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Antioxidative Propolis From Stingless Bees (Heterotrigona Itama) Preserves Endothelium-Dependent Aortic Relaxation of Diabetic Rats: The Role of Nitric Oxide and Cyclic Guanosine Monophosphate

Abstract

Propolis from stingless bees (Heterotrigona itama) is a resinous compound that exhibits antihyperglycaemia, free radical scavenging, and cardioprotective properties. The effect of propolis on diabetic vessels has not been investigated. Thus, this research aimed to determine the effect of propolis supplementation on the level of antioxidants and its mechanism of action in the aorta of diabetic rats. Male Sprague-Dawley rats were divided into five groups (n=8/group): healthy (control), untreated diabetes (DM), metformin-treated diabetes (DM+M, 300 mg/kg/day metformin), propolis-treated diabetes (DM+P, 300 mg/kg/day propolis extract) and diabetes with combined treatment (DM+M+P, dosage as former). Oral supplementation was conducted for four weeks immediately upon successful induction of diabetes by streptozotocin (60 mg/kg, intraperitoneal injection). At the end of the study, the rats were euthanised, and thoracic aorta was processed into tissue homogenates to determine the levels of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase-1 (GPx-1) and soluble receptor for advanced glycation end-products (sRAGE). Aorta segments were harvested to examine their relaxation response towards graded concentration of acetylcholine (Ach; 10-8-10-4) M following precontraction with phenylephrine (PE; 10-6 M). Vasorelaxation towards a cumulative dose of propolis (0.01-1.00%) using PE-precontracted healthy aorta (n=6/experiments) was investigated under various simulated conditions: physiological buffer, L-NAME (10-4 M), methylene blue (10-5 M), indomethacin (10-5 M) and elevated glucose (25 mM). Propolis maintained antioxidative enzymes and sRAGE decoy molecules in the aortic tissue of the diabetic rats. The amelioration of diabetes-induced impairment of endothelium-dependent relaxation by propolis was mediated through the nitric oxide(NO)-cyclic guanosine monophosphate (cGMP) pathway. This non-clinical study reports vasoprotective property of propolis in diabetes mellitus.

Keywords:
Propolis; Diabetes Mellitus; Antioxidants; Endothelium-Dependent Relaxation; Nitric Oxide; Cyclic Guanosine Monophosphate


INTRODUCTION

Diabetes mellitus is an endocrine abnormality that perpetuates metabolic derangement and heightens the risk of atherosclerotic cardiovascular disease (Wannamethee et al., 2011Wannamethee SG, Shaper AG, Whincup PH, Lennon L, Sattar N. Impact of diabetes on cardiovascular disease risk and all-cause mortality in older men: influence of age at onset, diabetes duration, and established and novel risk factors. Arch Intern Med. 2011;171(5):404-410.). Chronic hyperglycaemia in diabetes mellitus impairs mitochondrial function through accelerated production of free radicals in blood vessels. Sequentially, vascular oxidative stress overwhelms endogenous antioxidative defence and reduces the activity of endothelial NO synthase (Pitocco et al., 2013Pitocco D, Tesauro M, Alessandro R, Ghirlanda G, Cardillo C. Oxidative stress in diabetes: implications for vascular and other complications. Int J Mol Sci. 2013 Oct 30;14(11): 21525-50.). Reduced NO production is linked to the pro-inflammatory and proatherogenic states of vascular dysfunction in diabetes mellitus. Therefore, the search for an agent that hampers this pathophysiological cascade will provide a therapeutic opportunity for diabetic vascular disease (Förstermann, 2010Förstermann U. Nitric oxide and oxidative stress in vascular disease. Pflugers Arch - Eur J Physiol. 2010;459: 923.).

