Combination therapy with losartan and l -carnitine protects against endothelial dysfunction of streptozotocin-induced diabetic rats

European Journal of Pharmacology 744 (2014) 10–17

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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar

Cardiovascular pharmacology

Combination therapy with losartan and L-carnitine protects against endothelial dysfunction of streptozotocin-induced diabetic rats Mostafa Sleem a, Ashraf Taye a,n, Mohamed A. El-Moselhy a, Safwat A. Mangoura b a b

Department of Pharmacology & Toxicology, Faculty of Pharmacy, Minia University, Egypt Department of Pharmacology, Faculty of Medicine, Assiut University, Egypt

art ic l e i nf o

a b s t r a c t

Article history: Received 19 February 2014 Received in revised form 17 September 2014 Accepted 18 September 2014 Available online 2 October 2014

Endothelial dysfunction is a critical factor during the initiation of diabetic cardiovascular complications and angiotensin II appears to play a pivotal role in this setting. The present study aimed to investigate whether the combination therapy with losartan and the nutritional supplement, L-carnitine can provide an additional protection against diabetes-associated endothelial dysfunction and elucidate the possible mechanism(s) underlying this effect. Diabetes was induced by intraperitoneal injection of streptozotocin (STZ) (60 mg/kg) in rat. Effects of losartan (20 mg/kg, orally, 3 months) and L-carnitine (200 mg/kg, orally, 3 months) on tumor necrosis factor (TNF)-α, oxidative stress parameters, endothelial nitric oxide synthase expression (eNOS), and vascular function were evaluated. Our results showed a marked increase in aortic superoxide anion (O2
) production and serum malondialdehyde (MDA) level alongside attenuating antioxidant enzyme capacities in diabetic rats. This was associated with a significant increase in anigiotensin II type 1 receptor gene expression and TNF-α serum level of diabetic rats alongside reducing aortic eNOS gene expression and nitric oxide (NO) bioavailability. The single or combined administration of losartan and L-carnitine significantly inhibited these changes. Additionally, the vascular endothelium-dependent relaxation with acetylcholine (ACh) in aortic diabetic rat was significantly ameliorated by the single and combined administration of losartan or L-carnitine. Noteworthy, the combination therapy exhibited a more profound response over the monotherapy. Collectively, our results demonstrate that the combined therapy of losartan and L-carnitine affords additive beneficial effects against diabetes-associated endothelial dysfunction, possibly via normalizing the dysregulated eNOS and reducing the inflammation and oxidative stress in diabetic rats. & 2014 Elsevier B.V. All rights reserved.

Keywords: Endothelial dysfunction Losartan L-carnitine Oxidative stress Diabetes

1. Introduction Accumulating evidence indicates that the increased prevalence of cardiovascular diseases in diabetes has been attributed to the development of microvascular and macrovascular complications (Natali et al., 2000). Endothelial dysfunction plays a central role in diabetic vascular diseases (Liu et al., 2014). Conditions contributing to diabetic vascular remodeling and dysfunction include effects of oxidative stress and decreased nitric oxide (NO) bioavailability (De Vriese et al., 2000; Ceriello, 2006). NO production by the endothelial nitric oxide synthase (eNOS) is critically involved in maintaining the integrity and stability of the vascular endothelium, inhibiting platelet aggregation and leukocyte adhesion alongside maintaining blood

n Correspondence to: Department of Pharmacology and Toxicology, Faculty of Pharmacy, Minia University, 61511 Minia, Egypt. E-mail address: [email protected] (A. Taye).

http://dx.doi.org/10.1016/j.ejphar.2014.09.032 0014-2999/& 2014 Elsevier B.V. All rights reserved.

