J Physiol Biochem (2014) 70:247–254 DOI 10.1007/s13105-013-0299-7
ORIGINAL PAPER
Propolis protects against high glucose-induced vascular endothelial dysfunction in isolated rat aorta Mohammed S. El-Awady & Dina S. El-Agamy & Ghada M. Suddek & Manar A. Nader
Received: 30 June 2013 / Accepted: 29 October 2013 / Published online: 15 November 2013 # University of Navarra 2013
Abstract While propolis is known to have abundant bioactive constituents and a variety of biological activities, it is not clear whether propolis has beneficial effects on high glucose-mediated vascular endothelial impairment. The aim of the present study was to investigate the potential protective effect of propolis extract against the acute vascular endothelial dysfunction resulting from exposure to high glucose load and to elucidate its underlying mechanism. Rat aortic rings were incubated with normal glucose (11 mM), high glucose (44 mM), or mannitol (44 mM) for 3 h with or without propolis extract (400 μg/ml). Contraction to phenylephrine (Phe, 10−9–10−5 M) and relaxation to acetylcholine (ACh, 10−9–10−5 M) and sodium nitroprusside (SNP, 10−9–10−5 M) were measured before and after incubation. Changes in malondialdehyde (MDA), reduced glutathione (GSH), and superoxide dismutase (SOD) were also measured. Phe-induced contraction was impaired by high glucose as the Emax decreased from 138.87±11.43 to 103.65±11.5 %. In addition, ACh-induced relaxation was impaired as the Emax decreased from 99.80 ± 7.25 to 39.20 ± 6.5 %. SNPinduced relaxation was not affected. Furthermore, high glucose decreased the levels of both SOD (by 6 U/ml) and GSH (by 68 %) and increased levels of MDA (by 85 %). Propolis extract prevented high glucose-induced M. S. El-Awady : D. S. El-Agamy : G. M. Suddek (*) : M. A. Nader Department of Pharmacology and Toxicology, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt e-mail: [email protected]
impairment of Phe and ACh responses and increased both SOD and GSH, leading to decreased MDA levels. In conclusion, propolis can protect against high glucoseinduced vascular dysfunction by reducing oxidative stress. Keywords Propolis . High glucose . Rat aorta . Vascular endothelial dysfunction . Oxidative stress
Introduction Diabetes is a metabolic disorder characterized by hyperglycemia followed by micro- and macrovascular complications [7, 22]. Of the several metabolic disorders in diabetes, hyperglycemia has been suggested to be the main factor responsible for vascular dysfunctions [7, 25]. Both impairment of vascular contraction and endothelial dysfunction have been reported in diabetes. It has been shown that phenylephrine (Phe)-induced contraction was either enhanced [2] or impaired [30] in streptozotocin-induced diabetic rats. Prolonged exposure to high glucose in vitro or in vivo has been reported to inhibit acetylcholine (ACh)-induced endotheliumdependent relaxation [15, 23, 27] and to generate reactive oxygen species [10, 13]. Oxidative stress induced by hyperglycemia plays a key role in the pathogenesis of vascular complications [9, 32]. Propolis is a resinous material that honeybees collect from different plants and mix with wax and other secretions [24]. The chemical compositions of propolis are phenolic acids, terpenes, lignans, cinnamic acid, caffeic
248
acid, several esters, and flavonoid [17, 34]. Propolis has been used as a folk medicine as an antioxidant, antimicrobial, anti-inflammatory, and hepatoprotective, and in cancer treatment [3–5]. In addition, propolis has been shown to control metabolic disorders and production of free radicals in diabetic rats [14] and accelerates the tissue regeneration and repair of damaged pancreatic cell [8]. However, its effect on vascular complications induced by hyperglycemia is still unknown. The present study examined the effect of propolis against vascular endothelial dysfunction and oxidative stress induced by exposure of isolated rat aortic rings to high glucose. Our results indicate that the exposure of isolated rat aorta to high glucose decreased antioxidant parameters and impaired vascular contraction and endothelium-dependent relaxation, with no effect on endothelium-independent relaxation. Propolis extract prevented high glucose-induced dysfunctions in vascular contractility and endothelium-dependent relaxation and increased the levels of antioxidant parameters in aortic rings.
