Nitric oxide is an endogenous relaxing factor of the vascular system which may be measured in terms of serum nitrite levels. The decreased level of serum nitrite is an indication of vascular impairment. Serum nitrite concentration was measured spectrophotometrically (DU 640B Spectrophotometer; Beckman Coulter) at 545 nm, using the method of Sastry et al. (2002) and Sharma and Singh (2010).

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2.7.4. Estimation of brain acetyl cholinesterase (AChE) activity Acetylcholine (ACh) has been reported to be involved in the memory function, and the decrease in ACh level is indicated in memory loss. AChE is an enzyme which increases the breakdown of ACh and decreases the level of ACh. Its level in the brain is in correlation with the levels of Ach with an inverse relationship (Sharma et al., 2008a,b; Sharma and Singh, 2011a,b). The whole brain AChE activity was measured spectrophotometrically (DU 640B Spectrophotometer; Beckman Coulter) at 420 nm by the method of Ellman et al. (1961) with slight modification (Sharma and Singh, 2010).

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2.7.5. Estimation of brain total protein Brain protein levels are required to estimate the values of other parameters like AChE, TBARS and GSH. The brain total protein was determined by Lowry's method with slight modification (Lowry et al., 1951) using bovine serum albumin (BSA) as a standard. The protein content was determined spectrophotometrically (DU 640B Spectrophotometer, Beckman Coulter Inc., CA, USA) at 750 nm.

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2.7.6. Estimation of thiobarbituric acid reactive substances (TBARS) TBARS is the method of screening and monitoring lipid peroxidation, a major indicator of oxidative stress. The brain thiobarbituric acid reactive substances (TBARS) was measured spectrophotometrically (DU 640B Spectrophotometer; Beckman Coulter) at 532 nm according to the method of Okhawa et al. (1979).

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2.7.7. Estimation of reduced glutathione (GSH) GSH is an endogenous antioxidant; a decrease in the level of GSH is an indicator of oxidative stress. The GSH content in the brain was estimated spectrophotometrically (DU 640B Spectrophotometer; Beckman Coulter) at 412 nm using the method of Beutler et al. (1963).

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to estimate acetyl cholinesterase (AChE) activity, thiobarbituric acid reactive substances (TBARS), reduced glutathione (GSH) level and protein content. Blood samples for biochemical estimation were collected just before sacrificing the rats. The blood was kept at room temperature for 30 min and then centrifuged at 4000 rpm for 15 min to separate serum. Serum was used to estimate serum glucose level and serum nitrite/nitrate concentration.

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followed by exposure to Morris water maze on the 52nd day of STZ administration. The treatment was continued during acquisition (from the 52nd to 55th day) and retrieval trials (on the 56th day) on Morris water maze.

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2.6. Assessment of spatial learning and memory by Morris water maze

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Morris water maze (MWM) test was employed to assess spatial learning and memory of rats (Morris, 1984; Parle and Singh, 2004; Sharma and Singh, 2010, 2011a,b). The MWM procedure was based on a principle where the animal is placed in a large pool of water; as the animals dislike swimming, their tendency to escape from the water is being accomplished by finding an escape platform. MWM consisted of a large circular pool (150 cm in diameter, 45 cm in height, filled to a depth of 30 cm with water at 28 °C). The water was made opaque with white colored dye. The tank was divided into four equal quadrants with the help of two threads: the first thread was used to divide the tank in half and the second thread was placed right angle of the first thread. Both threads are fixed on the rim of the tank. A submerged platform (10 cm2), painted white, was placed inside the target quadrants of this pool 1 cm below surface of the water. The position of the platform was kept unaltered throughout the training session. Each animal was subjected to four consecutive trials on each day with a gap of 5 min. The rat was gently placed in the water of the pool between quadrants, facing the wall of the pool with drop location changing for each trial, and allowed 120 s to locate the submerged platform. Then, it was allowed to stay on the platform for 20 s. If it failed to find the platform within 120 s, it was guided gently onto the platform and allowed to remain there for 20 s. Escape latency time (ELT) to locate the hidden platform in water maze was noted as the index of acquisition or learning. The animal was subjected to acquisition trials for four consecutive days. On the fifth day, the platform was removed and each rat was allowed to explore the pool for 120 s. The mean time spent in all four quadrants was noted. The mean time spent by the animal in the target quadrant searching for the hidden platform is noted as the index of retrieval. All the trials were completed between 09.00 and 18.00 h.

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2.8. Assessment of vascular endothelial function using isolated rat aortic 302 ring preparation 303

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2.7.1. Collection of sample For biochemical estimation in the brain tissue, the animals were sacrificed. The brains were removed and homogenized in phosphate buffer (pH 7.4, 10% w/v) using Telfon homogenizer. The clear supernatant, obtained after centrifugation at 3000 rpm for 15 min, was used

The rats were decapitated, and the descending thoracic aorta was dissected. The ring of thoracic aorta approximately 3–5 mm long was carefully excised and mounted in tissue bath chamber containing Krebs–Henseleit solution (NaCl, 119 mM; KCl, 4.7 mM; NaHCO3, 25 mM; MgSO4, 1.0 mM; glucose, 11.1 mM; KH2PO4, 1.2 mM and

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2.5.8. Group VIII — STZ and donepezil Donepezil (1 mg/kg, p.o., daily) was administered to the STZ (50 mg/kg, i.p.) treated rats, starting from the 30th day of STZ treatment followed by exposure to Morris water maze on the 52nd day of STZ administration. The treatment was continued during acquisition (from the 52nd to 55th day) and retrieval trials (on the 56th day) on Morris water maze.

