Article
Flaxseed oil reduces oxidative stress and enhances brain monoamines release in streptozotocin-induced diabetic rats
Human and Experimental Toxicology 1–6 ª The Author(s) 2015 Reprints and permission: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0960327115571765 het.sagepub.com
EA Badawy, WI Rasheed, TR Elias, J Hussein, M Harvi, S Morsy and YA El-Latif Mahmoud
Abstract This study was performed to investigate the biochemical effect of flaxseed oil on oxidative stress and brain monoamines release in streptozotocin-induced diabetic rats. Sixty male albino rats were divided into following four groups (15 for each group): control group, flaxseed oil group, diabetic group, and flaxseed oil-treated diabetic group. Serum glucose, insulin, pentosidine, plasma advanced oxidation protein products (AOPPs), and plasma total antioxidant capacity were estimated. Brain neurotransmitters, malondialdehyde (MDA), and nitric oxide (NO) were also determined. The mean values of serum pentosidine and plasma AOPP showed a significant decrease in treated diabetic group as compared to their values in the diabetic group. Also, brain neurotransmitters levels were improved after treatment with flaxseed. Brain MDA and NO were increased significantly in the diabetic group, while they were significantly decreased after treatment. Brain NO and brain MDA had a significant positive correlation with pentosidine, AOPP, and neurotransmitters. We concluded that flaxseed oil supplementation may be useful in the treatment of brain dysfunction in diabetes. Keywords Flaxseed oil, brain monoamines, oxidative stress, diabetes
Introduction Diabetes mellitus is a complex disease associated with peripheral and central complications. These complications include retinopathy, nephropathy, and neuropathy.1 Several investigations have confirmed the role of oxidative stress in the development of diabetic-mediated disorders, possibly via the formation of free radicals.2 Hyperglycemia induces vascular damage probably through a single common pathway, increased intracellular oxidative stress, linking four major mechanisms, namely, the polyol pathway, advanced glycation end products (AGEs) formation, and the protein kinase C—diacylglycerol and the hexosamine pathways.3 Due to prolonged high glucose levels in diabetes, reactive derivatives are formed via nonenzymatic condensation reaction named Millard reaction between reducing glucose and amine residues of proteins, lipids, or nucleic acids that undergo a series of
complex reactions such as rearrangement, dehydration, and condensation to give an irreversible crosslinked complex group of compounds termed AGEs.4 Examples of some of the best chemically characterized AGE-modified sites in humans with diabetes are carboxymethyllysine, carboxyethyllysine, and pentosidine, which act as markers for the formation and accumulation of AGEs.5 Diabetes is associated with marked changes in brain monoamines content and transport in both rodents and humans6; however, conflicting results
Department of Medical Biochemistry, National Research Center, Doki, Giza, Egypt Corresponding author: TR Elias, Department of Medical Biochemistry, National Research Center, Doki, Giza, Egypt. Email: [email protected]
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have been reported. Chronic antioxidant therapy may be useful in decreasing the risk of diabetic complications. As an antioxidant, flaxseed oil can cause a significant reduction in blood glucose level7 and an increase in insulin level in diabetic-treated rats compared to diabetic rats by scavenging reactive oxygen species (ROS). Flaxseed (Linum usitatissimum L.) is a multipurpose crop and its consumption is beneficial for human health. The nutritional components of flaxseed are oil, proteins, lignans, fibers, and vitamins. Additionally, diet supplement with flaxseed ameliorated the antioxidant enzyme activities observed in diabetic rats and significantly decreased malondialdehyde (MDA) levels.8 This study aimed to investigate the brain oxidative stress induced by streptozotocin (STZ) in rats with consequent changes in brain neurotransmitters and examined the potential protective role of flaxseed oil against the changes due to diabetes.
Materials and methods Materials Chemicals. Norepinephrine, dopamine, serotonin, pentosidine high-performance liquid chromatography (HPLC) standards, and STZ were purchased from Sigma Aldrich Chemicals Company (St Louis, Missouri, USA). Flaxseed oil was purchased from local market, Egypt. All other chemicals were of HPLC grade and purchased from Sigma. Experimental animals. Sixty male Sprague Dawley rats weighing 100–120 g were obtained from the animal house of the National Research Centre (NRC), Giza, Egypt. The animals were housed in individual suspended stainless steel cages at the temperature range of 22 + 2 C, under a 12-h light/12-h dark cycle, and allowed to acclimatize for a period of 15 days to the experiment; rats were allowed free access to food and water. The guidelines of the ethical care and treatment of the animals followed the regulations of the ethical committee of NRC. The number of ethical committee permission is (10/219). The study was conducted in concordance with relevant European directives.