Material and methods Animals Male Sprague–Dawley rats weighing 150–200 g, purchased from the Nephrology and Urology Center, Mansoura University, Egypt, were used. The experiments were conducted in accordance with the ethical guidelines for investigations in laboratory animals and were approved by the Ethical Committee of Faculty of Pharmacy, Mansoura University, Egypt. Drugs and chemicals Phe, ACh, sodium nitroprusside (SNP), Ellman's reagent, thiobarbituric acid, trichloroacetic acid, 1,1,3,3tetramethoxypropane, pyrogallol, diethylenetriamine penta-acetic acid, tris-cacodylic acid, and catalase were obtained from Sigma Chemical Co. (Saint Louis, MO, USA). Chinese propolis powder was purchased from Dalion Garo International Trade Co., Ltd., China. Preparation of ethanolic extract of propolis [1, 17] One gram of propolis powder was added to 25 ml of 95 % ethanol and allowed to mix on a magnetic mixture
M.S. El-Awady et al.
for 24 h at room temperature (37 °C), and then, the obtained solution was filtered with a Whatman no. 1 filter paper. The filtrate was then adjusted to 25 ml by adding 80 % ethanol and stored in amber-colored bottles. Preparation of aortic rings Rats were humanely killed by cervical dislocation, and the descending thoracic aorta was quickly separated and placed in a cold oxygenated physiological salt solution (PSS) of the following composition (mmol/l): NaCl, 118; CaCl 2 , 2.5; KCl, 4.7; MgSO 4 ·7H 2 O, 1.2; KH2PO4, 1.2; NaHCO3, 25; and glucose, 11.1 (pH 7.4). The vessels were dissected free of connective tissue and fat and then cut into 2–4-mm rings. In vitro vascular reactivity Aortic rings were mounted between two stainless steel hooks in an organ bath filled with 18 ml of PSS at 37 °C and bubbled with a mixture of 95 % O2 and 5 % CO2. Rings were allowed to equilibrate under 1.5 g resting tension for 60 min, and during that time, the bath solution was replaced every 15 min. Isometric tension was measured using a force displacement transducer (K30, Hugosachs Elektronik, March, Germany) and recorded with the PowerLab unit linked to the PC running Chart v4.2 software (ADInstruments Pty Ltd., Australia). After the equilibration period, the responsiveness of arterial ring was assessed by measuring contraction to KCl (80 mM). Cumulative concentration–response curves (CRCs) were then constructed to Phe (10−9–10−5 M). To measure vasorelaxation, rings were first preconstricted with 1 μM Phe, and after reaching a steady state contraction (plateau), cumulative CRCs to ACh (10−9–10−5 M) or to SNP (10−9–10−5 M) were constructed. Rings were then incubated for 3 h in PSS containing either normal glucose (11 mM), high glucose (44 mM) [29], or mannitol (44 mM), and vascular contraction and relaxations were assessed again after the incubation period. In some experiments, endothelium was removed mechanically by gentle rubbing of endothelium along the long axis of the vessel, and the absence of endothelium was tested by measuring relaxation to ACh (10−6 M). Preparations that demonstrated greater than 50 % relaxation were considered to be endothelium intact, while those that did not relax to ACh were
Propolis prevents high glucose-induced vascular dysfunction
considered to be endothelium denuded. To examine the effect of propolis, propolis extract (100, 200, and 400 μg/ml) was added 30 min before glucose addition and kept during 3 h of incubations. Preparation of aortic homogenate The second set of experiments were undertaken to investigate the effect of propolis extract on high glucoseinduced changes in malondialdehyde (MDA) concentration, superoxide dismutase (SOD) activity, and reduced glutathione (GSH) concentration in rat aortic rings. Aortic rings were isolated and incubated for 3 h in normal glucose, high glucose, or propolis extract (400 μg/ml) plus high glucose as mentioned previously. Aortic tissues were collected after incubation, and a 5 % w/v tissue homogenate was made in ice-cold 0.9 % NaCl solution using