flow (Caldwell et al., 2010). High glucose greatly increases endothelial reactive oxygen species production leading to the reduction of NO production in human endothelial cells (Taye et al., 2010). On the other hand, activation of local renin–angiotensin system seems to be involved in the development of long-term complications of diabetes and large clinical trials confirming the beneficial effect of angiotensin converting enzyme inhibitors and angiotensin II receptor blockers in this setting (Hansson et al., 1999). Hypergly cemia stimulates production of angiotensin II, via up regulation of most of the cellular components of the renin–angiotensin system (Fiordaliso et al., 2000). A growing body of evidence suggests that the main effector peptide of the renin–angiotensin system, angiotensin II, induces inflammatory molecules and contributes to the pathophysiology of cardiovascular disease (Schiffrin and Touyz, 2003; Granger et al., 2004). More importantly, proinflammatory cytokines, such as tumor necrosis factor (TNF)-α play an important role in hypertension and heart diseases ( Jolda-Mydlowska and Salomon, 2003). TNF-α, can

M. Sleem et al. / European Journal of Pharmacology 744 (2014) 10–17

generate ROS and reactive nitrogen species, which may facilitate destruction of β-cells (Mathews et al., 2005). Likewise, TNF-α stimulates intracellular signaling cascades that promote apoptosis and matrix metalloproteinase expression (Sun et al., 2004). In addition, TNF-α is reported to downregulate eNOS expression in fat and muscle of obese rodents (Valerio et al., 2006). Recently, our group has shown that losartan could improve type 2 diabetes-associated metabolic abnormalities and inflammatory status via reducing TNF-α level (Mourad et al., 2012). L-carnitine (β-hydroxy-γ-4-ntrimethyl aminobuytric acid), a quaternary ammonium compound serves as a cofactor required for the transport of long chain fatty acids into the mitochondria for energy production in peripheral tissues. It has been reported that L-carnitine inhibits free radicals generation preventing the impairment of fatty acid beta-oxidation in mitochondria and protects tissues from damage by repairing oxidized membrane lipids (Calo et al., 2006; Gulcin, 2006). Although, the cardiovascular effects of L-carnitine and its analogs have already been reported by various experts in this field, its combinatory effect with losartan on diabetes-associated endothelial dysfunction has not been addressed. The present study was conducted to analyze whether the combination therapy with losartan and L-carnitine can confer additive protection against diabetes-associated endothelial dysfunction and the possible underlying mechanism(s) involved.

2. Materials and methods 2.1. Chemicals Losartan and L-carnitine were a kind gift from MEBACO Company, Egypt. Streptozotocin (STZ), acetylcholine (ACh) and phenylephrine were purchased from Sigma Chemical Company Inc., St Louis, MO, USA. 2.2. Animals Adult male Wistar rats (200–240 g, 10–12 weeks old) were housed at room temperature with 12:12 h light/dark cycles and were given food and water ad libitum. Experiments were conducted in accordance with the international ethical guidelines for animal care of the United States Naval Medical Research Centre, Unit No. 3, Abbaseya, Cairo, Egypt, accredited by the Association for Assessment and Accreditation of Laboratory Animal Care international (AAALAC international). The adopted guidelines are in accordance with “Principles of Laboratory Animals Care” (NIH publication no. 85-23, revised 1985). The study protocol was approved by members of “the research ethics committee” and by the pharmacology and toxicology department, faculty of pharmacy, Minia University, Egypt. STZ was freshly dissolved in 10 mmol/l citrate buffer, pH 4.5, and injected intraperitoneally at a single dose of 60 mg/kg for induction of diabetes in rat model (Coskun et al., 2005). The hyperglycemia was confirmed (2 days later) by measuring blood glucose levels using a SURESTEP Test Strip. Rats with blood glucose levels Z300 mg/dl were considered diabetics. Rats were randomly divided into five groups of eight rats each, as following: 1. Control group: rats were normal non-diabetic rats, received the same volume of the solvent. 2. Diabetic group (STZ): rats were treated with STZ as described above. 3. Diabetic rats treated with losartan (STZþ LOS): diabetic rats were orally treated with losartan in a dose of 20 mg/kg, for 3 months (Jin et al., 2013).