Methods Induction of diabetes. STZ was dissolved in 50 mM sodium citrate solution (pH 4.5) containing 150 mM sodium chloride. The solution (6 mg/100 g body weight (b.w.)) was subcutaneously administered in
rats. Fasting blood sugar was estimated after 3 days to confirm the development of diabetes mellitus.9 Experimental design. Sixty male rats were divided into 4 groups, with 15 rats in each group, and were classified as follows: group I (control group), healthy rats received 1.2 ml corn oil/kg b.w./day orally; group II (flaxseed oil group), healthy rats received 1.2 ml flaxseed oil/kg b.w./day orally; group III (diabetic group), diabetic rats received 1.2 ml corn oil/kg b.w./day orally; and group IV (flaxseed oil-treated diabetic group), diabetic rats received 1.2 ml flaxseed oil/kg b.w./day orally10 by stomach tube. Samples collection. After the experimental period (8 weeks), animals were kept fasting for 12 h, then anesthetized, and the blood was withdrawn from the retro-orbital venous plexus of the eye using heparinized capillary tubes. Blood samples were collected, left to clot, and then centrifuged at 3000 r/min for 15 min to separate the serum. The serum was stored at 20 C for pentosidine determination. Blood was centrifuged at 2000 r/min for 10 min using a cooling centrifuge. Plasma was separated and immediately frozen for other biochemical parameters. Brains were removed quickly and placed in iced normal saline, perfused with the same solution to remove blood cells, plotted on filter paper, and frozen at 80 C until use for estimation of brain monoamines, MDA, and nitric oxide (NO). Preparation of tissue homogenate. The frozen tissues were cut into small pieces and homogenized in phosphate buffer (pH 7.4), then centrifuged at 4000 r/min for 15 min at 4 C, and the supernatant was removed for estimation of chemical parameters.11 Biochemical assays. Freshly prepared serum was used for determination of blood glucose according to Trinder.12 Plasma insulin level was estimated by enzyme-linked immunosorbent assay (ELISA) kit.13 Reverse-phase HPLC was used for the determination of the plasma pentosidine content. Briefly, 50 ml of plasma were lyophilized and then hydrolyzed by 50 ml of 6 N hydrochloric acid at 110 C under nitrogen atmosphere for 16 h, subsequently neutralized with 100 ml of 5 N sodium hydroxide and 200 ml of 0.5 M phosphate buffer (pH 7.4), and then filtered through a 0.45-mm Millipore filter (Billerica, Massachusetts, USA) and diluted 20-fold with phosphatebuffered saline. Filtered samples (50 ml) were then
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Table 1. Blood glucose and insulin in the different studied groups. Groups Parameter
Control group (n ¼ 15)
Flaxseed oil (n ¼ 15)
Diabetic (n ¼ 15)
Diabetic flaxseed (n ¼ 15)
Glucose (mg/dl) Insulin (mIU/ml)
76.66 + 3.9a 10.5 + 0.8
77.51 + 2.4a 10.7 + 1.3
248.89 + 8.3b 8.5 + 1.1
181.4 + 4.7a,b 10.1 + 1.7
Values are expressed as mean + SE. n denotes number of rats in each group. a p < 0.05: significant difference compared with diabetic group. b p < 0.05: significant difference compared with the control group.
injected into C18 reverse phase analytical column (218TP104, Vydac; Separation Group, Hesperia, California, USA) using a fluorescence detector at excitation/emission wavelengths of 335/385 nm14 by gradient method using auto nitrile/water as mobile phase with a flow rate 1 ml/min. Brain MDA was measured using thiobarbituric acid reaction.15 The protein content of the homogenate was determined by the method of Lowry et al.16 using bovine serum albumin as standard. Total antioxidant capacity (TAC) was measured kinetically using commercial kits purchased from Biodiagnostics (Cairo, Egypt).17 Determination of advanced oxidation protein products (AOPPs)18 was done with double antibody sandwich ELISA. The kit was obtained from Glory Science Co. Ltd (Del Rio, Texas, USA). Determination of brain monoamines. Brain homogenate used for the determination of brain norepinephrine, dopamine, and serotonin was carried out using HPLC system, Agilent technologies 1100 series (Santa Clara, California, USA), equipped with a quaternary pump (G131A model). Separation was achieved on octadecylsilyl-reversed phase column (C18, 25 0.46 cm2; internal diameter: 5 mm). The mobile phase consisted of potassium phosphate buffer/methanol 97/3 (v/v) and was delivered at a flow rate of 1.5 ml/min. Ultraviolet detection was performed at 270 nm, and the injection volume was 20 ml. The concentration of both catecholamines and serotonin was determined by external standard method using peak areas. Serial dilutions of standards were injected and their peak areas were determined. A linear standard curve was constructed by plotting peak areas versus the corresponding concentrations. The concentration in samples was obtained from the curve.19
variance using Statistical Package for the Social Sciences (Version 12). Duncan’s new multiple range test was used to assess differences between means. Pearson’s correlation test was used to assess correlations between means. A significant difference was considered at the level of p < 0.05.
Results Diabetes was confirmed by estimating the blood glucose levels in the diabetic and the diabetic flaxseed groups. The diabetic and the diabetic flaxseed groups showed higher glucose levels than the control group (Table 1). There were no significant changes of plasma insulin level in all groups. As shown in Table 2, the serum level of pentosidine was significantly increased in the diabetic rats than in controls, but after treatment by flaxseed oil this value significantly decreased. Plasma AOPP level increases significantly in the diabetic group compared with the control group; on the other hand, plasma TAC level decreases significantly in diabetics group than controls, while the two parameter levels improved after treatment with flaxseed oil (Table 2). The levels of brain MDA and NO were significantly increased in the diabetic group compared with the control group; however, these increases were significantly attenuated after treatment with flaxseed oil. Compared with the control group, brain norepinephrine and dopamine levels were significantly increased in the diabetic group; however, flaxseed oil significantly decreased dopamine level and tended to decrease norepinephrine level compared with the diabetic group (Table 3). On the other hand, there was no significant change in serotonin level in all groups. Our study indicated that there is a positive correlation between MDA, NO, and all parameters in the study (Table 4). Serotonin has not been changed in treated groups.