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Please cite this article as: Singh G, et al, Efficacy of bosentan, a dual ETA and ETB endothelin receptor antagonist, in experimental diabetes induced vascular endothelial dys..., Pharmacol Biochem Behav (2014), http://dx.doi.org/10.1016/j.pbb.2014.05.002

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309 CaCl2, 2.5 mM) of bathing medium (pH 7.4), maintained at 37 °C and 310 Q14 bubbled with the carbogen (95% O2 and 5% CO2) (pH = 7.4). The ring

All the results of this study were statistically analyzed by software Sigma stat 3.5. The results were expressed as mean ± standard deviation of mean. The data for isolated aortic ring preparation was statistically analyzed using repeated measure ANOVA followed by Newman–Keuls test. The data for escape latency time was statistically analyzed using three way ANOVA followed by Bonferroni post hoc test. The rest of the data obtained from various groups was statistically analyzed using one-way ANOVA followed by Tukey's multiple range test. The P b 0.05 was considered to be statistically significant.

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Fig. 1. Effect of various pharmacological interventions on endothelium dependent relaxation using aortic ring preparation. Abbreviations: CMC — carboxymethyl cellulose (0.5% w/v, p.o.); STZ — streptozotocin (50 mg/kg, i.p.); Bosen LD — bosentan low dose (50 mg/kg/day, p.o.); Bosen HD — bosentan high dose (100 mg/kg/day, p.o.); DON — donepezil (1 mg/kg/day, p.o.). Responses are expressed as percentage of precontraction induced by 3 × 10−6 M phenylephrine. Values are mean ± standard deviation of mean (STDEV), n = 6, repeated measure analysis of variance (ANOVA) followed by Newman–Keuls test. aP b 0.05 versus control. b P b 0.05 versus streptozotocin treated group.

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3.1. Effect of various pharmacological interventions on endothelium 342 dependent and independent relaxation 343

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Fig. 2. Effect of various pharmacological interventions on endothelium independent relaxation using aortic ring preparation. Abbreviations: CMC — carboxymethyl cellulose (0.5% w/v, p.o.); STZ — streptozotocin (50 mg/kg, i.p.); Bosen LD — bosentan low dose (50 mg/kg/day, p.o.); Bosen HD — bosentan high dose (100 mg/kg/day, p.o.); DON — donepezil (1 mg/kg/day, p.o.). Responses are expressed as percentage of precontraction induced by 3 × 10−6 M phenylephrine. Values are mean ± standard deviation of mean (STDEV), n = 6, repeated measure analysis of variance (ANOVA) followed by Newman– Keuls test.

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was held with the help of two opposite parallel L-shaped stainless steel loops in opposite directions. One loop was connected with force– 313 displacement transducer (Ft-2147) which was connected to Physiograph 314 (INCO, Ambala, India) and another was connected with a holder act as an 315 anchor, submerged in bath chamber. The preparation was allowed to be 316 stretched with 1.5 g tension followed by equilibration for 90 min with 317 continuous washing with bath solution after every 10–15 min. The iso318 metric contractile force was measured (Pieper, 1997; Sharma and 319 Q15 Singh, 2010, 2011a,b) with a force–displacement transducer. The aortic 320 ring preparation incubated with Krebs–Henseleit solution was primed 321 with 80 mM KCl to check its functional integrity and to improve its con322 tractility. The aortic ring preparation was stimulated with phenylephrine 323 (3 × 10−6 M) until the contractile response reached a steady tension and 324 cumulative dose responses of acetylcholine (ACh; 10−8 to 10−4 M) or so325 dium nitroprusside (SNP; 10−8 to 10−4 M) were recorded with intact or 326 denuded endothelium, respectively at 30 minute intervals. The intimal 327 layer of aortic ring was rubbed gently with a moistened filter paper for 328 30 s to obtain endothelium free preparation (Sharma and Singh, 2010, −6 329 Q16 2011a,b). Loss of ACh (1 × 10 M) induced relaxation confirmed the ab330 sence of vascular endothelium.

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Administration of a single dose of streptozotocin (50 mg/kg, i.p.) significantly attenuated acetylcholine (10−8 to 10−4) induced endothelium dependent relaxation, when compared to control rats (Fig. 1). However it did not affect sodium nitroprusside (10−8 to 10−4) induced endothelium independent relaxation (Fig. 2). Administration of CMC (0.5%, 10 ml/kg/day, p.o.; 26 day) did not show any significant effect on endothelium dependent as well as independent relaxations (Figs. 1 and 2). Administration of bosentan (50 mg/kg/day, p.o./100 mg/kg/day, p.o.; 26 days)/donepezil (1 mg/kg p.o., 26 days) to streptozotocin treated rats significantly abolished streptozotocin induced impairment of endothelial dependent relaxation (Fig. 1). These interventions did not show any effect on endothelium independent relaxation (Fig. 2). Further, no per se effect of these drugs on endothelium dependent as well as independent relaxation was observed (Figs. 1 and 2).