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4. Diabetic rats treated with L-carnitine (STZþ LC): diabetic rats were orally treated with L-carnitine in a dose of 200 mg/kg, for 3 months (Yurekli et al., 2011). 5. Diabetic rats treated with a combination of losartan (20 mg/kg, orally, 3 months) and L-carnitine (200 mg/kg, orally, 3 months) (STZ þLOSþLC). The rats were killed and then blood samples were collected and centrifuged at 8000  g for 10 min to obtain clear sera. The sera were used to determine blood glucose, TNF-α, MDA and antioxidant enzyme activities. The thoracic aorta was dissected, and carefully cleaned from adhering tissue with special care. The first 2–3-mm segment of the aorta was used for vascular reactivity studies. A portion of the thoracic aorta was fixed in 10% neutral-buffered formalin and was used in histopatholgical analysis. The remaining portion of aorta was homogenized in cold potassium phosphate buffer (0.05 mmol/l, pH 7.4) and was centrifuged at 5000  g for 10 min at 4 1C. The supernatant was kept at
80 1C for subsequent measurements. Total protein concentration was also determined using a bicinchoninic acid (BCA) protein assay kit (Pierce Chemicals). 2.3. Measurement of antioxidant enzyme activities, oxidative stress biomarkers, and nitric oxide bioavailability The reduced glutathione (GSH) serum content was determined spectrophotometrically as previously described (Spooner et al., 1981). Serum superoxide dismutase (SOD) activity was evaluated as previously described (Ouedraogo et al., 2007). Serum MDA level was obtained spectrophotometrically as previously described (Satoh et al., 2005). Aortic tissues were homogenized in a cold Krebs-HEPES buffer (10 mmol/l glucose, 0.02 mmol/l Ca-Tritriplex, 25 mmol/l NaHCO3, 1.2 mmol/l KH2PO4, 120 mmol/l NaCl, 1.6 mmol/l CaCl2  2H2O, 1.2 mmol/l MgSO4  7 HO, and 5 mmol/l KCl, pH 7.4). Superoxide anion (O2
) was produced using lucigenin-derived chemiluminescence as described previously (Taye et al., 2010). O2
level was measured in the presence of lucigenin, (5 mmol/l) to minimize artifactual O2
production due to the redox cycling, incubated for 20 min. The reaction was started by the addition of NADPH (100 mmol/l), and the relative light units (RLU) of chemiluminescences were measured over a period of 30 min in a lumencense spectrometer. Results are expressed as count per min and normalized to the protein content in each sample. Finally, the aortic nitrite/nitrate ratio (as an indicator of nitric oxide bioavailability) was assessed spectrophotometrically as previously described (Tsikas, 2007). 2.4. TNF-α estimation Serum TNF-α level was assessed in this study using enzyme linked immunosorbent assay quantitative detection ELISA using a microplate reader as previously described (Intiso et al., 2004). 2.5. Determination of aortic eNOS and angiotensin II type 1 receptor gene expression by using reverse transcriptase-polymerase chain reaction (RT-PCR) Total RNA was isolated aortic homogenates using RNeasy Purification Reagent (Qiagen, Valencia, CA) according to manufacturer's instruction. The mRNAs were reverse transcribed into cDNA by using an Oligo(dT)12–18 primer and Superscript™ II RNase Reverse Transcriptase. This mixture was incubated at 42 1C for 1 h; the kit was supplied by SuperScript Choice System (Life Technologies, Breda, the Netherlands). eNOS gene was amplified and analyzed by the following forward primer: 50 -CATACAGAACCCAGGATGGGCT-30 , reverse primer: 50 -TCCTCAGGAGGTCTTGCACATA-30 .

