Mahdi Alyari Gavaher1, Daryoush Babazadeh2*, Alireza Sadeghi3, Veghar Hejazi3, Farhang Sasani4, Arman Moshavery1, and Pouria Ahmadi Simab5
- Faculty of Veterinary Medicine, Karaj Branch, Islamic Azad University, Karaj, Iran
- School of Veterinary Medicine, Shiraz University, Shiraz, Iran
- Tabriz University of Medical Sciences, Aras Branch, Tabriz, Iran
- Department of Veterinary Pathology, Faculty of Veterinary Medicine, Tehran University, Tehran, Iran
- Faculty of Veterinary Medicine, Sanandaj Branch, Islamic Azad University, Sanandaj, Iran
* Corresponding author: Daryoush Babazadeh, School of Veterinary Medicine, Shiraz University, Shiraz, Iran. Email: daryoush.babazadeh@shirazu.ac.ir
A B S T R A C T
Introduction: Antidiabetic effects of Portulaca oleracea (PO) plant, omega-3 and combination of Selenium and Vitamin E have been reported which could compensate defective insulin release and enhance antioxidant level. The purpose of the present study was comparative of serum glucose level and histopathological effects on PO, Omega-3 and combination of Selenium and Vitamin E in pancreas of adult male diabetic rats.
Materials and methods: 96 adults male Wistar rats, weighing approximately 220 g were used. The rats divided into four groups with 4 replicates for each group accidently. The rats were diabetic via single Injection of streptozotocin solution (60 mg/kg, IP injection). The groups contained the control group of diabetic rats, which received the standard ration daily, the second group of diabetic rats were fed standard ration plus Portulaca oleracea extract (1.5 mg/kg/day/orally) , the third group of diabetic rats were fed standard ration plus omega 3 (500 mg/kg/day/orally), the fourth group of diabetic rats were fed standard ration plus Vitamin E (400 iu/kg/day/orally) and Selenium (0.5 mg/kg/day/orally) for a period of 14 days. At the end of the study, the samples were taken for histopathological investigation of pancreas and serum glucose levels. The mean diameter of pancreatic islets and percentage of beta and alpha cells were calculated in all groups.
Results: The percentage of alpha cells in treatment groups were higher than the control group. The percentage of beta cells in the third group was higher than the fourth group and control group. The mean diameter of pancreatic islets in omega–3 treated rats was higher than other groups. The insulin level increased in treated rats in comparison with the rats were not treated significantly.
Conclusion: Diabetic male rats treated with omega-3 showed more positive effects on pancreatic islets and blood glucose compared to other treatment groups.
Keywords: Diabetes, Omega–3, Pancreas, Portulaca oleracea, Vitamin E
Introduction
Diabetes mellitus is a pathologic condition which causes extensive and non-physiological metabolic imbalance disorders, including an increase in blood glucose, and changes in carbohydrate, lipid, and protein metabolism in different body tissues, such as nail and hair1,2. An increase in blood glucose initiates a series of cascade reactions, which finally leads to an increase in free radicals’ production (including oxygen free radicals) in various body tissues3,4. The high potency of these compounds for chemical reactions damages cells and tissues. Several reports have been published concerning the involvement of Reactive Oxygen Species (ROS) in the tissue damages5 among which the high level of ROS in pancreatic islets and changes in oxidative stress markers in animals laboratory can be noted6. Aerobic cells can be protected against free radical particularly ROS by antioxidants compounds, such as glutathione, Vitamins E and C, as well as super Oxide Dismutase (SOD), glutathione Peroxidase (GPx), and catalase enzymes7,8. On the other hand, studies has also showed a significant decline both in non-enzymatic antioxidants ( including rehabilitated glutathione (GSH) and Vitamin E) and enzymatic antioxidants (such as SOD, catalase, and GPx in diabetic rat)9,10. It is also shown that the free radicals can cause diabetic damages in different organs like pancreas and liver by declining SOD, catalase, and antioxidants activities11,10. Free radicals can also damage the unsaturated fatty acid in cell membranes12. The combination of fatty acids in cell membranes can also affect cell membrane-related phenomena such as the interaction between insulin and its receptors13. In addition, it has been indicated that fatty acid composition of membrane phospholipids in insulin target tissues like liver and skeletal muscles, which is an important factor affecting both insulin secretion and its biological activity14. Red blood cells are also susceptible to oxidative damage, due to the presence of fatty acid in their membrane and high concentration of oxygen and hemoglobin11. Hence, it is beneficial to use antioxidants compounds (particularly natural antioxidants) and omega3 fatty acid to prevent the oxidative damage.
Vitamin E plus Selenium is one of the important foods compounds which not only have high antioxidant properties but it can also affect different biological processes of the body. Shamsi et al.15 has also shown that Vitamin E decreases blood glucose in diabetic rats and reduces diabetic disorders. It was also reported that Vitamin E declines Malondialdehyde (MDA) and increases GSH and SOD in diabetic rats16.
Vitamin E prevents lipid peroxidation and protect cells against peroxide radicals thus it is the most important antioxidant in biological membrane which can neutralize free radicals17. Selenium is the only trace element which enters the genetic code as selenocysteine. This element can be extensively found in selenoproteins, namely GPx enzyme, through which Selenium antioxidant effect is activated18. Reports available on the efficacy of Selenium in the diabetes have indicate a decline in the effect of streptozotocin (STZ) by Selenium and its positive effect on GPX enzyme activity in laboratory rats19,20.