mini handheld homogenizer (Omni International, USA). Tissue homogenates were centrifuged (1,000 g, 4 °C, 10 min), and supernatants were collected to measure the biochemical parameters. Determination of lipid peroxidation As described by Wang et al. [35], lipid peroxidation was determined by measuring the MDA concentration (thiobarbituric acid-reactive substances (TBARS)) in the aorta homogenate. Briefly, 1.0 ml of 20 % trichloroacetic acid and 1.0 ml of 1 % TBARS reagent were added to 100 μl supernatant and then mixed and incubated for 80 min at 100 °C. After cooling, samples were centrifuged for 20 min at 1,000 g, and the absorbance was read at 532 nm. TBARS results were expressed as MDA equivalents using tetramethoxypropane as a standard. Determination of reduced GSH The content of GSH in the aorta was assayed colormetrically, based on its reaction with Ellman's reagent according to the method described earlier by Ellman [12]. The absorbance was measured at 412 nm, and the concentrations were expressed as micromoles per milliliter. Determination of SOD As described by Li et al. [20], SOD activity in aortic homogenates was measured spectrophotometrically by
249
monitoring the SOD-induced inhibition of pyrogallol auto-oxidation. The reaction mixture (4.5 ml) consisted of 0.2 mmol/l of pyrogallol, 1 mmol/l of diethylenetriamine penta-acetic acid, 50 mmol/l of tris-cacodylic acid buffer (pH 8.2), and 4 μg of catalase. The rate of increase in absorbance was recorded at 420 nm. One unit of SOD activity is defined as the amount of the enzyme causing 50 % inhibition of auto-oxidation of pyrogallol. The SOD activity was expressed as units per milliliter aortic homogenates. Data analysis Data are expressed as mean ± standard error of mean (SEM). Contraction was calculated as percentage of maximal contraction induced by 80 mM KCl, while rela xation wa s calcu lated a s p erce ntag e of preconstriction induced by 1 μM Phe. Emax is the highest response obtained. Nonlinear regression analysis was carried out using the GraphPad Prism software (GraphPad Software, Inc., San Diego, USA). Significant differences between groups were determined using paired Student's t test (for E max), one-way ANOVA with Tukey–Kramer's multiple comparisons post hoc test (for oxidative stress parameters), or twoway ANOVA followed by Bonferroni posttest (for concentration–response curves).
Results Constituents of ethanolic extract of propolis Ethanolic extract of propolis contains the following constituents (chrysin, 2.3 %; galangin, 1.75 %; caffeic acid phenethyl ester, 1.08 %; pinocembrin, 8.2 %; kaempferol, 3.5). Propolis effect on high glucose-induced changes in vasoconstriction to Phe Incubation of rat aortic rings with PSS containing normal glucose for 3 h had no effect on Phe-induced contraction (Fig. 1a). Conversely, incubation of the aortic rings with high glucose (44 mM) for 3 h produced a significant reduction in Phe-induced contraction as manifested by the decrease in Emax (from 138.87±11.43 to 103.65 ± 11.5 %) when value after incubation was
250
M.S. El-Awady et al.
compared to their corresponding value before incubations (Fig. 1b). Similar to the control group, incubation with mannitol (44 mM) had no effect on contraction (data not shown), thus excluding the effect of increased osmolarity of high glucose on Phe-induced contraction. The role of endothelium was tested by incubation of endothelium-denuded rings with high glucose. The impaired contraction to Phe induced by acute exposure to high glucose was abolished by removal of endothelium (Fig. 1c), indicating that high glucose-induced effects are endothelium dependent. Coincubation of aortic rings with propolis extract (400 μg/ml) significantly prevented the high glucoseinduced attenuation of Phe-induced contraction (Fig. 1d). Lower concentrations of propolis extract
(100 and 200 μg/ml) did not significantly prevent high glucose-induced reduction of contraction to Phe (data not shown).