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2.8. Histopathological examination

The amplification products of eNOS gene were detected at 399 bp. The angiotensin II type 1 receptor gene expression was analyzed by the following forward primer: 50 -TCG AAT TCC ACC TAT GTA AGA TCG CTT C-30 , reverse primer:50 -TCG GAT CCG CAC AAT CGC CAT AAT TAT CC-30 . The amplification product of angiotensin II type 1 receptor gene was detected at 439 bp. β-actin forward primer: 5-ACTGCCGCATCCTCTTCCTC-30 , reverse primer: 50 -ACTCCTGCTTGCTGATCCACAT-30 . Forty cycles were performed under the following conditions: annealing temperature 60 1C, 65 1C, 55 1C, respectively. 95 1C, 1 min denaturation, 63 1C, 1 min annealing, 72 1C, 2 min elongation, with an additional 10 min incubation at 72 1C after completion of the last cycle. At the end of the amplification process, the DNA product was detected using agarose gel electrophoresis as previously described by Dahiya et al. (1997).

Data are expressed as mean 7 S.E.M. (standard error of the mean) and were analyzed using the one-way ANOVA followed by the Tukey–Kramer post analysis test for multiple comparisons. A probability value (P) below 0.05 was considered statistically significant. Statistical analysis was performed using GraphPad Prism 5.

2.6. Real-time quantitative PCR

3. Results

Real-time PCR amplification was carried out using 10 μl amplification mixtures containing Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA), equivalent to 8 ng of reverse-transcribed RNA and 300 nM primers, the sequences of PCR primer pairs used for each gene. Reactions were run on an ABI PRISM 7900 HT detection system (Applied Biosystems). PCR reactions consisted of 95 1C for 10 min (1 cycle), 94 1C for 15 s, and 60 1C for 1 min (40 cycles). Data were analyzed with the ABI Prism sequence detection system software and quantified using the v1  7 Sequence Detection Software from PE Biosystems (Foster City, CA). The relative expression levels of eNOS and angiotensin II type 1 receptor mRNA were calculated by the comparative threshold cycle (Ct) method as previously described (Livak and Schmittgen, 2001). All values were normalized to β-actin gene expression and expressed as gene fold changes.

3.1. General parameters

2.7. Vascular reactivity The analysis of vascular reactivity was performed as described previously (Crews et al., 2000). Briefly, the periadventitial tissue of the descending thoracic aorta was dissected into the Krebs–Henseleit buffer (KHB; 118.4 mmol/l NaCl, 4.7 mmol/l KCl, 2.5 mmol/l CaCl2, 1.2 mmol/l KH2PO4, 1.2 mmol/l MgSO4, 25 mmol/l NaHCO3, and 11.1 mmol/l glucose) and cut transversely into  2.0-mm-long ring segments. The vessel rings were mounted onto two parallel stainless-steel pins through the lumen and placed in a chamber containing a 20 ml KHB buffer (pH 7.4) for isometric tension recording (Panlab, Barcelona, Spain ADInstrument). Vessels were contracted with a highpotassium solution (80 mmol/l KCl) to determine their variability. Thereafter, cumulative concentration–response curves to acetylcholine (ACh; 10
9–0
5 M) were generated after submaximal precontraction of the vessel with phenyephrine.

Formalin fixed aorta embedded in paraffin wax was serially sectioned with a microtome and stained with hematoxylin and eosin, for assessment of histopathological changes. 2.9. Statistical analysis

Injection of STZ produced a marked increase in the blood glucose level above 300 mg/dl and this was associated with a marked reduction in the body weight. The single and the combined administration of losartan and L-carnitine did not produce any significant change in either the blood glucose level or the body weight in diabetic rats. 3.2. Effects of losartan, L-carnitine and their combination on oxidative stress parameters and NO bioavailability STZ-induced diabetic rats showed a marked decrease in the serum antioxidant enzyme capacities of SOD and GSH alongside an increase in the MDA level (Table 1). In addition, the aortic O2
production level as a measure of NADPH oxidase activity was found to be elevated in diabetic rats. However, treatment with either of losartan, L-carnitine or their dual combination significantly normalized the reduced antioxidant enzyme activities and inhibited the observed elevated MDA (Table 1) and O2
levels (Fig. 1A) in the diabetic rats. Notably, effect of the combined administration was more pronounced as compared to the use of either drug alone. Moreover, there was a significant attenuation in nitrite level, as an indicator of NO bioavailability relative to control group. However, treatment with losartan or L-carnitine, solely or concurrently significantly increased the attenuated nitrite level in diabetic rats (Fig. 1B). It is noteworthy that the combination therapy of losartan or L-carnitine significantly revealed a greater response than in their monotherapy. 3.3. Effects of losartan, L-carnitine and their combination on the serum TNF-α level Serum TNF-α level of diabetic rate was significantly (Po0.01) increased compared to control rats. However, the individual and