Propolis is a sticky resin that forms a protective barrier in stingless bees hive. Propolis produced by H. itama shows anti-hyperglycaemic (Usman et al., 2017Usman UZ, Bakar AB, Zin AA, Mohamed M. LC-MS analysis and effects of Malaysian propolis on insulin, glucagon, pancreas and oxidative stress status in streptozotocin-induced diabetic rats. J Med Biomed Res. 2017;16(1):15-27.), free radical scavenging (Akhir, Bakar, Sanusi, 2017Akhir RA, Bakar MF, Sanusi SB. Antioxidant and antimicrobial activity of stingless bee bread and propolis extracts. AIP Conf. Proc. 2017 Oct 3 (Vol. 1891, No. 1, p. 020090). AIP Publishing.) and cardioprotective (Ahmed et al., 2017Ahmed R, Tanvir EM, Hossen M, Afroz R, Ahmmed I, Rumpa NE, et al. Antioxidant properties and cardioprotective mechanism of Malaysian propolis in rats. Evid Based Complement Alternat Med. 2017;2017.) properties. These reported bioactivities address the pathogenesis of diabetic angiopathy and lead to the hypothesis that propolis protects against vascular complication and potential multi-target therapeutics in diabetes mellitus. However, there is a lack of data on the effect of H. itama propolis on vasculature affected by diabetes mellitus. Therefore, this study aims to determine the level of antioxidants and endothelialdependent relaxation in the aorta of diabetic rats with propolis supplementation and its mechanism of action.

MATERIAL AND METHODS

Extraction of propolis

Extraction was performed in accord with the published protocol (Usman et al., 2017Usman UZ, Bakar AB, Zin AA, Mohamed M. LC-MS analysis and effects of Malaysian propolis on insulin, glucagon, pancreas and oxidative stress status in streptozotocin-induced diabetic rats. J Med Biomed Res. 2017;16(1):15-27.). Raw propolis (H. itama) was obtained from Kelantan, Malaysia (6.090432, 102.291131). Then, it was cleaned and grounded before being dissolved into 70% ethanol (100 mL/30 g). The resultant solution was filtered sequentially, and the filtrate was concentrated in a rotatory evaporator at 60 ºC. The ethanolic extract of propolis was collected, lyophilised and stored at -20 ºC for experimental use.

Research animals and induction of diabetes

The use of adult male Sprague Dawley rats aged 10-12 weeks old was approved by the Universiti Sains Malaysia (USM) Institutional Animal Care and Use Committee (Approval Number: USM/IACUC/2018/[112)] [922]). The experiment was conducted in accordance with the Guide for the Care and Use of Laboratory Animals. Intraperitoneal streptozotocin (60 mg/kg) was used to induce diabetes, and 1mL normal saline (vehicle) was injected to the rats in the non-diabetic groups. The animals were assessed after 72 hours post-induction for the attainment of diabetes mellitus (FBG > 11.1 mM).

Experimental design

The animals were divided into five groups (n=8/ group): healthy (control), untreated diabetes (DM), metformin-treated diabetes (DM+M, 300 mg/kg/day metformin), propolis-treated diabetes (DM+P, 300 mg/ kg/day propolis extract) and diabetes with combined treatment (DM+M+P, dosage as former). Treatment was administered via oral gavage for four weeks immediately after successful induction of diabetes. All groups received the same volume of 1mL per gavage. The rats were euthanised at the end of the experiment using sodium pentobarbital (200 mg/kg), and blood was collected to determine fasting blood glucose (FBG). Thoracic aorta was dissected and processed into 10% tissue homogenates using ice-cold phosphate buffer saline. Segments of the aorta (2-4 mm) were reserved for tissue bath assay.