Fig. 1 Propolis prevents high glucose-induced impairment of aortic contraction to Phe. Rat aortic contraction to Phe (10−9– 10−5 M) was measured before and after 3 h of incubation with PSS containing a normal glucose (11 mM), b high glucose (44 mM), c high glucose in the absence of endothelium, and d
high glucose in the presence of propolis extract (400 μg/ml). Data are expressed as means ± SEM, n=5. ***p
ORIGINAL PAPER
Propolis protects against high glucose-induced vascular endothelial dysfunction in isolated rat aorta Mohammed S. El-Awady & Dina S. El-Agamy & Ghada M. Suddek & Manar A. Nader
Received: 30 June 2013 / Accepted: 29 October 2013 / Published online: 15 November 2013 # University of Navarra 2013
Abstract While propolis is known to have abundant bioactive constituents and a variety of biological activities, it is not clear whether propolis has beneficial effects on high glucose-mediated vascular endothelial impairment. The aim of the present study was to investigate the potential protective effect of propolis extract against the acute vascular endothelial dysfunction resulting from exposure to high glucose load and to elucidate its underlying mechanism. Rat aortic rings were incubated with normal glucose (11 mM), high glucose (44 mM), or mannitol (44 mM) for 3 h with or without propolis extract (400 μg/ml). Contraction to phenylephrine (Phe, 10−9–10−5 M) and relaxation to acetylcholine (ACh, 10−9–10−5 M) and sodium nitroprusside (SNP, 10−9–10−5 M) were measured before and after incubation. Changes in malondialdehyde (MDA), reduced glutathione (GSH), and superoxide dismutase (SOD) were also measured. Phe-induced contraction was impaired by high glucose as the Emax decreased from 138.87±11.43 to 103.65±11.5 %. In addition, ACh-induced relaxation was impaired as the Emax decreased from 99.80 ± 7.25 to 39.20 ± 6.5 %. SNPinduced relaxation was not affected. Furthermore, high glucose decreased the levels of both SOD (by 6 U/ml) and GSH (by 68 %) and increased levels of MDA (by 85 %). Propolis extract prevented high glucose-induced M. S. El-Awady : D. S. El-Agamy : G. M. Suddek (*) : M. A. Nader Department of Pharmacology and Toxicology, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt e-mail: [email protected]
impairment of Phe and ACh responses and increased both SOD and GSH, leading to decreased MDA levels. In conclusion, propolis can protect against high glucoseinduced vascular dysfunction by reducing oxidative stress. Keywords Propolis . High glucose . Rat aorta . Vascular endothelial dysfunction . Oxidative stress
Introduction Diabetes is a metabolic disorder characterized by hyperglycemia followed by micro- and macrovascular complications [7, 22]. Of the several metabolic disorders in diabetes, hyperglycemia has been suggested to be the main factor responsible for vascular dysfunctions [7, 25]. Both impairment of vascular contraction and endothelial dysfunction have been reported in diabetes. It has been shown that phenylephrine (Phe)-induced contraction was either enhanced [2] or impaired [30] in streptozotocin-induced diabetic rats. Prolonged exposure to high glucose in vitro or in vivo has been reported to inhibit acetylcholine (ACh)-induced endotheliumdependent relaxation [15, 23, 27] and to generate reactive oxygen species [10, 13]. Oxidative stress induced by hyperglycemia plays a key role in the pathogenesis of vascular complications [9, 32]. Propolis is a resinous material that honeybees collect from different plants and mix with wax and other secretions [24]. The chemical compositions of propolis are phenolic acids, terpenes, lignans, cinnamic acid, caffeic
248
acid, several esters, and flavonoid [17, 34]. Propolis has been used as a folk medicine as an antioxidant, antimicrobial, anti-inflammatory, and hepatoprotective, and in cancer treatment [3–5]. In addition, propolis has been shown to control metabolic disorders and production of free radicals in diabetic rats [14] and accelerates the tissue regeneration and repair of damaged pancreatic cell [8]. However, its effect on vascular complications induced by hyperglycemia is still unknown. The present study examined the effect of propolis against vascular endothelial dysfunction and oxidative stress induced by exposure of isolated rat aortic rings to high glucose. Our results indicate that the exposure of isolated rat aorta to high glucose decreased antioxidant parameters and impaired vascular contraction and endothelium-dependent relaxation, with no effect on endothelium-independent relaxation. Propolis extract prevented high glucose-induced dysfunctions in vascular contractility and endothelium-dependent relaxation and increased the levels of antioxidant parameters in aortic rings.