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3.2. Effect of various pharmacological interventions on escape latency time 359 (ELT) and mean time spent in target quadrant (TSTQ), using Morris water 360 maze (MWM) 361 Before exposure to MWM, all the animals have been exposed to rotarod and actophotometer to study the effect of various agents on the motor coordination and activity where no significant effects of these agents have been observed (data not shown). Control rats showed a downward trend in their ELT. There was a significant [F (3, 224) = 14,423.344, P b 0.001] drop in day 4 ELT, when compared to day 1 ELT of these rats, reflecting normal learning ability (Fig. 3). Further on day 5 a significantly higher TSTQ was observed, when compared to time spent in other quadrants of control rats, reflecting normal retrieval (memory) as well (Fig. 4). Administration of carboxy methyl cellulose (CMC) (0.5%, 10 ml/kg/day, p.o.; 26 days) did not show any significant effect on day 4 ELT (Fig. 3) and day 5 TSTQ (Fig. 4). Administration of a single dose of streptozotocin (50 mg/kg, i.p.) produced a significantly [F (1, 224) = 6806.250, P b 0.001] slower

Please cite this article as: Singh G, et al, Efficacy of bosentan, a dual ETA and ETB endothelin receptor antagonist, in experimental diabetes induced vascular endothelial dys..., Pharmacol Biochem Behav (2014), http://dx.doi.org/10.1016/j.pbb.2014.05.002

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when compared to both the doses of bosentan. Also there was a significant interaction between drug treatments and days [F (9, 224) = 45.192], which suggest a significant day wise (day 1 to day 4) difference between the drug treatments. Further, these drugs also prevented streptozotocin induced decrease in day 5 TSTQ in a significant manner, thereby indicating amelioration of streptozotocin induced impairment of memory (Fig. 4). Bosentan as well as donepezil did not show any significant per se effect on day 4 ELT (Fig. 3) and day 5 TSTQ of control animals (Fig. 4).

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3.3. Effect of various pharmacological interventions on serum glucose level 395 and bodyweight 396

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Administration of a single dose of streptozotocin (50 mg/kg, i.p.) produced a significant increase in serum glucose level and a significant decrease in body weight as compared to control rats (Tables 1 and 2) Administration of CMC (0.5%, 10 ml/kg/day, p.o.; 26 days) did not show any significant effect on serum glucose level and body weight as compared to control rats (Tables 1 and 2). Administration of bosentan (50 mg/kg/day, p.o./100 mg/kg/day, p.o.; 26 days)/donepezil (1 mg/kg p.o., 26 days) did not show any significant change in streptozotocin induced increase in serum glucose level and decrease in body weight (Tables 1 and 2). Further, these drugs did not show any per se effect on serum glucose level and body weight of the animals (Tables 1 and 2).

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Administration of a single dose of streptozotocin (50 mg/kg, i.p.) produced a significant decrease in serum nitrite/nitrate levels, when compared to control rats (Fig. 5). Administration of CMC (0.5%, 10 ml/kg/day, p.o.; 26 days) did not show any significant effect on serum nitrite/nitrate level (Fig. 5). Administration of bosentan (50 mg/kg/day, p.o./100 mg/kg/day, p.o.; 26 days)/donepezil (0.5 mg/kg i.p., 26 days) to streptozotocin treated rats attenuated streptozotocin induced decrease in serum nitrite/nitrate levels in a

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rate of acquisition of the Morris water maze test compared to control animals (Fig. 3). Further, these animals showed a significant reduction in day 5 TSTQ when compared to day 5 TSTQ of control rats, indicating impairment of memory as well (Fig. 4). Administration of bosentan (50 mg/kg/day, p.o./100 mg/kg/day, p.o.; 26 days)/donepezil (1 mg/kg p.o., 26 days) treatment significantly [F (9, 224) = 41.523, P b 0.001] counteracts the STZ-induced learning impairment (Fig. 3). There was no significant dose dependent effect on ELT of bosentan but donepezil has provided significantly [F (3, 224) = 45.720, P b 0.001] better effect in STZ treated animals

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Fig. 3. Effect of various pharmacological interventions on day 1 to day 4 escape latency time (ELT) using Morris water maze. Abbreviations: STZ — streptozotocin (50 mg/kg, i.p.); Bosen LD — bosentan low dose (50 mg/kg/day, p.o.); Bosen HD — bosentan high dose (100 mg/kg/day, p.o.); DON — donepezil (1 mg/kg/day, p.o.). Values are expressed as mean ± standard deviation of mean (STDEV), n = 6, three way ANOVA followed by Bonferroni post hoc tests. aVersus previous day ELT in respective group; bVersus respective day ELT in control group; cVersus respective day ELT in STZ treated group; dVersus respective day ELT in bosentan treated group.

397 398

Fig. 4. Effect of various pharmacological interventions on mean time spent in target quadrant (TSTQ) using Morris water maze. Abbreviations: CMC — carboxymethyl cellulose (0.5% w/v, p.o.); STZ — streptozotocin (50 mg/kg, i.p.); Bosen LD — bosentan low dose (50 mg/kg/day, p.o.); Bosen HD — bosentan high dose (100 mg/kg/day, p.o.); DON — donepezil (1 mg/kg/day, p.o.). Values are expressed as mean ± standard deviation of mean (STDEV), n = 6, two way ANOVA followed by Bonferroni post hoc tests. [F (1, 40) = 532.22 for days, P b 0.0001 and F (7, 40) = 9.05 for treatment, P b 0.0001]. aP b 0.05 versus mean time spent in other quadrants in control. bP b 0.05 versus mean time spent in target quadrant in control group. cP b 0.05 versus mean time spent in target quadrant in STZ treated group.