Determination of the level of antioxidants in aorta

Aortic tissue homogenates were centrifuged at 10,000 × g for 10 minutes, and the supernatant was aliquoted for further assay on protein concentration, SOD, CAT, GPx-1 and sRAGE. Tissue SOD activity was determined on the basis of reaction with riboflavin/nitrotetrazolium blue upon photoactivation (Cheng et al., 2015Cheng CW, Chen LY, Chou CW, Liang JY. Investigations of riboflavin photolysis via coloured light in the nitro blue tetrazolium assay for superoxide dismutase activity. J Photochem Photobiol B. 2015 Jul 1;148:262-7.). A protein assay kit (QCPR-500, QuantiChromTM, BioAssay System), Catalase Assay Kit (E-BC-K30, Elabscience®, the United States), Rat GPx-1 ELISA Kit (E-EL-R2491, Elabscience®, the United States) and Rat Advanced Glycosylation End Product Specific Receptor ELISA Kit (E-EL-R0643, Elabscience®, the United States) were used to determine the tissue level of CAT (Hadwan, Abed, 2016Hadwan MH, Abed HN. Data supporting the spectrophotometric method for the estimation of catalase activity. Data Brief. 2016 Mar 1;6:194-9.), GPx-1 (Luo et al., 2015Luo Y, Fu C, Wang Z, Zhang Z, Wang H, Liu Y. Asiaticoside attenuates the effects of spinal cord injury through antioxidant and anti-inflammatory effects, and inhibition of the p38-MAPK mechanism. Mol Med Rep. 2015 Dec 1;12(6):8294-300.) and s-RAGE (Greco et al., 2014Greco R, Tassorelli C, Mangione AS, Levandis G, Certo M, Nappi G, et al. Neuroprotection by the PARP inhibitor PJ34 modulates cerebral and circulating RAGE levels in rats exposed to focal brain ischemia. Eur J Pharmacol. 2014 Dec 5;744:91-7.). The SOD/(CAT+GPx-1) ratio was calculated as previously reported (Hadzi-Petrushev et al., 2018Hadzi-Petrushev N, Bogdanov J, Krajoska J, Ilievska J, Bogdanova-Popov B, Gjorgievska E, et al. Comparative study of the antioxidant properties of monocarbonyl curcumin analogues C66 and B2BrBC in isoproteranol induced cardiac damage. Life Sci. 2018 Mar 15;197:10-8.).

Tissue bath assay set-up

An automated organ bath (PanLab, LE01026, the United States), force transducer (ADInstrument, MLT050/D, New Zealand), PowerLab 8/30 (ADInstrument, ML870, New Zealand) and LabChart® Reader software were assembled for tissue bath assay. Then, the set-up was calibrated using a 1 g weight. KrebsRinger bicarbonate solution (NaCl, 118.6 mM; KCl, 4.8 mM; CaCl2, 2.5 mM; MgSO4, 1.2 mM; KH2PO4, 0.2 mM; NaHCO3, 25.1 nM; glucose, 11.0 mM) was utilised as the physiological buffer.

At the beginning of the assay, the aorta was mounted and equilibrated in the tissue bath chamber filled with buffer for 60 mins at a resting tension of 1 g. The buffer was replaced every 20 mins. Throughout the experiment, the tissue bath chamber was equilibrated at 37 ºC with continuous carbogen (95% oxygen, 5% carbon dioxide) aeration. The viability of aorta was confirmed by the consistent tension increment following two rounds of stimulation with KCL (60 mM) before the experiment (Ulu et al., 2010Ulu N, Gurdal H, Landheer SW, Duin M, Guc MO, Buikema H, et al. α1-Adrenoceptor-mediated contraction of rat aorta is partly mediated via transactivation of the epidermal growth factor receptor. Br J Pharmacol. 2010 Nov;161(6):1301-10.).

Determination of Endothelium-Dependent Relaxation

The cumulative tension reductions towards graded concentration of Ach (10-8-10-4 M) in aorta segments precontracted with PE (10-6 M) were recorded (Hassan et al., 2011Hassan Z, Dewa A, Asmawi MZ, Sattar A, Munavvar Z. Assessment of vascular reactivity at different time-course on streptozotocin-induced diabetic rats. J Exp Integr Med. 2011 Jul 1;1(3).).

Determination of the Mechanism of Action of Propolis

Thoracic aorta of healthy rats was dissected and divided into segments. The integrity of the endothelial layer in PE-precontracted viable aorta segments was confirmed by the presence of a 60-80% reduction in tension towards Ach (10-6 M) (Ulu et al., 2010Ulu N, Gurdal H, Landheer SW, Duin M, Guc MO, Buikema H, et al. α1-Adrenoceptor-mediated contraction of rat aorta is partly mediated via transactivation of the epidermal growth factor receptor. Br J Pharmacol. 2010 Nov;161(6):1301-10.). Only viable aortic tissues with intact endothelium were used for further assay. Then, the aortic segments (n=6/experiment) were incubated for 30 minutes with enzyme inhibitors before precontraction with PE (10-6 M): L-NAME, 10-4 M; methylene blue, 10-5 M; indomethacin, 10-5 M; elevated glucose, 25 mM (Fatehi-Hassanabad et al., 2005Fatehi-Hassanabad Z, Jafarzadeh M, Tarhini A, Fatehi M. The antihypertensive and vasodilator effects of aqueous extract from Berberis vulgaris fruit on hypertensive rats. Phytother. Res. 2005 Mar;19(3):222-5.). Subsequently, relaxation responses towards graded concentration of propolis extract (0.01-1.00%) under each conditions were recorded.