Portulaca oleracea is a rich source of omega-3 polyunsaturated fatty acids (alpha-linolenic acid), different vitamins (A, C, and E), and minerals which has different pharmacological (such as antioxidant, anticancer, anti-inflammatory, and antimicrobial) properties21.
Although bioactive compounds of Portulaca oleracea can have beneficial effects against the diabetes22. There is a few research addressing the anti-diabetic effects of this plant in previous years23,24. Thus, the aim of present study was to compare the effect of consumption of Portulaca oleracea extracts with natural antioxidants (vitamin E + selenium) and omega3 fatty acids on the serum levels of glucose, blood insulin, and histopathology of the pancreatic tissues.
Materials and Methods
Animals
96 male Wistar rats aged 2-3 months, with average weight of 220 g have been bought from Razi institute, Iran, and kept in laboratory conditions with free access to water and commercial food daily. Experimental animals have been kept in standard cages with minimum 50 percent humidity, 24°C temperature and 12 hours dark/light cycle with appropriate ventilation in a particular cage. Out-and-out functions on animals, coincided on university morality committee. The rats divided into four groups with 4 replicates for each group accidently. The groups contained the control group of diabetic rats, which received the standard ration daily, the second group of diabetic rats were fed standard ration plus Portulaca oleracea extract (1.5 mg/kg/day) via gastric feeding tube daily, the third group of diabetic rats were fed standard ration plus omega 3 (500 mg/kg/day) via gastric feeding tube daily, the fourth group of diabetic rats were fed standard plus Vitamin E (400 iu/kg/day) and Selenium (0.5 mg/kg/day) via gastric feeding tube daily.
Extraction of Portulaca oleracea
Aforementioned atmospheric parts of Portulaca oleracea prepared from the farm of Islamic Azad University, Tabriz, Iran and kept in a dark glass battle at 10°C temperature away from direct sunlight. 250 g of the intended powder was extracted by ethanol-water solvent (70% ethanol-30% water) three times at normal laboratory temperature based on the method of Abdullah and Kusumaningtyas25. The extracts were mixed and condensed with reduced pressure so that its volume reached to 500 ml, which was equal to 0.5 g of the powder per milliliter was soluble. For further investigation, the extract was divided into equal volumes (25 ml) and stored at -20°C temperature.
Diabetes infusion
The rats were diabetic via IP Injection of STZ solution at dosage of 60 mg/kg which dissolved in buffer citrate 0.1 at pH=4.5. At second day, blood samples were collected through tail vein from animals, under anesthesia with chloroform. Rats with fasting blood sugar higher than 250 mg/dl were considered diabetic and were used in present study.
Data analysis method
Micrometric perusal methods
The pancreas tissue incisions were done and at least 10 islets were collected from each group. Then the large parts of the islets were measured by using graded eye lens and calibrated slide. The percentage proportion of the alfa and beta cells in each islet was counted.
Blood sample
The last day of treatment was done on day 28 and the samples of blood and pancreas tissue were collected under euthanasia with chloroform to determine serum glucose and blood insulin levels. The tissue samples were placed in 10% formalin and immediately referred to pathology laboratory, Islamic Azad University, Karaj, Iran for investigating the pathological changes.
Statistical analysis
The obtained data from histopathological study was analyzed by using SPSS software (version 19). The results were analyzed in one-way variance analysis (ANOVA) and reported as mean +_standard error. The statistical differences between the treatments and the control groups were checked via Tukey test at the significance level of p < 0.05.
Ethical approval
All procedures were approved by the Animal Care Committee of Veterinary Medicine, Karaj Branch, Islamic Azad University. The principles of laboratory animal care were followed, and specific international laws were observed.
Results
Blood sugar and insulin levels
The blood glucose level in control rats were significantly higher than treated rats with Portulaca oleracea and Vitamin E + Selenium on day 14 (p < 0.05, Table 1). The blood glucose level in treated rats were significantly lower than control group on day 28 (p < 0.05, Table 1).
Blood insulin in treated rats increased significantly in comparison with the rats of control group on days 14 and 28 (p < 0.05, Table 1).
Weight changes
After treatment of diabetic rats with Portulaca oleracea for four weeks, there was a significant difference (180 g) in weight of diabetic rats compared to omega-3 (165 g), Vitamin E + Selenium (172 g) and control groups (142 g,
p < 0.05, Table 1).
Histopathologic findings
In present study, Portulaca oleracea, omega-3 and Vitamin E + Selenium had crucial role in pancreatic islets regeneration. The minimum islets diameter in omega-3 group on day 14 was significantly more than other groups (p < 0.05). The regenerated pancreatic islets had increased significantly in treated diabetic rats with Portulaca oleracea in comparison with control group at fourth week (p < 0.05, Table 2). The diameter of pancreatic islets in control group decreased significantly in comparison with treated groups on day 28 (p < 0.05, Figure 1).
In treated diabetic rats with omega-3 and Portulaca oleracea, the percentage of beta cells was significantly more than Vitamin E+ Selenium and control groups on day 14 (p < 0.05, Table 2). In control group, significant degenerative changes were seen in number of beta cells on day 28 compared to other groups (p < 0.05, Figures 1–4).
The percentage of alpha cells in treated rats with Portulaca oleracea on day 14 was significantly more than the other groups (p < 0.05, Table 2). At end of the study the percentage of alpha cells in the control group showed a significant increase (p < 0.05) compared to other groups on day 28 (Table 2).