Please cite this article as: Singh G, et al, Efficacy of bosentan, a dual ETA and ETB endothelin receptor antagonist, in experimental diabetes induced vascular endothelial dys..., Pharmacol Biochem Behav (2014), http://dx.doi.org/10.1016/j.pbb.2014.05.002

410 411 412 413 414 415 416 417

6

Serum glucose (mg/dl)

– 10 ml (0.5% w/v), p.o. 100 mg, p.o. 1 mg, p.o. 50 mg, i.p. 50 mg, i.p. + 50 mg, p.o. 50 mg, i.p. + 100 mg, p.o. 50 mg, i.p. + 1 mg, p.o.

96.0 ± 95..3 ± 99.0 ± 97.0 ± 98.6 ± 94.5 ± 94.8 ± 96.6 ±

94.8 ± 95.1 ± 97.3 ± 98.1 ± 392.1 ± 385.0 ± 389.0 ± 383.0 ±

3.3 3.5 3.2 3.7 3.0 4.5 3.9 4.2

2.9 3.8 2.9 2.5 3.9a 2.8 4.3 3.0

Values are expressed as mean ± standard deviation of mean (STDEV), n = 6, two way ANOVA followed by Bonferroni post hoc tests. [F (1, 40) = 7809.38 for days, P b 0.0001 and F (7, 40) = 1124.07 for treatment, P b 0.0001]. a P b 0.05 versus final values in control group.

418 419

significant manner (Fig. 5). Further, these drugs did not show any per se effect on serum nitrite/nitrate level (Fig. 5).

420 421

3.5. Effect of various pharmacological interventions on brain acetyl cholinesterase (AChE) activity

422

Administration of a single dose of streptozotocin (50 mg/kg, i.p.) produced a significant increase in brain acetyl cholinesterase (AChE) activity, when compared to control rats (Fig. 5). Administration of CMC (0.5%, 10 ml/kg/day, p.o.; 26 days) did not show any significant effect on AChE activity (Fig. 6). Administration of bosentan (50 mg/kg/day, p.o./100 mg/kg/day, p.o.; 26 days)/donepezil (1 mg/kg p.o., 26 days) to streptozotocin treated rats prevented streptozotocin induced increase in AChE activity in a significant manner (Fig. 6). Further, these drugs did not show any per se effect on brain acetyl cholinesterase activity (Fig. 6).

429 430 431

432 433 434

C

427 428

3.6. Effect of various pharmacological interventions on brain thiobarbituric acid reactive substances (TBARS) level

E

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423 424

t2:1 t2:2

Table 2 Effect of various pharmacological interventions on body weight.

t2:3

t2:13 t2:14 t2:15 t2:16

446 447

4. Discussion

455

Morris water maze employed in the present study is one of the most widely accepted models to evaluate spatial learning and memory of the rodents (Morris, 1984; Parle and Singh, 2007). Control untreated animals in our study have shown marked reduction in day 4 escape latency time (ELT) as compared to their day 1 ELT during acquisition trial, suggesting

456

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Group

Dose/kg

t2:4 t2:5 t2:6 t2:7 t2:8 t2:9 t2:10 t2:11 t2:12

Administration of a single dose of streptozotocin (50 mg/kg, i.p.) produced a significant decrease in brain reduced glutathione (GSH) level, when compared to control rats (Fig. 8). Administration of CMC (0.5%, 10 ml/kg/day, p.o.; 26 days) did not show any significant effect on brain GSH level (Fig. 8). Administration of bosentan (50 mg/kg/day, p.o./100 mg/kg/day, p.o.; 26 days)/donepezil (1 mg/kg p.o., 26 days) to streptozotocin treated rats prevented streptozotocin induced decrease in brain GSH level in a significant manner (Fig. 8). Further, these drugs did not show any per se effect on brain GSH level (Fig. 8).

C

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3.7. Effect of various pharmacological interventions on brain reduced form 444 of glutathione (GSH) level 445

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Administration of a single dose of streptozotocin (50 mg/kg, i.p.) produced a significant increase in brain thiobarbituric acid reactive substances (TBARS) level, when compared to control rats (Fig. 7). Administration of CMC (0.5%, 10 ml/kg/day, p.o.; 26 days) did not show any significant effect on brain TBARS level (Fig. 7). Administration of bosentan (50 mg/kg/day, p.o./100 mg/kg/day, p.o.; 26 days)/donepezil (1 mg/kg p.o., 26 days) to streptozotocin treated rats prevented streptozotocin induced increase in brain TBARS level in a significant manner (Fig. 7). Further, these drugs did not show any per se effect on brain TBARS level (Fig. 7).