Statistical analysis

Statistical Package for the Social Sciences version 24 and GraphPad Prism version 7.0a were used for statistical analysis. Numerical data were presented as median (interquartile range). Differences between groups were analysed using the Kruskal-Wallis H test with post-hoc Bonferroni correction. The percentage of tension increment from resting baseline and that of tension decrement from plateau were derived from vascular tension tracing. Non-linear regression was then performed to produce dose-response curves, from which the potency, pEC50 and maximal tension reduction, Emax were derived. The pEC50 and Emax differences of the propolis extract in the presence of inhibitors compared with the physiological buffer were analysed using the Wilcoxon signed ranks test.

RESULTS AND DISCUSSION

Anti-hyperglycaemic activity of propolis

At the end of this study, the DM group had significantly higher FBG compared with the control group (27.0 [5.8) vs 4.1 [0.3] mM). Meanwhile, the DM+M, DM+P and DM+M+P groups showed glycaemic improvement compared with the DM group, with FBG values of 8.9 (2.7), 11.9 (0.5) and 5.6 (0.8) mM, respectively. Chronic elevation of blood glucose, as the disease hallmark of diabetes mellitus, is the key trigger of oxidative stress generation and end-organ damage (King, Loeken, 2004King GL, Loeken MR. Hyperglycemia-induced oxidative stress in diabetic complications. Histochem Cell Biol. 2004;122: 333.). The current findings reinforce the anti-hyperglycaemic effect of propolis in diabetic rats (Usman et al., 2017Usman UZ, Bakar AB, Zin AA, Mohamed M. LC-MS analysis and effects of Malaysian propolis on insulin, glucagon, pancreas and oxidative stress status in streptozotocin-induced diabetic rats. J Med Biomed Res. 2017;16(1):15-27.) and invites further study to elucidate its glucose-lowering mechanism.

H. itama propolis can inhibit alpha-glucosidase, an intestinal enzyme that facilitates carbohydrate hydrolysis and glucose absorption in the gastrointestinal tract (Ibrahim et al., 2016Ibrahim N, Niza NF, Rodi MM, Zakaria AJ, Ismail Z, Mohd KS. Chemical and biological analyses of Malaysian stingless bee propolis extracts. Malaysian Journal of Analytical Sciences. 2016;20(2):413-22.). Acarbose is the marketed alphaglucosidase inhibitor used to manage diabetes mellitus (DiNicolantonio, Bhutani, O’Keefe, 2015DiNicolantonio JJ, Bhutani J, O’Keefe JH. Acarbose: safe and effective for lowering postprandial hyperglycaemia and improving cardiovascular outcomes. Open heart. 2015 Oct 1;2(1):e000327.). However, the alpha-glucosidase inhibition of H. itama propolis has higher potency compared with that of acarbose (Ibrahim et al., 2016Ibrahim N, Niza NF, Rodi MM, Zakaria AJ, Ismail Z, Mohd KS. Chemical and biological analyses of Malaysian stingless bee propolis extracts. Malaysian Journal of Analytical Sciences. 2016;20(2):413-22.).

Nevertheless, metformin was chosen as the positive control in this study for two reasons: (1) Metformin reduces aortic oxidative stress and improves ACh-induced aortic relaxation through the involvement of NO in rats with streptozotocin-induced diabetes (Majithiya, Balaraman, 2006Majithiya JB, Balaraman R. Metformin reduces blood pressure and restores endothelial function in aorta of streptozotocininduced diabetic rats. Life Sci. 2006 Apr 25;78(22):2615-24.). (2) Metformin provides cardiovascular benefits for patients with type 1 diabetes mellitus (Petrie et al., 2017Petrie JR, Chaturvedi N, Ford I, Brouwers MC, Greenlaw N, Tillin T, et al. Cardiovascular and metabolic effects of metformin in patients with type 1 diabetes (REMOVAL): a double-blind, randomised, placebo-controlled trial. The Lancet Diabetes & endocrinology. 2017 Aug 1;5(8):597-609.).