Statistical analysis
Discussion
All data were expressed as mean + standard error. Data were analyzed using one-way analysis of
In this study, the rats treated with STZ developed hyperglycemia. The increase in the serum glucose
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Table 2. Oxidants/antioxidants parameters in the different studied groups. Groups Parameter
Control group (n ¼ 15)
Brain MDA (nmol/g tissue) Serum pentosidine (pM) Plasma AOPP (ng/ml) Brain NO (mmol/g tissue) Plasma TAC (mM/L)
29.73 0.19 10.33 3.03 1.92
Flaxseed oil (n ¼ 15)
+ 1.52a + 0.01a + 0.24a + 0.19a + 0.07a
33.64 + 0.08 + 10.43 + 4.16 + 1.79 +
1.57a 0.01a,b 0.15a 0.16a,b 0.03a
Diabetic (n ¼ 15) 73.28 + 1.05 + 15.08 + 8.04 + 1.23 +
Diabetic flaxseed (n ¼ 15)
2.92b 0.02b 0.11b 0.20b 0.04b
46.49 0.88 13.19 5.68 1.57
+ 1.49a,b + 0.02a,b + 0.32a,b + 0.26a,b + 0.04a,b
MDA: malondialdehyde; AOPP: advanced oxidation protein product; NO: nitric oxide; SE: standard error; n: number of rats in each group; TAC: total antioxidant capacity. Values are expressed as mean + SE. a p < 0.05: significant difference compared with diabetic group. b p < 0.05: significant difference compared with the control group.
Table 3. Brain monoamines parameters in the different studied groups. Groups Parameter
Control group (n ¼ 15)
Flaxseed oil (n ¼ 15)
Diabetic (n ¼ 15)
Diabetic flaxseed (n ¼ 15)
Norepinephrine (ng/g tissue) Dopamine (ng/g tissue) Serotonin (mg/g tissue)
731 + 35a 31.00 + 1.63a 3.73 + 0.25
705 + 29.65a 30.7 + 3.01a 3.80 + 0.25
940 + 10.68b 52.2 + 2.82b 4.40 + 0.14
892 + 36.41b 40.8 + 2.78a,b 4.2 + 0.47
Values are expressed as mean + SE. n denotes number of rats in each group. a p < 0.05: significant difference compared with diabetic group. b p < 0.05: significant difference compared with the control group.
Table 4. Correlation between MDA, NO, and all parameters in the study. NO
MDA
r ¼ 0.908 p ¼ 0.000 MDA r ¼ 0.908 – p ¼ 0.000 – NO
–
Total antioxidants capacity Serotonin Dopamine Norepinephrine pentosidine r¼ p¼ r¼ p¼
0.827 0.000 0.879 0.000
r¼ p¼ r¼ p¼
0.348 r 0.028 P 0.282 r 0.078 p
¼ 0.692 ¼ 0.000 ¼ 0.728 ¼ 0.000
r p r p
¼ 0.664 ¼ 0.000 ¼ 0.598 ¼ 0.000
r p r p
¼ 0.861 ¼ 0.000 ¼ 0.834 ¼ 0.000
AOPP r p r p
¼ 0.883 ¼ 0.000 ¼ 0.871 ¼ 0.000
NO: nitric oxide; MDA: malondialdehyde; AOPP: advanced oxidation protein product. p < 0.05: correlation is significant.
could be due to a decrease in the levels of serum insulin as a result of destruction of the pancreas by STZ and generation of ROS.20 This study demonstrated that in diabetic rats brain neurotransmitters significantly increased, which may be related to oxidative stress since a positive correlation was observed in this study between MDA, NO, and neurotransmitters. This finding was confirmed by other studies.19,21 Araki and Nishikawa22 demonstrated that increased oxidative stress could be one of the common pathogenic factors of diabetic complications (like peripheral neuropathy) as well as in neurodegenerative disorders like Alzheimer’s disease and Parkinson’s disease.23 Oxidative damage is generally
attributed to the formation of highly reactive OH, which leads to severe oxidative damage of the cells’ components like lipids, proteins, and DNA.24 Efficiency of enzymatic and nonenzymatic antioxidative systems could be detected by the determination of single components of this system or by the so-called TAC.17 Recent study demonstrated that neurons develop hyperexcitability in STZ-induced diabetic rats; this may be attributed to certain disturbances in neurotransmitters pattern.21 Consequently, tissue cells isolated from hyperglycemic animals demonstrated increase in norepinephrine and dopamine production/secretion.25
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Firoozrai et al.26 reported that MDA is produced by breakdown of lipids under oxidative stress conditions. Evidence of lipid peroxidation has been reported to be present in a number of diabetic complications27 and it significantly contributes to endothelial dysfunction, which is a key event in the pathogenesis of diabetic microvascular disease. In this study, the lipid peroxidation marker (MDA) was elevated in diabetic rats as reported previously.28 The increase in lipid peroxidation might be a reflection in enzymatic and nonenzymatic antioxidants of defense systems.8 This study demonstrated that diabetic rats that were treated with flaxseed oil enriched with omega-3 significantly decrease brain norepinephrine and dopamine levels. In addition, there was a significant decrease in glucose, MDA, and NO levels that indicate a decrease in oxidative stress. The result of this study suggested that flaxseed oil contains a free radical scavenging activity, which could exert a beneficial effect against pathological alterations caused by the presence of O 2 and OH . Oxidative stress plays a central role in the pathogenesis of metabolic diseases like diabetes mellitus and its complications (like peripheral neuropathy) due to its great oxygen consumption, high lipid and poor antioxidants defense. Oxygen free radicals attack objects on the polyunsaturated components of membranes and may cause a serious organizational dysfunction within cells and tissues. It has been suggested that