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Fig. 5. Effect of various pharmacological interventions on serum nitrite/nitrate level. Abbreviations: CMC — carboxymethyl cellulose (0.5% w/v, p.o.); STZ — streptozotocin (50 mg/kg, i.p.); Bosen LD — bosentan low dose (50 mg/kg/day, p.o.); Bosen HD — bosentan high dose (100 mg/kg/day, p.o.); DON — donepezil (1 mg/kg/day, p.o.). Values are expressed as mean ± standard deviation of mean (STDEV), n = 6, one way ANOVA followed by Tukey's multiple range test. [F (7, 40) = 13.095, P b 0.05]. aP b 0.05 versus serum nitrite/nitrate of control. bP b 0.05 versus serum nitrite of STZ treated group.

T

t1:13 t1:14 t1:15 t1:16

F

Control Vehicle (CMC) treated Bosentan per se Donepezil per se STZ STZ + bosentan low dose STZ + bosentan high dose STZ + donepezil

Final value

O

t1:5 t1:6 t1:7 t1:8 t1:9 t1:10 t1:11 t1:12

Basal value

R O

Dose/kg

P

Group

t1:4

D

t1:3

Table 1 Effect of various pharmacological interventions on serum glucose level.

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t1:1 t1:2

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Control Vehicle (CMC) treated Bosentan per se Donepezil per se STZ STZ + bosentan low dose STZ + bosentan high dose STZ + donepezil

– 10 ml (0.5% w/w), p.o. 50 mg, p.o. 1 mg, p.o. 50 mg, i.p. 50 mg, i.p. + 10 mg, p.o. 50 mg, i.p. + 20 mg, p.o. 50 mg, i.p. + 1 mg, p.o.

Body weight (g) Basal value

Final value

214.0 215.0 216.0 220.0 222.0 223.0 224.0 219.0

220.0 ± 218.0 ± 219.0 ± 225.0 ± 167.0 ± 171.0 ± 174.0 ± 170.0 ±

± ± ± ± ± ± ± ±

5.4 6.5 7.5 6.8 5.0 6.6 4.9 9

5.1 6.3 7.6 7.2 6.0a 7.0 6.7 6.5

Values are expressed as mean ± standard deviation of mean (STDEV), n = 6, two way ANOVA followed by Bonferroni post hoc tests. [F (1, 40) = 54.18 for days, P b 0.0001 and F (7, 40) = 7.13 for treatment, P b 0.0001]. a P b 0.05 vs final body weight in control.

Fig. 6. Effect of various pharmacological interventions on brain acetylcholinesterase (AChE) activity. Abbreviations: CMC — carboxymethyl cellulose (0.5% w/v, p.o.); STZ — streptozotocin (50 mg/kg, i.p.); Bosen LD — bosentan low dose (50 mg/kg/day, p.o.); Bosen HD — bosentan high dose (100 mg/kg/day, p.o.); DON — donepezil (1 mg/kg/day, p.o.). Values are expressed as mean ± standard deviation of mean (STDEV), n = 6, one way ANOVA followed by Tukey's multiple range test. [F (7, 40) = 38.438, P b 0.05]. a P b 0.05 versus brain AChE activity of control. bP b 0.05 versus brain AChE activity of STZ treated group.

Please cite this article as: Singh G, et al, Efficacy of bosentan, a dual ETA and ETB endothelin receptor antagonist, in experimental diabetes induced vascular endothelial dys..., Pharmacol Biochem Behav (2014), http://dx.doi.org/10.1016/j.pbb.2014.05.002

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Fig. 8. Effect of various pharmacological interventions on brain reduced glutathione (GSH) level. Abbreviations: CMC — carboxymethyl cellulose (0.5% w/v, p.o.); STZ — streptozotocin (50 mg/kg, i.p.); Bosen LD — bosentan low dose (50 mg/kg/day, p.o.); Bosen HD — bosentan high dose (100 mg/kg/day, p.o.); DON — donepezil (1 mg/kg/day, p.o.). Values are expressed as mean ± standard deviation of mean (STDEV), n = 6, one way ANOVA followed by Tukey's multiple range test. [F (7, 47) = 45.870, P b 0.05]. aP b 0.05 versus brain GSH level of control. b P b 0.05 versus brain GSH level of STZ treated group.