Level of antioxidants in aorta

Figure 1A-D shows the level of aortic antioxidants in the experimental rats. The aortic SOD (Figure 1A) and CAT (Figure 1B) activities increased significantly in the DM group compared with those in control group. Superoxide dismutase detoxifies intracellular free radicals into hydrogen peroxide, which is then degraded by CAT into water and oxygen. Both antioxidative enzyme activities increase as a compensatory mechanism to protect against heightened oxidative stress in the aorta of diabetic rats (Kakkar et al., 1996Kakkar R, Mantha SV, Kalra J, Prasad K. Time course study of oxidative stress in aorta and heart of diabetic rat. Clin Sci (Lond). 1996 Oct 1;91(4):441-8.). The DM+P group had significantly lower SOD activity compared with the DM group, whereas the CAT activity was higher in the DM+M+P group than in the control. Propolis prevents the reactive elevation of SOD activity in the aorta. Meanwhile, metformin treatment increases the aortic CAT level in diabetic rats (Chukwunonso Obi et al., 2016Chukwunonso Obi B, Chinwuba Okoye T, Okpashi VE, Nonye Igwe C, Olisah Alumanah E. Comparative study of the antioxidant effects of metformin, glibenclamide, and repaglinide in alloxan-induced diabetic rats. J Diabetes Res. 2016;2016.), and this beneficial effect was augmented by the addition of propolis in this study.

FIGURE 1
The level of antioxidants in the aorta of experimental rats (n=8/group) comprising: A superoxide dismutase, B catalase, C glutathione peroxidase 1, D SOD/(CAT/GPx1) ratio, E soluble receptor for advanced glycation end-product. Data were presented as median (interquartile range) and analysed using Kruskal-Wallis H test with post hoc Bonferroni correction. CAT, catalase; DM, diabetes mellitus; GPx1, glutathione peroxidase 1; M, metformin; P, propolis; SOD, superoxide dismutase.

The concentrations of GPx-1 (Figure 1C) and sRAGE (Figure 1D) in the aortic tissue were lower in the DM group than in the control group. All treatment groups showed the opposite changes. Glutathione peroxidase scavenges hydrogen peroxide into stable alcohol in the presence of reduced glutathione. Aortic tissue in the diabetic rats show a reduction in both glutathione peroxidase and reduced glutathione (Pari, Monisha, Jalaludeen, 2012Pari L, Monisha P, Jalaludeen AM. Beneficial role of diosgenin on oxidative stress in aorta of streptozotocin induced diabetic rats. Eur J Pharmacol. 2012 Sep 15;691(1-3):143-50.). Meanwhile, sRAGE is a decoy receptor that blocks the downstream AGE-RAGE signalling-mediated pro-inflammatory, pro-oxidative and atherogenic vasculopathy. Exogenous administration of sRAGE attenuates chronic vascular inflammation and atherosclerosis in diabetic mice (Wendt et al., 2006Wendt T, Harja E, Bucciarelli L, Qu W, Lu Y, Rong LL, et al. RAGE modulates vascular inflammation and atherosclerosis in a murine model of type 2 diabetes. Atherosclerosis. 2006 Mar 1;185(1):70-7.), and propolis and metformin restore the levels of GPx-1 and sRAGE in the aorta as defence against oxidative stress. The SOD/(CAT+GPx-1) ratio represents the capability of the first line antioxidative enzymes to remove hydrogen peroxide, which is a source of cellular damage (HadziPetrushev et al., 2018). Both DM+P and DM+M+P groups had low SOD/(CAT+GPx-1) ratios (Figure 1E), representing an in vivo antioxidative potential of propolis in preventing the accumulation of hydrogen peroxide.

Endothelium-dependent relaxation of aorta

FIGURE 2
Dose-response curve towards acetylcholine (10-8-10-4 M) as the indicator of the degree of endothelium-dependent relaxation among experimental rats in tissue bath assay (n=8/group). Non-linear regression of aortic tension to plot fourparameter variable sloops. All data were presented as median (interquartile range) and analysed using Kruskal-Wallis H test with post hoc Bonferroni correction. ACh, acetylcholine; DM, diabetes mellitus; Emax, maximal relaxation; M, metformin; P, propolis; pEC50, potency.