the use of omega-3 polyunsaturated fatty acids (PUFAs) may have ameliorating effect on such damage by two possible ways. First, omega-3 PUFA may increase the level of catalase within the peroxisome and in the cytoplasm resulting in enhanced defense against free oxygen radicals. Second, omega-3 PUFAs, which have been supplemented, may be replaced with PUFA components of the membranes that had been attacked by oxygen free radicals.21 In our study, pentosidine is significantly higher in diabetic rats than the controls. This result is in agreement with Kerkeni et al.29 who demonstrated that pentosidine might accelerate the development of microvascular complications both by accumulation of AGEs in the vessel walls and by causing endothelial dysfunction, as mediated by the AGE-AGEreceptor axis activity. The mean levels of pentosidine in diabetic group that was modulated with flaxseed oil orally were significantly decreased as compared to the diabetic group but still not reached the levels of the
control group. The effectiveness of flaxseed oil in diabetes could be because of its antioxidant activity.30 Recently, Yamagishi et al.31 showed the role of AGEs and oxidative stress in vascular complications in diabetes. The authors demonstrated that AGEs induce mesangial cell loss and dysfunction, glomerulosclerosis, and tubulointerstitial fibrosis, taking into account the fact that patients with diabetes are prone to microvascular disease. In the same line, the elevation of AOPP in diabetic rats of this study may be due to the generation of ROS, this result is in agreement with the results of Abou-Seif and Youssef32 who reported that AOPPs are formed during oxidative stress by the action of chloramines produced by myeloperoxidase in activated neutrophils and is accumulated in biological systems and thus caus damage to biological membranes.
Conclusion STZ-induced diabetes mellitus is mediated through ROS and prevention of diabetic complications by flaxseed oil is due to its antioxidant activity. Acknowledgment Authors are grateful to the National Research Centre, Giza, Egypt, for providing space to carry out this work.
Funding This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.
References 1. Ho N, Sommers MS and Lucki I. Effects of diabetes on hippocampal neurogenesis: links to cognition and depression. Neurosci Biobehav Rev 2013; 37: 1346–1362. 2. El-Khayat Z, Hussein J, Ramzy T, et al. Antidiabetic antioxidant effect of Panax ginseng. J Med Plants Res 2011; 5(18): 4616–4620. 3. Yamagishi S and Imaizumi T. Diabetic vascular complications: pathophysiology, biochemical basis and potential therapeutic strategy. Curr Pharm Des 2005; 11: 2279–2299. 4. Takeuchi M and Yamagishi S. Involvement of toxic AGEs (TAGE) in the pathogenesis of diabetic vascular complications and Alzheimer’s disease. J Alzheimers Dis 2009; 16: 845–858. 5. Ghanem AA, Elewa A and Arafa LF. Pentosidine and N-carboxymethyl-lysine: biomarkers for type 2 diabetic retinopathy. Eur J Ophthalmol 2011; 21: 48–54.
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6. Lackovic Z, Salkovic M, Kuci Z, et al. Effect of long-lasting diabetes mellitus on rat and human brain monoamines. J Nurochem 1990; 54: 143–147. 7. Rhee Y and Brunt A. Flaxseed supplementation improved insulin resistance in obese glucose intolerant people: a randomized crossover design. Nutr J 2011; 10: 1–7. 8. Hussein J, El-Khayat Z, Taha M, et al. Insulin resistance and oxidative stress in diabetic rats treated with flaxseed oil. J Med Plants Res 2012; 6(42): 5499–5506. 9. Uchiyama S and Yamaguchi M. Alteration in serum and bone component findings induced in streptozotocin-diabetic rats is restored by zinc acexamate. Int J Mol Med 2003; 12: 949–954. 10. Taylor G, Noto CD, Stringer DM, et al. Dietary milled flaxseed and flaxseed oil improve n-3 fatty acid status and do not affect glycemic control in individuals with well-controlled type 2 diabetes. J Am Coll Nutr 2010; 29(1): 72–80. 11. Manna F, Ahmed HH, Estefan SF, et al. Saccharomyces cerevisiae intervention for relieving flutamide induced hepatotoxicity in male rats. Pharmazie 2005; 60: 689–695. 12. Trinder P. Determination of glucose in blood using glucose oxidase with an alternative oxygen acceptor. Am Clin Biochem 1969; 6: 22–27. 13. Yalow R and Bauman WA. Insulin in health and disease. In: Ellenberg M and Rifkin H (eds) Diabetes Mellitus: Theory and Practice. New York: Excerpta Medica, 1983. pp. 119–150. 14. Odetti P, Fogarty J, Sell DR, et al. Chromatographic quantitation of plasma and erythrocyte pentosidine in diabetic and uremic subjects. Diabetes 1992; 1(2): 153–159. 15. Uchiyama M and Mihara M. Determination of malondialdehyde in tissue by thiobarbituric acid test. Anal Biochem 1978; 86: 271–278. 16. Lowry OH, Rosenbrough NJ, Farr AL, et al. Protein measurement with the Folin phenol reagent. J Boil Chem 1951; 193: 265–275. 17. Kankofer M, Lipko J and Zdunczyk S. Total antioxidant capacity of bovine spontaneously released and retained placenta. Pathophysiology 2005; 11(4): 215–219. 18. Witko-Sarsat V, Friedlander M, Capeille`re-Blandin C, et al. Advanced oxidation protein products as a novel marker of oxidative stress in uremia. Kidney Int 1996; 49(5): 1304–1313.