humans (Isingrini et al., 2009; Kearney-Schwartz et al., 2009; Bomboi et al., 2010). Endothelial dysfunction has also been reported to be related to higher oxidative stress levels (Javeshghani et al., 2009; Ozkul et al., 2010; Weseler and Bast, 2010). Moreover cerebrovascular changes, blood flow reductions, endothelial dysfunction and oxidative stress have already been reported to be involved in various kinds of dementias including Alzheimer's disease and vascular dementia. Lester-Coll et al. (2006) have reported that intracerebral streptozotocin results in chemical depletion of insulin and IGF signaling mechanisms combined with oxidative injury which is sufficient to cause AD-type neurodegeneration. Results of the present investigation state that streptozotocin (STZ) in a single dose of 50 mg kg−1 i.p. has produced hyperglycemia, vascular endothelial dysfunction, impairment of learning (indicated by higher ELT value as compared control group) as well as memory (indicated by decreased day 5 TSTQ), along with increase in brain oxidative stress (increased TBARS, and decreased GSH levels), reduction serum nitrite/ nitrate and rise in brain AChE activity, which is in consonance with our earlier studies (Sharma and Singh, 2010, 2011a,b). In the present investigation treatment of bosentan (50 mg/kg and 100 mg/kg) a dual (ETA and ETB) endothelin receptor blocker as well as donepezil (1 mg/kg p.o.) an acetylcholinesterase inhibitor has attenuated the effect of STZ diabetes on the learning and memory of rats. In addition these agents have also improved STZ induced endothelial dysfunction and associated biochemical changes. Donepezil has served as the positive control in this study. We have used this agent in our previous publications on VaD (Sharma and Singh, 2010, 2011a,b, 2012a,b,c). We have used the doses of bosentan (Rossi et al., 2000; Abdelsaid et al., 2014) and donepezil (Gawel et al., 2014) on the bases of our preliminary findings and on the bases of previously published reports. Results of this study suggest that donepezil have provided more benefits as compared to bosentan in ELT and brain AChE activity. In all other parameters bosentan has shown almost similar effect as that of donepezil, although a much higher dose of bosentan was required. Therefore, at least in terms of AChE activity and task acquisition, donepezil may be considered better than both the doses of bosentan. Endothelin (ET) being the most potent endothelium derived contracting factor has been demonstrated to exist in three isoforms i.e. ET-1, ET-2, and ET-3. Out of the three isoforms ET-1 is the most widely studied and considered to be an important mediator of a variety of physiological and pathophysiological functions (Khimji and Rockey, 2010). The biological effects of ET-1 are transduced by two pharmacologically distinguishable receptor subtypes, ETA and ETB receptors, respectively (Kawanabe and Nauli, 2011). In the vasculature, the ETA receptor is mainly located on vascular smooth muscle cells and mediates potent vasoconstriction. The ETB receptor is primarily located on endothelial cells, but may also be present on vascular smooth muscle cells. Stimulation of the endothelial ETB receptor results in the release of NO which causes vasodilatation, whereas the stimulation of the vascular smooth muscle cell ETB receptor results in vasoconstriction. Thus, the net effect produced by ET-1 is determined on the receptor localization and the balance between ETA and ETB receptors. Under physiological conditions, the net effect is vasoconstriction mediated by the ETA receptor, which is partly counteracted by ETB receptor mediated release of NO. Under pathophysiological conditions, ET-1 production is increased, and NO production is reduced, resulting in a greater shift in the balance towards vasoconstriction (Bohm and Pernow, 2007). In endothelial dysfunction there is increased expression of ET-1 in smooth muscle cells. There is also increased expression of ETB receptors on smooth muscle cells mediating vasoconstriction. ET-1 may decrease endothelial NO synthase (eNOS) expression, thereby reducing NO production. Both the ETA and the ETB receptor on smooth muscle cells may mediate formation of superoxide (O2−) in endothelial dysfunction. Superoxide will decrease the biological activity of NO by forming peroxynitrate (ONOO −). Collectively the balance of effects is shifted towards more vasoconstriction, inflammation and oxidative stress in endothelial dysfunction (Bohm and Pernow, 2007).

O

Fig. 7. Effect of various pharmacological interventions on brain thiobarbituric acid reactive substances (TBARS) level. Abbreviations: CMC — carboxymethyl cellulose (0.5% w/v, p.o.); STZ — streptozotocin (50 mg/kg, i.p.); Bosen LD — bosentan low dose (50 mg/kg/day, p.o.); Bosen HD — bosentan high dose (100 mg/kg/day, p.o.); DON — donepezil (1 mg/kg/day, p.o.). Values are expressed as mean ± standard deviation of mean (STDEV), n = 6, one way ANOVA followed by Tukey's multiple range test. [F (7, 40) = 38.986, P b 0.05]. a P b 0.05 versus brain TBARS level of control. bP b 0.05 versus brain TBARS level of STZ treated group.

normal acquisition or learning ability. Further, these animals have shown significant increase in day 5 mean time spent in target quadrant (TSTQ) 463 when compared to time spent in other quadrants, indicating normal re464 trieval (memory) as well. These results are in line with previous studies 465 from our own lab (Koladiya et al., 2008; Rinwa et al., 2010; Sharma and 466 Q18 Singh, 2010, 2011a,b) as well as from other labs (Packard et al., 1996; 467 Camarasa et al., 2010; Frye et al., 2010). Vehicle (carboxymethyl cellulose) 468 employed in the study did not show any modification in the values of day 469 4 ELT and day 5 TSTQ of control. Moreover no per se effect of various 470 drugs used in the study was observed. 471 Single dose STZ administration is a very well documented and an 472 accepted model of diabetes in rats (Leung et al., 2010; Sokolovska 473 Q19 et al., 2010; Sharma and Singh, 2010, 2011a,b). STZ induced experimen474 tal diabetes is commonly used for the assessment of diabetic conditions 475 and its secondary complications including endothelial dysfunction 476 (Chopra et al., 2010; Feng et al., 2010; Huynh et al., 2010; Marotta et al., 477 2010; Olukman et al., 2010; Yohannes et al., 2010; Zhang et al., 2010). 478 Further, endothelial dysfunction (vascular defects) has been reported to 479 induce varying degrees of memory impairment in animals as well as in

7

Please cite this article as: Singh G, et al, Efficacy of bosentan, a dual ETA and ETB endothelin receptor antagonist, in experimental diabetes induced vascular endothelial dys..., Pharmacol Biochem Behav (2014), http://dx.doi.org/10.1016/j.pbb.2014.05.002

480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 Q20 498 499 500 501 502 503 504 505 Q21 Q22 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545

G. Singh et al. / Pharmacology, Biochemistry and Behavior xxx (2014) xxx–xxx

endothelial dysfunction and associated dementia and that ET-1 receptor 612 can be considered an important target to develop drug therapy of vascular 613 endothelial dysfunction associated dementia. 614 615

Role of funding source

616

None.