Figure 2 depicts the dose-response curves towards ACh and the corresponding pEC50and Emax in the aorta of the rats. The DM group had reduced relaxation response towards ACh compared with the control group. Diabetic animals have impaired ACh-mediated endothelialdependent relaxation (Oyama et al., 1986Oyama Y, Kawasaki H, Hattori Y, Kanno M. Attenuation of endothelium-dependent relaxation in aorta from diabetic rats. Eur J Pharmacol. 1986 Dec 2;132(1):75-8.). A recent study demonstrated the duration-dependent deterioration of endothelial function in diabetic rats (Hassan et al., 2011Hassan Z, Dewa A, Asmawi MZ, Sattar A, Munavvar Z. Assessment of vascular reactivity at different time-course on streptozotocin-induced diabetic rats. J Exp Integr Med. 2011 Jul 1;1(3).). Endothelial dysfunction in streptozotocin-induced diabetes mellitus affects both small and large vessels (Leo, Hart, Woodman, 2011Leo C, Hart J, Woodman O. Impairment of both nitric oxidemediated and EDHF-type relaxation in small mesenteric arteries from rats with streptozotocin-induced diabetes. British Journal of Pharmacology. 2011;162: 365-377.; Ali, Woodman, 2019Ali SF, Woodman OL. Tocomin restores endotheliumdependent relaxation in the diabetic rat aorta by increasing NO bioavailability and improving the expression of eNOS. Front Physiol. 2019;10:186.). In the present study, all treatment arms exhibited a significant improvement in ACh-induced relaxation compared with that in the DM group. Metformin mediates the restoration of endothelial function in rats with streptozotocin-induced diabetes via NO signalling involving potassium channels (Majithiya, Balaraman, 2006Majithiya JB, Balaraman R. Metformin reduces blood pressure and restores endothelial function in aorta of streptozotocininduced diabetic rats. Life Sci. 2006 Apr 25;78(22):2615-24.).

Notably, there was no significant difference in the ACh potency of aortic tissues between the DM+P and DM+M groups. Thus, propolis supplementation preserved endothelial function in the diabetic rats. The DM+M and DM+M+P groups achieved statistically higher percentages of relaxation compared with the DM group. The pleiotropic effects of metformin on the improvement of systemic oxidative stress and NO bioavailability, besides glycaemic control, may support this finding. Additionally, there is no significant difference in maximal relaxation response towards ACh between the control and DM+P groups. Therefore, H. itama propolis normalised vascular function.

Mechanism of action of propolisinduced aortic relaxation

The pEC50 and Emax of the propolis extract were 2.88 (0.79)% and 80 (28) %, respectively, as shown in Figure 3A. This study is the first to produce the pharmacodynamic data of dose-dependent vasorelaxation by propolis extract (H. itama) in rat aorta. Propolis extract of the Australian stingless bee, Tetragonula carbonaria has been found to mediate endothelium-independent vasorelaxation in coronary arteries (Massaro et al., 2013Massaro FC, Brooks PR, Wallace HM, Nsengiyumva V, Narokai L, Russell FD. Effect of Australian propolis from stingless bees (Tetragonula carbonaria) on pre-contracted human and porcine isolated arteries. PLoS One. 2013 Nov 15;8(11):e81297.). Nonetheless, research and development on propolis represent a niche in modern pharmaceutics. Further research warrants the delineation of the cardiovascular effect of stingless bee propolis from different regions and species with a focus on its vasoactive property.

Figure 3B-E displays the dose-response curves towards propolis extract with their pEC50 and Emax in healthy aorta segments under various conditions. The pEC50 (propolis) was significantly reduced after incubation with L-NAME (Figure 3B), methylene blue (Figure 3C) and elevated glucose. The reduced pEC50 (propolis) after exposure to inhibitors of NO synthase and guanylyl cyclase indicated the involvement of the NO-cGMP pathway in propolis-mediated relaxation. The lack of a significant reduction in pEC50 (propolis) with indomethacin (Figure 3D), which is an inhibitor of cyclooxygenase enzyme, suggested that the activity of propolis was independent of prostaglandin signalling (Fatehi-Hassanabad et al., 2005Fatehi-Hassanabad Z, Jafarzadeh M, Tarhini A, Fatehi M. The antihypertensive and vasodilator effects of aqueous extract from Berberis vulgaris fruit on hypertensive rats. Phytother. Res. 2005 Mar;19(3):222-5.). Short-term exposure to hyperglycaemia raised the aortic tissue level of hydroxyl radicals, as evidence of oxidative stress, and concomitantly decreased the level of NO and NO synthase activity (Qian et al., 2010Qian LB, Wang HP, Chen Y, Chen FX, Ma YY, Bruce IC, et al. Luteolin reduces high glucose-mediated impairment of endothelium-dependent relaxation in rat aorta by reducing oxidative stress. Pharmacol Res. 2010 Apr 1;61(4):281-7.). Particularly, acute hyperglycaemia (Figure 3E) reduced the potency of propolis, but not the maximal vasodilatory response.