19. Hussein J, El-Matty D, El-Khayat Z, et al. Brain neurotransmitters in diabetic rats treated with CO enzyme Q10. Int J Pharm Pharm Sci 2012; 4: 554–556. 20. Wilson JL, Patton NJ, McCord JM, et al. Mechanism of streptozotocin and alloxan-induced damage in rat B cells. Diabetologia 1984; 27: 587–591. 21. El-Saeed GS, Wafay H, EL-Waseif M, et al. Effect of fish oil on oxidative stress and monoamine of brain tissue in STZ mice. World J Med Sci 2013; 8(4): 401–407. 22. Araki E and Nishikawa T. Oxidative stress: a cause and therapeutic target of diabetic complications. J Diabetes Investig 2010; 1: 90–96. 23. Eggers AE. Why do Alzheimer’s disease and Parkinson’s disease target the same neurons? Med Hypotheses 2009; 72(6): 698–700. 24. Mckillop IH and Schrum LW. Alcohol and liver cancer. Alcohol 2005; 35(3): 195–203. 25. Marco GS, Colucci JA, Fernandes FB, et al. Diabetes induces changes of catecholamines in primary mesangial cells. Int J Biochem Cell Biol 2008; 40(4): 747–754. 26. Firoozrai M, Nourbakhsh M and Razzaghy-Azar M. Erythrocyte susceptibility to oxidative stress and antioxidant status in patients with type 1 diabetes. Diabetes Res Clin Pract 2007; 77: 427–432. 27. Martin-Galla´n P, Carrascosa A, Gussinye´ M, et al. Biomarkers of diabetes-associated oxidative stress and antioxidant status in young diabetic patients with or without subclinical complications. Free Radic Biol Med 2003; 34: 1563–1574. 28. Murugan P and Pari L. Influence of tetrahydrocurcumin on erythrocyte membrane and antioxidant status in experimental type 2 diabetic rats. J Ethnopharmacol 2007; 113: 479–486. 29. Kerkeni M, Saı¨di A, Bouzidi H, et al. Pentosidine as a biomarker for microvascular complications in type 2 diabetic patients. Diabetes Vasc Dis Res 2013; 10(3): 239–245. 30. Prasad K. Secoisolariciresinol diglucoside from flaxseed delays the development of type 2 diabetes in Zucker rat. J Lab Clin Med 2001; 138: 32–39. 31. Yamagishi SI, Maeda S, Matsui T, et al. Role of advanced glycation end products (AGEs) and oxidative stress in vascular complications in diabetes. Biochim Biophys Acta 2012; 1820: 663–671. 32. Abou-Seif A and Youssef A. Evaluation of some biochemical changes in diabetic patients. Clin Chim Acta 2004; 346: 161–170.
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Flaxseed oil reduces oxidative stress and enhances brain monoamines release in streptozotocin-induced diabetic rats
Human and Experimental Toxicology 1–6 ª The Author(s) 2015 Reprints and permission: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0960327115571765 het.sagepub.com
EA Badawy, WI Rasheed, TR Elias, J Hussein, M Harvi, S Morsy and YA El-Latif Mahmoud
Abstract This study was performed to investigate the biochemical effect of flaxseed oil on oxidative stress and brain monoamines release in streptozotocin-induced diabetic rats. Sixty male albino rats were divided into following four groups (15 for each group): control group, flaxseed oil group, diabetic group, and flaxseed oil-treated diabetic group. Serum glucose, insulin, pentosidine, plasma advanced oxidation protein products (AOPPs), and plasma total antioxidant capacity were estimated. Brain neurotransmitters, malondialdehyde (MDA), and nitric oxide (NO) were also determined. The mean values of serum pentosidine and plasma AOPP showed a significant decrease in treated diabetic group as compared to their values in the diabetic group. Also, brain neurotransmitters levels were improved after treatment with flaxseed. Brain MDA and NO were increased significantly in the diabetic group, while they were significantly decreased after treatment. Brain NO and brain MDA had a significant positive correlation with pentosidine, AOPP, and neurotransmitters. We concluded that flaxseed oil supplementation may be useful in the treatment of brain dysfunction in diabetes. Keywords Flaxseed oil, brain monoamines, oxidative stress, diabetes
Introduction Diabetes mellitus is a complex disease associated with peripheral and central complications. These complications include retinopathy, nephropathy, and neuropathy.1 Several investigations have confirmed the role of oxidative stress in the development of diabetic-mediated disorders, possibly via the formation of free radicals.2 Hyperglycemia induces vascular damage probably through a single common pathway, increased intracellular oxidative stress, linking four major mechanisms, namely, the polyol pathway, advanced glycation end products (AGEs) formation, and the protein kinase C—diacylglycerol and the hexosamine pathways.3 Due to prolonged high glucose levels in diabetes, reactive derivatives are formed via nonenzymatic condensation reaction named Millard reaction between reducing glucose and amine residues of proteins, lipids, or nucleic acids that undergo a series of
complex reactions such as rearrangement, dehydration, and condensation to give an irreversible crosslinked complex group of compounds termed AGEs.4 Examples of some of the best chemically characterized AGE-modified sites in humans with diabetes are carboxymethyllysine, carboxyethyllysine, and pentosidine, which act as markers for the formation and accumulation of AGEs.5 Diabetes is associated with marked changes in brain monoamines content and transport in both rodents and humans6; however, conflicting results
Department of Medical Biochemistry, National Research Center, Doki, Giza, Egypt Corresponding author: TR Elias, Department of Medical Biochemistry, National Research Center, Doki, Giza, Egypt. Email: [email protected]
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Human and Experimental Toxicology
have been reported. Chronic antioxidant therapy may be useful in decreasing the risk of diabetic complications. As an antioxidant, flaxseed oil can cause a significant reduction in blood glucose level7 and an increase in insulin level in diabetic-treated rats compared to diabetic rats by scavenging reactive oxygen species (ROS). Flaxseed (Linum usitatissimum L.) is a multipurpose crop and its consumption is beneficial for human health. The nutritional components of flaxseed are oil, proteins, lignans, fibers, and vitamins. Additionally, diet supplement with flaxseed ameliorated the antioxidant enzyme activities observed in diabetic rats and significantly decreased malondialdehyde (MDA) levels.8 This study aimed to investigate the brain oxidative stress induced by streptozotocin (STZ) in rats with consequent changes in brain neurotransmitters and examined the potential protective role of flaxseed oil against the changes due to diabetes.