Table 1 Effects of the single and combined administration of losartan (20 mg/kg, p.o.) and L-carnitine (200 mg/kg, p.o.) on the general and oxidative stress characteristics. Parameter Body weight (g) Blood glucose level (mg/dl) Serum SOD (U/l) Serum GSH (U/l) Serum MDA (nmol/l)

Control 402 721.1 105.2 78.5 287.2 715.8 66.8 75.31 8.3 72.9

Values represent the mean7 S.E.M., n¼ 8 rats/group. a

P o0.01 vs. control group. Po 0.05 vs. STZ-treated group. c Po 0.05 vs. STZþLOS-treated group. d Po 0.05 vs. STZþLC-treated group. b

STZ a

230 7 25 370.37 19a 86.7 7 8.5a 13.7 7 2.5a 29.6 7 4.1a

STZþLOS

STZþLC

STZþ LOSþ LC

2517 15.6 3357 18 201.7 7 17.8b 58.9 7 2.1b 15.5 7 1.9b

264 7 20.3 321.7 7 20.2 171.9 7 16.8b 41.6 7 4.5b 16.17 1.7b

269 7 12.4 317.3 7 21 263.3 7 19.1b,c,d 56.17 2.4b,c,d 11.7 7 2.2b,c,d

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Fig. 1. Effects of the single and combined administration of losartan (20 mg/kg, p.o.) and L-carnitine (200 mg/kg, p.o.) on aortic superoxide anion production and nitric oxide bioavailability. (A) The aortic superoxide anion production in diabetes was increased compared to that the control rats and treatment with either losartan or L-carnitine inhibited this effect. (B) Aortic tissue of diabetic rats exhibited a significant attenuation in nitric oxide production compared to control normal rats. Losartan and L-carnitine treatment significantly increased nitric oxide bioavailability. Values represent the mean 7S.E.M., n¼8 rats; nPo 0.01 vs. control group; #Po 0.01 vs. STZ-treated group; ƒ Po 0.05 vs. STZþ LOS-treated group; δPo 0.01 vs. STZþ LC-treated group.

3.5. Effects of losartan, L-carnitine and their combination on the vascular function

Fig. 2. Effects of the single and combined administration of losartan (20 mg/kg, p.o.) and L-carnitine (200 mg/kg, p.o.) on serum TNF-α level of STZ-induced diabetic rats. TNF-α serum level of diabetic rat was significantly increased as compared to the control rats and losartan, L-carnitine and their combination significantly inhibited these effects. Values represent the mean 7S.E.M., n ¼8 rats; nPo 0.01 vs. control group; #Po 0.01 vs. STZ-treated group; ƒPo 0.05 vs. STZþ LOS-treated group; δPo 0.01 vs. STZþ LC-treated group.

combined administration of losartan and L-carnitine markedly (P o0.05) prevented this change (Fig. 2).

3.4. Effects of losartan, L-carnitine and their combination on eNOS and angiotensin II type 1 receptor gene expression The aortic eNOS gene expression of diabetic rat exhibited a significant (Po0.05) downregulation compared to the control group (Fig. 3A). However, treatment with either losartan or L-carnitine, solely or concurrently, markedly corrected the dysregulated eNOS mRNA expression in the diabetic rats. On the other hand, gene expression of angiotensin II type 1 receptor was found to be upregulated in aortic tissues of STZ-induced as compared to the control group. However, treatment with losartan or L-carnitine as well as their combination revealed a marked reduction in angiotensin II type 1 receptor gene expression in diabetic rat aorta. Of note, the combination therapy exhibited a more profound effect relative to the monotherapy (Fig. 3B). In addition, eNOS and angiotensin II type 1 receptor gene expression levels were quantitatively estimated using the real-time PCR (Fig. 3C and D).