In sum, propolis of H. itama possesses antihyperglycaemic, antioxidative effects and maintains the level of AGE-scavenging sRAGE in the aorta of rats with streptozotocin-induced diabetes. Functionally, propolis alleviates the impairment of endothelium-dependent relaxation in diabetic rats via the endothelium-dependent NO-cGMP pathway. This non-clinical animal study demonstrated the vasoprotective property of propolis in streptozotocin-induced diabetes mellitus.

FIGURE 3
Dose-response curve towards propolis extract under various conditions in the phenylephrine-precontracted aorta (n=6/experiment): A physiological buffer as control, B L-NAME (10-4 M) as nitric oxide synthase inhibitor, C methylene blue (10-5 M) as guanylyl cyclase inhibitor, D indomethacin (10-5 M) as cyclooxygenase inhibitor and E elevated glucose. Data presented as median (interquartile range) and analysed using Wilcoxon signed ranks test to compare with the control. Emax, maximal relaxation; L-NAME, Nomega-Nitro-L-arginine methyl ester; pEC50, potency.

ACKNOWLEDGEMENTS

Universiti Sains Malaysia Short-Term Research Grant (Account number: 304/PPSP/6315123) funded this research. We acknowledge the financial support from USM School of Medical Sciences Key Performance Index Allocation to present this study at Young Investigator Award session in the National Heart Association of Malaysia (NHAM) Annual Scientific Meeting 2018. We are cognizant of honorarium to the first author from the NHAM and Taiwan Society of Cardiology (TSOC) for the International Young Investigator presentation in 49th Annual Convention & Scientific Session of TSOC. We thank Mr Victor Udo NNa and Madam Aminah Che Romli for generously providing technical assistance in the laboratory.