Materials and methods Materials Chemicals. Norepinephrine, dopamine, serotonin, pentosidine high-performance liquid chromatography (HPLC) standards, and STZ were purchased from Sigma Aldrich Chemicals Company (St Louis, Missouri, USA). Flaxseed oil was purchased from local market, Egypt. All other chemicals were of HPLC grade and purchased from Sigma. Experimental animals. Sixty male Sprague Dawley rats weighing 100–120 g were obtained from the animal house of the National Research Centre (NRC), Giza, Egypt. The animals were housed in individual suspended stainless steel cages at the temperature range of 22 + 2 C, under a 12-h light/12-h dark cycle, and allowed to acclimatize for a period of 15 days to the experiment; rats were allowed free access to food and water. The guidelines of the ethical care and treatment of the animals followed the regulations of the ethical committee of NRC. The number of ethical committee permission is (10/219). The study was conducted in concordance with relevant European directives.
Methods Induction of diabetes. STZ was dissolved in 50 mM sodium citrate solution (pH 4.5) containing 150 mM sodium chloride. The solution (6 mg/100 g body weight (b.w.)) was subcutaneously administered in
rats. Fasting blood sugar was estimated after 3 days to confirm the development of diabetes mellitus.9 Experimental design. Sixty male rats were divided into 4 groups, with 15 rats in each group, and were classified as follows: group I (control group), healthy rats received 1.2 ml corn oil/kg b.w./day orally; group II (flaxseed oil group), healthy rats received 1.2 ml flaxseed oil/kg b.w./day orally; group III (diabetic group), diabetic rats received 1.2 ml corn oil/kg b.w./day orally; and group IV (flaxseed oil-treated diabetic group), diabetic rats received 1.2 ml flaxseed oil/kg b.w./day orally10 by stomach tube. Samples collection. After the experimental period (8 weeks), animals were kept fasting for 12 h, then anesthetized, and the blood was withdrawn from the retro-orbital venous plexus of the eye using heparinized capillary tubes. Blood samples were collected, left to clot, and then centrifuged at 3000 r/min for 15 min to separate the serum. The serum was stored at 20 C for pentosidine determination. Blood was centrifuged at 2000 r/min for 10 min using a cooling centrifuge. Plasma was separated and immediately frozen for other biochemical parameters. Brains were removed quickly and placed in iced normal saline, perfused with the same solution to remove blood cells, plotted on filter paper, and frozen at 80 C until use for estimation of brain monoamines, MDA, and nitric oxide (NO). Preparation of tissue homogenate. The frozen tissues were cut into small pieces and homogenized in phosphate buffer (pH 7.4), then centrifuged at 4000 r/min for 15 min at 4 C, and the supernatant was removed for estimation of chemical parameters.11 Biochemical assays. Freshly prepared serum was used for determination of blood glucose according to Trinder.12 Plasma insulin level was estimated by enzyme-linked immunosorbent assay (ELISA) kit.13 Reverse-phase HPLC was used for the determination of the plasma pentosidine content. Briefly, 50 ml of plasma were lyophilized and then hydrolyzed by 50 ml of 6 N hydrochloric acid at 110 C under nitrogen atmosphere for 16 h, subsequently neutralized with 100 ml of 5 N sodium hydroxide and 200 ml of 0.5 M phosphate buffer (pH 7.4), and then filtered through a 0.45-mm Millipore filter (Billerica, Massachusetts, USA) and diluted 20-fold with phosphatebuffered saline. Filtered samples (50 ml) were then
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Table 1. Blood glucose and insulin in the different studied groups. Groups Parameter
Control group (n ¼ 15)
Flaxseed oil (n ¼ 15)
Diabetic (n ¼ 15)
Diabetic flaxseed (n ¼ 15)
Glucose (mg/dl) Insulin (mIU/ml)
76.66 + 3.9a 10.5 + 0.8
77.51 + 2.4a 10.7 + 1.3
248.89 + 8.3b 8.5 + 1.1
181.4 + 4.7a,b 10.1 + 1.7
Values are expressed as mean + SE. n denotes number of rats in each group. a p < 0.05: significant difference compared with diabetic group. b p < 0.05: significant difference compared with the control group.