The endothelium-dependent vasodilator ACh elicited the concentration-dependent relaxation of phenylephrine preconstricted isolated aorta of all animals. Aortic rings of diabetic rat exerted a markedly impaired endothelium-dependent relaxation as compared to control (Fig. 4). Notably, the maximum relaxation response (Emax) induced by ACh was significantly reduced in aorta of diabetic rats when compared to the control rats. Both of losartan and L-carnitine significantly enhanced ACh-mediated relaxation in aorta of the diabetic rats. The combination therapy of losartan and L-carnitine markedly revealed a more pronounced response in ACh-mediated relaxation over the use of either drug alone. Measuring the relaxation ability of the rat aorta is determined by inducing maximum contraction by the addition of 1 μmol/l phenylephrine that produce the maximum aorta tissue pretension that represent 100% contraction. 3.6. Histopathological analysis Thoracic aortic media thickness was assessed by image analysis of H&E-stained sections at  400 magnification. Three different areas of each aorta were examined and quantified using NIH image software, and the results were averaged. Histopathological examination of aortic tissues from the control non-diabetic rats showed normal histological structures with no marked alterations in the tunica intima or endothelium. However, aorta of diabetic rats revealed hypertrophy in the tunica media and edema in the tunica adventitia. Diabetic rats treated with losartan and L-carnitine exhibited a reduction in the media thickness and less endothelial changes compared to the control group (Fig. 5).

4. Discussion The present study revealed that the combination therapy with losartan and L-carnitine exhibited additive beneficial responses towards the inflammatory and oxidative stress markers as well as endothelial dysfunction in STZ-induced diabetic rats over the use of either drug alone. In fact, a considerable body of evidence implicates oxidative stress as a critical pathogenic element in diabetic endothelial dysfunction. Excessive generation of O2
in the endothelium by hyperglycemia has been considered as one of the

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eNOS mRNA

AT1-R mRNA

β-actin

β-actin

Fig. 3. Effects of the single and combined administration of losartan (20 mg/kg, p.o.) and L-carnitine (200 mg/kg, p.o.) on eNOS and angiotensin II type 1 receptor (AT1-R) gene expression. (A) Representative gene eNOS bands in the aortic tissue of the control and diabetic rats. Bar graphs indicate results of densitometric analysis of the bands as normalized to the quantity of β-actin gene. Values represent the mean 7 S.E.M. for five separate experiments; nPo 0.01 vs. control group; #P o 0.01 vs. STZ-treated group. (B) Representative AT1-R bands in the aortic tissue of the control and diabetic rats. Bar graphs indicate results of densitometric analysis of the bands as normalized to the quantity of β-actin gene. (C) Quantitative analysis of endothelial nitric oxide synthase (eNOS) gene by real-time PCR expressed in Ct values relative to housekeeping gene. (D) Quantitative analysis of AT1-R gene expressed in Ct values relative to housekeeping gene. Values represent the mean7 S.E.M. for five separate experiments; nP o 0.01 vs. control group; #Po 0.05 vs. STZ-treated group; ƒP o 0.05 vs. STZþLOS-treated group; δP o0.05 vs. STZþ LC-treated group.