Material and methods Animals Male Sprague–Dawley rats weighing 150–200 g, purchased from the Nephrology and Urology Center, Mansoura University, Egypt, were used. The experiments were conducted in accordance with the ethical guidelines for investigations in laboratory animals and were approved by the Ethical Committee of Faculty of Pharmacy, Mansoura University, Egypt. Drugs and chemicals Phe, ACh, sodium nitroprusside (SNP), Ellman's reagent, thiobarbituric acid, trichloroacetic acid, 1,1,3,3tetramethoxypropane, pyrogallol, diethylenetriamine penta-acetic acid, tris-cacodylic acid, and catalase were obtained from Sigma Chemical Co. (Saint Louis, MO, USA). Chinese propolis powder was purchased from Dalion Garo International Trade Co., Ltd., China. Preparation of ethanolic extract of propolis [1, 17] One gram of propolis powder was added to 25 ml of 95 % ethanol and allowed to mix on a magnetic mixture
M.S. El-Awady et al.
for 24 h at room temperature (37 °C), and then, the obtained solution was filtered with a Whatman no. 1 filter paper. The filtrate was then adjusted to 25 ml by adding 80 % ethanol and stored in amber-colored bottles. Preparation of aortic rings Rats were humanely killed by cervical dislocation, and the descending thoracic aorta was quickly separated and placed in a cold oxygenated physiological salt solution (PSS) of the following composition (mmol/l): NaCl, 118; CaCl 2 , 2.5; KCl, 4.7; MgSO 4 ·7H 2 O, 1.2; KH2PO4, 1.2; NaHCO3, 25; and glucose, 11.1 (pH 7.4). The vessels were dissected free of connective tissue and fat and then cut into 2–4-mm rings. In vitro vascular reactivity Aortic rings were mounted between two stainless steel hooks in an organ bath filled with 18 ml of PSS at 37 °C and bubbled with a mixture of 95 % O2 and 5 % CO2. Rings were allowed to equilibrate under 1.5 g resting tension for 60 min, and during that time, the bath solution was replaced every 15 min. Isometric tension was measured using a force displacement transducer (K30, Hugosachs Elektronik, March, Germany) and recorded with the PowerLab unit linked to the PC running Chart v4.2 software (ADInstruments Pty Ltd., Australia). After the equilibration period, the responsiveness of arterial ring was assessed by measuring contraction to KCl (80 mM). Cumulative concentration–response curves (CRCs) were then constructed to Phe (10−9–10−5 M). To measure vasorelaxation, rings were first preconstricted with 1 μM Phe, and after reaching a steady state contraction (plateau), cumulative CRCs to ACh (10−9–10−5 M) or to SNP (10−9–10−5 M) were constructed. Rings were then incubated for 3 h in PSS containing either normal glucose (11 mM), high glucose (44 mM) [29], or mannitol (44 mM), and vascular contraction and relaxations were assessed again after the incubation period. In some experiments, endothelium was removed mechanically by gentle rubbing of endothelium along the long axis of the vessel, and the absence of endothelium was tested by measuring relaxation to ACh (10−6 M). Preparations that demonstrated greater than 50 % relaxation were considered to be endothelium intact, while those that did not relax to ACh were
Propolis prevents high glucose-induced vascular dysfunction
considered to be endothelium denuded. To examine the effect of propolis, propolis extract (100, 200, and 400 μg/ml) was added 30 min before glucose addition and kept during 3 h of incubations. Preparation of aortic homogenate The second set of experiments were undertaken to investigate the effect of propolis extract on high glucoseinduced changes in malondialdehyde (MDA) concentration, superoxide dismutase (SOD) activity, and reduced glutathione (GSH) concentration in rat aortic rings. Aortic rings were isolated and incubated for 3 h in normal glucose, high glucose, or propolis extract (400 μg/ml) plus high glucose as mentioned previously. Aortic tissues were collected after incubation, and a 5 % w/v tissue homogenate was made in ice-cold 0.9 % NaCl solution using mini handheld homogenizer (Omni International, USA). Tissue homogenates were centrifuged (1,000 g, 4 °C, 10 min), and supernatants were collected to measure the biochemical parameters. Determination of