injected into C18 reverse phase analytical column (218TP104, Vydac; Separation Group, Hesperia, California, USA) using a fluorescence detector at excitation/emission wavelengths of 335/385 nm14 by gradient method using auto nitrile/water as mobile phase with a flow rate 1 ml/min. Brain MDA was measured using thiobarbituric acid reaction.15 The protein content of the homogenate was determined by the method of Lowry et al.16 using bovine serum albumin as standard. Total antioxidant capacity (TAC) was measured kinetically using commercial kits purchased from Biodiagnostics (Cairo, Egypt).17 Determination of advanced oxidation protein products (AOPPs)18 was done with double antibody sandwich ELISA. The kit was obtained from Glory Science Co. Ltd (Del Rio, Texas, USA). Determination of brain monoamines. Brain homogenate used for the determination of brain norepinephrine, dopamine, and serotonin was carried out using HPLC system, Agilent technologies 1100 series (Santa Clara, California, USA), equipped with a quaternary pump (G131A model). Separation was achieved on octadecylsilyl-reversed phase column (C18, 25 0.46 cm2; internal diameter: 5 mm). The mobile phase consisted of potassium phosphate buffer/methanol 97/3 (v/v) and was delivered at a flow rate of 1.5 ml/min. Ultraviolet detection was performed at 270 nm, and the injection volume was 20 ml. The concentration of both catecholamines and serotonin was determined by external standard method using peak areas. Serial dilutions of standards were injected and their peak areas were determined. A linear standard curve was constructed by plotting peak areas versus the corresponding concentrations. The concentration in samples was obtained from the curve.19
variance using Statistical Package for the Social Sciences (Version 12). Duncan’s new multiple range test was used to assess differences between means. Pearson’s correlation test was used to assess correlations between means. A significant difference was considered at the level of p < 0.05.
Results Diabetes was confirmed by estimating the blood glucose levels in the diabetic and the diabetic flaxseed groups. The diabetic and the diabetic flaxseed groups showed higher glucose levels than the control group (Table 1). There were no significant changes of plasma insulin level in all groups. As shown in Table 2, the serum level of pentosidine was significantly increased in the diabetic rats than in controls, but after treatment by flaxseed oil this value significantly decreased. Plasma AOPP level increases significantly in the diabetic group compared with the control group; on the other hand, plasma TAC level decreases significantly in diabetics group than controls, while the two parameter levels improved after treatment with flaxseed oil (Table 2). The levels of brain MDA and NO were significantly increased in the diabetic group compared with the control group; however, these increases were significantly attenuated after treatment with flaxseed oil. Compared with the control group, brain norepinephrine and dopamine levels were significantly increased in the diabetic group; however, flaxseed oil significantly decreased dopamine level and tended to decrease norepinephrine level compared with the diabetic group (Table 3). On the other hand, there was no significant change in serotonin level in all groups. Our study indicated that there is a positive correlation between MDA, NO, and all parameters in the study (Table 4). Serotonin has not been changed in treated groups.
Statistical analysis
Discussion
All data were expressed as mean + standard error. Data were analyzed using one-way analysis of
In this study, the rats treated with STZ developed hyperglycemia. The increase in the serum glucose
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Human and Experimental Toxicology
Table 2. Oxidants/antioxidants parameters in the different studied groups. Groups Parameter
Control group (n ¼ 15)
Brain MDA (nmol/g tissue) Serum pentosidine (pM) Plasma AOPP (ng/ml) Brain NO (mmol/g tissue) Plasma TAC (mM/L)
29.73 0.19 10.33 3.03 1.92
Flaxseed oil (n ¼ 15)
+ 1.52a + 0.01a + 0.24a + 0.19a + 0.07a
33.64 + 0.08 + 10.43 + 4.16 + 1.79 +
1.57a 0.01a,b 0.15a 0.16a,b 0.03a
Diabetic (n ¼ 15) 73.28 + 1.05 + 15.08 + 8.04 + 1.23 +
Diabetic flaxseed (n ¼ 15)
2.92b 0.02b 0.11b 0.20b 0.04b
46.49 0.88 13.19 5.68 1.57
+ 1.49a,b + 0.02a,b + 0.32a,b + 0.26a,b + 0.04a,b
MDA: malondialdehyde; AOPP: advanced oxidation protein product; NO: nitric oxide; SE: standard error; n: number of rats in each group; TAC: total antioxidant capacity. Values are expressed as mean + SE. a p < 0.05: significant difference compared with diabetic group. b p < 0.05: significant difference compared with the control group.
Table 3. Brain monoamines parameters in the different studied groups. Groups Parameter
Control group (n ¼ 15)
Flaxseed oil (n ¼ 15)
Diabetic (n ¼ 15)
Diabetic flaxseed (n ¼ 15)
Norepinephrine (ng/g tissue) Dopamine (ng/g tissue) Serotonin (mg/g tissue)
731 + 35a 31.00 + 1.63a 3.73 + 0.25
705 + 29.65a 30.7 + 3.01a 3.80 + 0.25
940 + 10.68b 52.2 + 2.82b 4.40 + 0.14
892 + 36.41b 40.8 + 2.78a,b 4.2 + 0.47
Values are expressed as mean + SE. n denotes number of rats in each group. a p < 0.05: significant difference compared with diabetic group. b p < 0.05: significant difference compared with the control group.