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Authors are thankful to Department of Pharmaceutical Sciences and Drug Research, Faculty of Medicine, Punjabi University, Patiala, Punjab, India for providing all the necessary facilities and funding to conduct this research.

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Abdelsaid M, Kaczmarek J, Coucha M, Ergul A. Dual endothelin receptor antagonism with bosentan reverses established vascular remodeling and dysfunctional angiogenesis in diabetic rats: relevance to glycemic control. Life Sci 2014. http://dx.doi.org/10.1016/j. lfs.2014.01.008. [Jan 18. pii: S0024-3205(14)00071-X, [Epub ahead of print]]. Anthoni C, Mennigen RB, Rijcken EJ, Laukötter MG, Spiegel HU, Senninger N, et al. Bosentan, an endothelin receptor antagonist reduces leucocyte adhesion and inflammation in a murine model of inflammatory bowel disease. Int J Colorectal Dis 2006; 21(5):409–18. Araki A. Dementia and insulin resistance in patients with diabetes mellitus. Nippon Rinsho 2010;68(3):569–74. Beutler RG, Duron O, Kelly B. Reduced glutathion estimation. J Lab Clin Med 1963;61:82. Bohm F, Pernow J. The importance of endothelin-1 for vascular dysfunction in cardiovascular disease. Cardiovasc Res 2007;76(1):8–18. Bomboi G, Castello L, Cosentino F, Giubilei F, Orzi F, Volpe M. Alzheimer's disease and endothelial dysfunction. Neurol Sci 2010;31(1):1–8. Brosky G, Logothetopoulos J. Streptozotocin diabetes in the mouse and guinea pig. Diabetes 1969;18(9):606–11. Burnier M, Forni V. Endothelin receptor antagonists: a place in the management of Essential hypertension. Nephrol Dial Transplant 2012;27(3):865–8. Camarasa J, Rodrigo T, Pubill D, Escubedo E. Memantine is a useful drug to prevent the spatial and non-spatial memory deficits induced by methamphetamine in rats. Pharmacol Res 2010;62(5):450–6. Capone C, Faraco G, Coleman C, Young CN, Pickel VM, Anrather J, et al. Endothelin 1dependent neurovascular dysfunction in chronic intermittent hypoxia. Hypertension 2012;60(1):106–13. Chen Z, Zhong C. Decoding Alzheimer's disease from perturbed cerebral glucose metabolism: implications for diagnostic and therapeutic strategies. Prog Neurobiol 2013; 108:21–43. Chen Y, Hanaoka M, Droma Y, Chen P, Voelkel NF, Kubo K. Endothelin-1 receptor antagonists prevent the development of pulmonary emphysema in rat. Eur Respir J 2010; 35(4):904–12. Chopra K, Tiwari V, Arora V, Kuhad A. Sesamol suppresses neuroinflammatory cascade in experimental model of diabetic neuropathy. J Pain 2010;11(10):950–7. de Bruin NM, Prickaerts J, van Loevezijn A, Venhorst J, de Groote L, Houba P, et al. Two novel 5-HT6 receptor antagonists ameliorate scopolamine-induced memory deficits in the object recognition and object location tasks in Wistar rats. Neurobiol Learn Mem 2011;96(2):392–402. Ekonomou A, Ballard CG, Pathmanaban ON, Perry EK, Kalaria RN, Minger SL. Increased neural progenitors in vascular dementia. Neurobiol Aging 2011;32(12):2152–61. Elesber AA, Bonetti PO, Woodrum JE, Zhu XY, Lerman LO, Younkin SG, et al. Bosentan preserves endothelial function in mice overexpressing APP. Neurobiol Aging 2006;27(3): 446–50. Ellman GL, Courtney KD, Valentino A, Featherstone RM. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol 1961;7:88–95. Ergul A. Endothelin-1 and diabetic complications: focus on the vasculature. Pharmacol Res 2011;63(6):477–82. Esiri MM. The interplay between inflammation and neurodegeneration in CNS disease. J Neuroimmunol 2007;184(1–2):4–16. Feng B, Chen S, George B, Feng Q, Chakrabarti S. MiR133a regulates cardiomyocyte hypertrophy in diabetes. Diabetes Metab Res Rev 2010;26(1):40–9. Frye CA, Edinger KL, Lephart ED, Walf AA. 3Alpha-androstanediol, but not testosterone, attenuates age-related decrements in cognitive, anxiety, and depressive behavior of male rats. Front Aging Neurosci 2010;2:15. [8]. Gawel K, Labuz K, Jenda M, Silberring J, Kotlinska JH. Influence of cholinesterase inhibitors, donepezil and rivastigmine on the acquisition, expression, and reinstatement of morphine-induced conditioned place preference in rats. Behav Brain Res 2014. http://dx.doi.org/10.1016/j.bbr.2014.04.019. [Apr 19. pii: S0166-4328(14)00237-X, [PMID: 24755308]]. Gupta SK, Saxena A, Singh U, Arya DS. Bosentan, the mixed ETA–ETB endothelin receptor antagonist, attenuated oxidative stress after experimental myocardial ischemia and reperfusion. Mol Cell Biochem 2005;275(1–2):67–74. Huynh K, McMullen JR, Julius TL, Tan JW, Love JE, Cemerlang N, et al. Cardiac-specific IGF1 receptor transgenic expression protects against cardiac fibrosis and diastolic dysfunction in a mouse model of diabetic cardiomyopathy. Diabetes 2010;59(6):1512–20.