lipid peroxidation As described by Wang et al. [35], lipid peroxidation was determined by measuring the MDA concentration (thiobarbituric acid-reactive substances (TBARS)) in the aorta homogenate. Briefly, 1.0 ml of 20 % trichloroacetic acid and 1.0 ml of 1 % TBARS reagent were added to 100 μl supernatant and then mixed and incubated for 80 min at 100 °C. After cooling, samples were centrifuged for 20 min at 1,000 g, and the absorbance was read at 532 nm. TBARS results were expressed as MDA equivalents using tetramethoxypropane as a standard. Determination of reduced GSH The content of GSH in the aorta was assayed colormetrically, based on its reaction with Ellman's reagent according to the method described earlier by Ellman [12]. The absorbance was measured at 412 nm, and the concentrations were expressed as micromoles per milliliter. Determination of SOD As described by Li et al. [20], SOD activity in aortic homogenates was measured spectrophotometrically by
249
monitoring the SOD-induced inhibition of pyrogallol auto-oxidation. The reaction mixture (4.5 ml) consisted of 0.2 mmol/l of pyrogallol, 1 mmol/l of diethylenetriamine penta-acetic acid, 50 mmol/l of tris-cacodylic acid buffer (pH 8.2), and 4 μg of catalase. The rate of increase in absorbance was recorded at 420 nm. One unit of SOD activity is defined as the amount of the enzyme causing 50 % inhibition of auto-oxidation of pyrogallol. The SOD activity was expressed as units per milliliter aortic homogenates. Data analysis Data are expressed as mean ± standard error of mean (SEM). Contraction was calculated as percentage of maximal contraction induced by 80 mM KCl, while rela xation wa s calcu lated a s p erce ntag e of preconstriction induced by 1 μM Phe. Emax is the highest response obtained. Nonlinear regression analysis was carried out using the GraphPad Prism software (GraphPad Software, Inc., San Diego, USA). Significant differences between groups were determined using paired Student's t test (for E max), one-way ANOVA with Tukey–Kramer's multiple comparisons post hoc test (for oxidative stress parameters), or twoway ANOVA followed by Bonferroni posttest (for concentration–response curves).
Results Constituents of ethanolic extract of propolis Ethanolic extract of propolis contains the following constituents (chrysin, 2.3 %; galangin, 1.75 %; caffeic acid phenethyl ester, 1.08 %; pinocembrin, 8.2 %; kaempferol, 3.5). Propolis effect on high glucose-induced changes in vasoconstriction to Phe Incubation of rat aortic rings with PSS containing normal glucose for 3 h had no effect on Phe-induced contraction (Fig. 1a). Conversely, incubation of the aortic rings with high glucose (44 mM) for 3 h produced a significant reduction in Phe-induced contraction as manifested by the decrease in Emax (from 138.87±11.43 to 103.65 ± 11.5 %) when value after incubation was
250
M.S. El-Awady et al.
compared to their corresponding value before incubations (Fig. 1b). Similar to the control group, incubation with mannitol (44 mM) had no effect on contraction (data not shown), thus excluding the effect of increased osmolarity of high glucose on Phe-induced contraction. The role of endothelium was tested by incubation of endothelium-denuded rings with high glucose. The impaired contraction to Phe induced by acute exposure to high glucose was abolished by removal of endothelium (Fig. 1c), indicating that high glucose-induced effects are endothelium dependent. Coincubation of aortic rings with propolis extract (400 μg/ml) significantly prevented the high glucoseinduced attenuation of Phe-induced contraction (Fig. 1d). Lower concentrations of propolis extract
(100 and 200 μg/ml) did not significantly prevent high glucose-induced reduction of contraction to Phe (data not shown).
Fig. 1 Propolis prevents high glucose-induced impairment of aortic contraction to Phe. Rat aortic contraction to Phe (10−9– 10−5 M) was measured before and after 3 h of incubation with PSS containing a normal glucose (11 mM), b high glucose (44 mM), c high glucose in the absence of endothelium, and d
high glucose in the presence of propolis extract (400 μg/ml). Data are expressed as means ± SEM, n=5. ***p