Table 4. Correlation between MDA, NO, and all parameters in the study. NO
MDA
r ¼ 0.908 p ¼ 0.000 MDA r ¼ 0.908 – p ¼ 0.000 – NO
–
Total antioxidants capacity Serotonin Dopamine Norepinephrine pentosidine r¼ p¼ r¼ p¼
0.827 0.000 0.879 0.000
r¼ p¼ r¼ p¼
0.348 r 0.028 P 0.282 r 0.078 p
¼ 0.692 ¼ 0.000 ¼ 0.728 ¼ 0.000
r p r p
¼ 0.664 ¼ 0.000 ¼ 0.598 ¼ 0.000
r p r p
¼ 0.861 ¼ 0.000 ¼ 0.834 ¼ 0.000
AOPP r p r p
¼ 0.883 ¼ 0.000 ¼ 0.871 ¼ 0.000
NO: nitric oxide; MDA: malondialdehyde; AOPP: advanced oxidation protein product. p < 0.05: correlation is significant.
could be due to a decrease in the levels of serum insulin as a result of destruction of the pancreas by STZ and generation of ROS.20 This study demonstrated that in diabetic rats brain neurotransmitters significantly increased, which may be related to oxidative stress since a positive correlation was observed in this study between MDA, NO, and neurotransmitters. This finding was confirmed by other studies.19,21 Araki and Nishikawa22 demonstrated that increased oxidative stress could be one of the common pathogenic factors of diabetic complications (like peripheral neuropathy) as well as in neurodegenerative disorders like Alzheimer’s disease and Parkinson’s disease.23 Oxidative damage is generally
attributed to the formation of highly reactive OH, which leads to severe oxidative damage of the cells’ components like lipids, proteins, and DNA.24 Efficiency of enzymatic and nonenzymatic antioxidative systems could be detected by the determination of single components of this system or by the so-called TAC.17 Recent study demonstrated that neurons develop hyperexcitability in STZ-induced diabetic rats; this may be attributed to certain disturbances in neurotransmitters pattern.21 Consequently, tissue cells isolated from hyperglycemic animals demonstrated increase in norepinephrine and dopamine production/secretion.25
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Badawy et al.
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Firoozrai et al.26 reported that MDA is produced by breakdown of lipids under oxidative stress conditions. Evidence of lipid peroxidation has been reported to be present in a number of diabetic complications27 and it significantly contributes to endothelial dysfunction, which is a key event in the pathogenesis of diabetic microvascular disease. In this study, the lipid peroxidation marker (MDA) was elevated in diabetic rats as reported previously.28 The increase in lipid peroxidation might be a reflection in enzymatic and nonenzymatic antioxidants of defense systems.8 This study demonstrated that diabetic rats that were treated with flaxseed oil enriched with omega-3 significantly decrease brain norepinephrine and dopamine levels. In addition, there was a significant decrease in glucose, MDA, and NO levels that indicate a decrease in oxidative stress. The result of this study suggested that flaxseed oil contains a free radical scavenging activity, which could exert a beneficial effect against pathological alterations caused by the presence of O 2 and OH . Oxidative stress plays a central role in the pathogenesis of metabolic diseases like diabetes mellitus and its complications (like peripheral neuropathy) due to its great oxygen consumption, high lipid and poor antioxidants defense. Oxygen free radicals attack objects on the polyunsaturated components of membranes and may cause a serious organizational dysfunction within cells and tissues. It has been suggested that the use of omega-3 polyunsaturated fatty acids (PUFAs) may have ameliorating effect on such damage by two possible ways. First, omega-3 PUFA may increase the level of catalase within the peroxisome and in the cytoplasm resulting in enhanced defense against free oxygen radicals. Second, omega-3 PUFAs, which have been supplemented, may be replaced with PUFA components of the membranes that had been attacked by oxygen free radicals.21 In our study, pentosidine is significantly higher in diabetic rats than the controls. This result is in agreement with Kerkeni et al.29 who demonstrated that pentosidine might accelerate the development of microvascular complications both by accumulation of AGEs in the vessel walls and by causing endothelial dysfunction, as mediated by the AGE-AGEreceptor axis activity. The mean levels of pentosidine in diabetic group that was modulated with flaxseed oil orally were significantly decreased as compared to the diabetic group but still not reached the levels of the
control group. The effectiveness of flaxseed oil in diabetes could be because of its antioxidant activity.30 Recently, Yamagishi et al.31 showed the role of AGEs and oxidative stress in vascular complications in diabetes. The authors demonstrated that AGEs induce mesangial cell loss and dysfunction, glomerulosclerosis, and tubulointerstitial fibrosis, taking into account the fact that patients with diabetes are prone to microvascular disease. In the same line, the elevation of AOPP in diabetic rats of this study may be due to the generation of ROS, this result is in agreement with the results of Abou-Seif and Youssef32 who reported that AOPPs are formed during oxidative stress by the action of chloramines produced by myeloperoxidase in activated neutrophils and is accumulated in biological systems and thus caus damage to biological membranes.
Conclusion STZ-induced diabetes mellitus is mediated through ROS and prevention of diabetic complications by flaxseed oil is due to its antioxidant activity. Acknowledgment Authors are grateful to the National Research Centre, Giza, Egypt, for providing space to carry out this work.
Funding This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.
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