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It has been demonstrated that the expression of ETB receptors on VSMCS is increased in diabetes and that ETB receptor stimulation subse548 quently enhances production of reactive oxygen species (Pernow et al., 549 2012). Diabetic conditions have shown an increased sensitivity to ET-1 550 and decreased relaxation in basilar artery (Li et al., 2011). A significant 551 role of ET-1 is suggested in the endothelial dysfunction of an individual 552 with insulin resistance and type-2 diabetes (Pernow et al., 2012). Dual 553 ETA/ETB receptor blockers have been shown to improve diabetes associ554 ated complications including endothelial dysfunction and diabetic ne555 phropathy (Rafnsson et al., 2012; Sen et al., 2012). ET has also been 556 shown to have a proinflammatory role in the brain (Pittman, 2006). 557 ET mediated disruption of blood brain barrier (BBB) paves a path for 558 the entry of inflammatory cells in the brain. ET-1 promotes leukocyte 559 adhesion through intercellular adhesion molecule-1 (ICAM-1) and 560 vascular cell adhesion molecule-1 (VCAM-1). ET-1 can promote leuko561 cyte endothelium interactions by increasing the expression of E-selectin 562 on endothelium. ET also influences the release of certain key inflammato563 ry mediators which are known to be involved in ischemic brain damage 564 (Kaundal et al., 2012). Endothelin receptor antagonists including 565 bosentan have been demonstrated to exert anti-inflammatory action 566 (Esiri, 2007). Bosentan is noted to reduce inflammation by inhibiting leu567 cocyte adhesion, a crucial step in the recruitment of leucocytes to the in568 Q23 flamed tissue (Anthoni et al., 2006). Furthermore, bosentan has also been 569 documented to possess neuroprotective and antioxidative actions (May 570 et al., 1996; Gupta et al., 2005). Recent reports have also indicated a 571 role of endothelin in dementia of AD (Palmer and Love, 2011). The amy572 loid beta (Aβ) causes excessive production and release of ET-1 in nervous 573 system via upregulation of endothelin-converting enzyme-2 (ECE-2). 574 This enzyme is responsible for the breakdown of proendothelin (inactive 575 Q24 precursor) and leads to formation of endothelins especially ET-1. Based 576 on this report this indicates that Aβ-ET-1 axis contributes to the develop577 ment of AD via reduction of cerebral blood flow (CBF) (Palmer and Love, 578 2011). In addition, endothelin receptor antagonists have also been shown 579 to have an ameliorative potential in AD (Palmer and Love, 2011). 580 Bosentan has been noted to preserve aortic and carotid endothelial func581 tion in transgenic mice (Tg2576 mice) overexpressing the amyloid pre582 cursor protein, beneficial in maintaining cerebral blood flow in AD 583 (Elesber et al., 2006). The findings in this paper further suggest that 584 these Aβ associations could involve the endothelin-system, as it has 585 been previously shown that Aβ upregulates ET-1 production (Palmer 586 et al., 2012), and the findings here involving bosentan are further 587 evidence for the involvement of the endothelin system. The present find588 ings suggest that endothelin receptors can be considered potential targets 589 in various dementia conditions including vascular dementia and 590 Alzheimer's disease. Moreover involvement of Ab, enhanced oxidative 591 stress, acetyl cholinesterase activity, perturbed cerebral glucose levels, 592 neuronal degeneration, cognitive deficits etc. have been reported as im593 portant features of Alzheimer's disease (Chen and Zhong, 2013). Thus, 594 at this point it is possible to speculate that endothelin receptor modula595 tion probably plays an important role in dementia including vascular de596 mentia and dementia of AD. 597 Therefore with support from literature and data in hand it may be 598 suggested that bosentan a dual ETA/ETB endothelin receptor blocker 599 has shown an ameliorative effect in STZ diabetes induced endothelial 600 dysfunction and associated dementia by virtue of its multiple effect in601 cluding ETA/ETB receptor blocking effect and antioxidative activity. 602 Bosentan is a selective antagonist of endothelin receptors, so the role 603 of endothelin could not be ruled out for this protective effect however 604 direct effect on the levels of endothelin-1 required to be measured in 605 the future studies. Perhaps it is the first report indicating beneficial 606 effect of bosentan in diabetes induced vascular endothelial dysfunction 607 and associated dementia, further research is required to explore the full 608 potential of endothelin receptors and their modulators in various de609 mentia conditions. 610 Hence, it is concluded that bosentan, a dual ETA/ETB receptor blocker, 611 has shown efficacy in a rat model of STZ diabetes induced vascular

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Please cite this article as: Singh G, et al, Efficacy of bosentan, a dual ETA and ETB endothelin receptor antagonist, in experimental diabetes induced vascular endothelial dys..., Pharmacol Biochem Behav (2014), http://dx.doi.org/10.1016/j.pbb.2014.05.002

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