REFERENCES

  • Ahmed R, Tanvir EM, Hossen M, Afroz R, Ahmmed I, Rumpa NE, et al. Antioxidant properties and cardioprotective mechanism of Malaysian propolis in rats. Evid Based Complement Alternat Med. 2017;2017.
  • Akhir RA, Bakar MF, Sanusi SB. Antioxidant and antimicrobial activity of stingless bee bread and propolis extracts. AIP Conf. Proc. 2017 Oct 3 (Vol. 1891, No. 1, p. 020090). AIP Publishing.
  • Ali SF, Woodman OL. Tocomin restores endotheliumdependent relaxation in the diabetic rat aorta by increasing NO bioavailability and improving the expression of eNOS. Front Physiol. 2019;10:186.
  • Cheng CW, Chen LY, Chou CW, Liang JY. Investigations of riboflavin photolysis via coloured light in the nitro blue tetrazolium assay for superoxide dismutase activity. J Photochem Photobiol B. 2015 Jul 1;148:262-7.
  • Chukwunonso Obi B, Chinwuba Okoye T, Okpashi VE, Nonye Igwe C, Olisah Alumanah E. Comparative study of the antioxidant effects of metformin, glibenclamide, and repaglinide in alloxan-induced diabetic rats. J Diabetes Res. 2016;2016.
  • DiNicolantonio JJ, Bhutani J, O’Keefe JH. Acarbose: safe and effective for lowering postprandial hyperglycaemia and improving cardiovascular outcomes. Open heart. 2015 Oct 1;2(1):e000327.
  • Fatehi-Hassanabad Z, Jafarzadeh M, Tarhini A, Fatehi M. The antihypertensive and vasodilator effects of aqueous extract from Berberis vulgaris fruit on hypertensive rats. Phytother. Res. 2005 Mar;19(3):222-5.
  • Förstermann U. Nitric oxide and oxidative stress in vascular disease. Pflugers Arch - Eur J Physiol. 2010;459: 923.
  • Greco R, Tassorelli C, Mangione AS, Levandis G, Certo M, Nappi G, et al. Neuroprotection by the PARP inhibitor PJ34 modulates cerebral and circulating RAGE levels in rats exposed to focal brain ischemia. Eur J Pharmacol. 2014 Dec 5;744:91-7.
  • Hadwan MH, Abed HN. Data supporting the spectrophotometric method for the estimation of catalase activity. Data Brief. 2016 Mar 1;6:194-9.
  • Hadzi-Petrushev N, Bogdanov J, Krajoska J, Ilievska J, Bogdanova-Popov B, Gjorgievska E, et al. Comparative study of the antioxidant properties of monocarbonyl curcumin analogues C66 and B2BrBC in isoproteranol induced cardiac damage. Life Sci. 2018 Mar 15;197:10-8.
  • Hassan Z, Dewa A, Asmawi MZ, Sattar A, Munavvar Z. Assessment of vascular reactivity at different time-course on streptozotocin-induced diabetic rats. J Exp Integr Med. 2011 Jul 1;1(3).
  • Ibrahim N, Niza NF, Rodi MM, Zakaria AJ, Ismail Z, Mohd KS. Chemical and biological analyses of Malaysian stingless bee propolis extracts. Malaysian Journal of Analytical Sciences. 2016;20(2):413-22.
  • Kakkar R, Mantha SV, Kalra J, Prasad K. Time course study of oxidative stress in aorta and heart of diabetic rat. Clin Sci (Lond). 1996 Oct 1;91(4):441-8.
  • King GL, Loeken MR. Hyperglycemia-induced oxidative stress in diabetic complications. Histochem Cell Biol. 2004;122: 333.
  • Leo C, Hart J, Woodman O. Impairment of both nitric oxidemediated and EDHF-type relaxation in small mesenteric arteries from rats with streptozotocin-induced diabetes. British Journal of Pharmacology. 2011;162: 365-377.
  • Luo Y, Fu C, Wang Z, Zhang Z, Wang H, Liu Y. Asiaticoside attenuates the effects of spinal cord injury through antioxidant and anti-inflammatory effects, and inhibition of the p38-MAPK mechanism. Mol Med Rep. 2015 Dec 1;12(6):8294-300.
  • Majithiya JB, Balaraman R. Metformin reduces blood pressure and restores endothelial function in aorta of streptozotocininduced diabetic rats. Life Sci. 2006 Apr 25;78(22):2615-24.
  • Massaro FC, Brooks PR, Wallace HM, Nsengiyumva V, Narokai L, Russell FD. Effect of Australian propolis from stingless bees (Tetragonula carbonaria) on pre-contracted human and porcine isolated arteries. PLoS One. 2013 Nov 15;8(11):e81297.
  • Oyama Y, Kawasaki H, Hattori Y, Kanno M. Attenuation of endothelium-dependent relaxation in aorta from diabetic rats. Eur J Pharmacol. 1986 Dec 2;132(1):75-8.
  • Pari L, Monisha P, Jalaludeen AM. Beneficial role of diosgenin on oxidative stress in aorta of streptozotocin induced diabetic rats. Eur J Pharmacol. 2012 Sep 15;691(1-3):143-50.
  • Petrie JR, Chaturvedi N, Ford I, Brouwers MC, Greenlaw N, Tillin T, et al. Cardiovascular and metabolic effects of metformin in patients with type 1 diabetes (REMOVAL): a double-blind, randomised, placebo-controlled trial. The Lancet Diabetes & endocrinology. 2017 Aug 1;5(8):597-609.
  • Pitocco D, Tesauro M, Alessandro R, Ghirlanda G, Cardillo C. Oxidative stress in diabetes: implications for vascular and other complications. Int J Mol Sci. 2013 Oct 30;14(11): 21525-50.
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Publication Dates

  • Publication in this collection
    26 Nov 2021
  • Date of issue
    2021

History

  • Received
    11 Mar 2019
  • Accepted
    06 June 2019
Universidade de São Paulo, Faculdade de Ciências Farmacêuticas Av. Prof. Lineu Prestes, n. 580, 05508-000 S. Paulo/SP Brasil, Tel.: (55 11) 3091-3824 - São Paulo - SP - Brazil
E-mail: bjps@usp.br