Fig. 4. Effects of the single and combined administration of losartan (20 mg/kg, p.o.) and L-carnitine (200 mg/kg, p.o.) on the vascular reactivity. ACh-mediated endothelial-dependent relaxation presented as the percentage of phenylephrine precontraction in endothelium intact thoracic aortic rings from the control and diabetic rats treated orally with losartan or L-carnitine. Values represent the mean 7S.E.M., n¼ 8 rats for each group; nPo 0.01 vs. control group; #Po 0.05 vs. STZ-treated group; ƒP o0.05 vs. STZþLOS-treated group; δPo 0.01 vs. STZþ LCtreated group.

major factors profoundly involved in accelerating vascular complications (Matsumoto et al., 2010a). Recently, we have demonstrated that the hyperglycemia caused an imbalance between the production of free radicals and the antioxidant defense system in diabetic cardiomyopathy (Taye et al., 2013). However, the precise

mechanism(s) by which the oxidative stress may accelerate the development of vascular complications in diabetes is only partly known. Herein, we observed that the diabetic aorta exhibited a significant increase in the O2
production and MDA level along with defective antioxidant enzyme activities compared to the control. The aforementioned increased O2
generation in aorta of diabetic rats may account for the reduction in NO bioavailability alongside downregulation of eNOS mRNA and angiotensin II type 1 receptor mRNA expression. In this context, it has been previously reported that oxidative stress is a major cause of reduced endothelial NO bioavailability in diabetes (Landmesser et al., 2003). It has been reported that drugs interfering with the renin– angiotensin–aldosterone system enhances eNOS phosphorylation and improves NO bioavailability (Imanishi et al., 2008). In the current study, aorta of diabetic rats exhibited enhanced O2
generation and this was associated with decreased eNOS expression. However, treatment with either losartan or L-carnitine markedly enhanced NO availability via increasing eNOS expression levels. Likewise, it has been reported that angiotensin II type 1 receptor antagonism improved NO production and enhanced eNOS in hearts from a rat model of insulin resistance (Huisamen et al., 2011).

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Fig. 5. A panel of micrographs of the thoracic aortic sections in the control group, diabetic group or diabetic rats treated with a single or combined administration of losartan (20 mg/kg, p.o.) and L-carnitine (200 mg/kg, p.o.). i) Representative images of aortas stained with hematoxylin and eosin (H&E) to evaluate and measure aortic media thickness. (A) Representative image of the control non-diabetic rat aorta showing a normal ascending aortic wall thickness (arrows) and no histological changes in tunica intima or endothelium, tunica media and tunica adventitia. (B) The wall thickening is apparent in micrograph of the rat diabetic aorta (arrows), in addition to the presence of inflammatory cells compared to the control non-diabetic rats. (C) Micrograph of aortic tissue of diabetic rats treated with losartan revealed a significant decrease in the medial thickness (arrows) relative to the diabetic aorta. (D) Micrograph of aortic tissue of diabetic rats treated with L-carnitine showed a partial reduction in the aortic wall thickness (arrows) compared to that of STZ-treated rats. (E) Representative images illustrate that the aorta of diabetic rats treated with losartan þ L-carnitine has quite a reduced aortic wall thickness (arrows) as compared to control. Sections were stained with haematoxylin and eosin, original magnification is  400. Scale bar, 50 μm. ii) Calculation of media thickness. Values represent average aortic wall thickness7S.E.M., n¼ 5 rats for each group; nPo 0.01 vs. control group; #Po 0.05 vs. STZ-treated group; ƒPo 0.05 vs. STZþLOS-treated group; δP o 0.01 vs. STZþ LC-treated group.

Recently, L-carnitine has been reported to inhibit oxidative stress in STZ-induced diabetic rats (ElGendy and Abbas, 2014) supporting its crucial role as antioxidant. On the other hand, TNF-α upregulation has been shown to activate the NADPH oxidase with subsequent increased O2
generation, thereby contributing to endothelial dysfunction in type 2 diabetes (Gao et al., 2007). Moreover, a quite recent report showed that the new angiotensin II receptor blocker, azilsartan, was able to restore endothelial function by normalizing eNOS function and reducing inflammation and oxidative stress in diabetic mice (Matsumoto et al., 2014). Strategies to reduce oxidative stress in diabetes mellitus may exert favorable effects on its progression. Going back to our results, the observed elevated TNF-α serum level in STZ induced diabetic tended to be increased in monotherapy treatment with either losartan or L-carnitine and its level was profoundly reduced in the their combination therapy. Thus, it is tempting to postulate that the ability of the combined therapy of losartan and L-carnitine to preserve eNOS expression and NO bioavailability is probably mediated through more potent inhibition of TNF-α. Due to the increasing amount of evidence linking endothelial dysfunction to the onset and progression of cardiovascular complication of diabetes (Hamilton and Watts, 2013; Liao et al., 2013), it is important to have a method of testing substances that may have potential to protect endothelial function. Endothelial dysfunction, as indicated by impaired endothelium-dependent, reflects the inability of the vascular endothelium to generate adequate amounts of NO and to produce NO-mediated relaxation, which has been suggested

to be an early event in diabetic atherosclerosis and is associated with coronary artery disease risk (Creager et al., 2003). In the current study, aorta of diabetic rat exerted a marked reduction in NO-dependent vasorelaxation in response to ACh, which is in a harmony with studies of large and resistance arteries of STZ diabetic animals (Chakraphan et al., 2005). Previous studies have investigated effects of L-carnitine administration on cardiovascular diseases, including hypertension-related complications (Bueno et al., 2005; de Sotomayor et al., 2007; O’Brien et al., 2010). In addition, losartan also improved the endothelial function in various vessels in subjects with type 2 diabetes (Cheetham et al., 2001; Matsumoto et al., 2010a, 2010b). Herein, we showed that the single or combinatory administration of L-carnitine and losartan significantly ameliorated ACh-mediated vasorelaxation and this was associated with downregulation of angiotensin II type 1 receptor gene expression. Importantly, effect of the combined therapy of losartan and L-carnitine exhibited a more pronounced over the use of either drug alone. In the light of these findings, it is convincible to speculate that, at least, some of the beneficial effects achieved by losartan and L-carnitine are mediated through an action of the angiotensin II type 1 receptor. In this context, the angioprotective effects of L-carnitine in diabetes- associated endothelial dysfunction have been already documented (Bueno et al., 2005; de Sotomayor et al., 2007). Several reports have indicated that TNF-α-induced O2
production may play a crucial role in ventricular remodeling (Moe et al., 2011) and aortic smooth muscle cells hypertrophy (Goetze et al., 2001).

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Notably, the diabetic rat aortic exhibited hypertrophy of tunica media and edema in the tunica adventitia and the combined therapy of losartan and L-carnitine could inhibit these abnormalities, indicating an essential role of TNF-α and O2
-mediated medial hypertrophy in aorta of diabetic rats. Since the single or combined administration of losartan and L-carnitine could not produce any significant change on the body weight or the blood glucose level of diabetic rats, our data suggest that STZ-induced diabetes may elicit endothelial dysfunction properly through the enhanced oxidative stress. Therefore, it is quite possible to speculate that the correction of diabetes-associated endothelial dysfunction is not related to their effects on blood glucose levels per se but could instead be a consequence of combating oxidative stress and inflammatory responses. A plausible explanation for this assumption is that the combination therapy of losartan with L-carnitine resulted in normalizing eNOS function and downregulation of angiotensin-II type 1 receptor through inhibition of TNF-αinduced O2
production, and thereby ameliorating the vascular function. Our findings support a previous study demonstrating that TNF-α increased angiotensin II type 1 receptor gene expression in rats given TNF-α, which led to upregulation of the NADPH oxidase-driven O2
formation (Mariappan et al., 2012). In conclusion, our results demonstrate that the combination therapy with losartan and L-carnitine could exhibit additive beneficial effects against diabetes-associated endothelial dysfunction in rat model, presumably via correcting the dysregulated eNOS expression and reducing the inflammation and oxidative status. Nonetheless, further studies will have to investigate whether this form of pharmacological intervention can be passed into the human pathophysiology of diabetes.

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