Comparative Assessment of Portulaca oleracea , Omega - 3 Fatty Acids, and Combination of Selenium Plus Vitamin E on Histopathology of Pancreas in Diabetic Rats

present study was to compare the serum glucose levels and histopathological effects on PO, Omega-3, and a combination of Selenium and Vitamin E in the pancreas of adult male diabetic rats. Materials and methods: A total of 96 adult male Wistar rats, weighing approximately 220 g were used. The rats were divided into four groups with 4 replicates for each group accidentally. The rats were diabetic via a 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 was fed the standard ration plus Portulaca oleracea extract (1.5 mg/kg/day/orally), the third group of diabetic rats was fed standard ration plus omega 3 (500 mg/kg/day/orally), the fourth group of diabetic rats was 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 the 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 the treatment groups was higher than in the control group. The percentage of beta cells in the third group was higher than in the fourth group and control group. The mean diameter of pancreatic islets in omega - 3 - treated rats was higher than in other groups. The insulin level increased in treated rats in comparison with the rats that 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.


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 nails and hair 1,2 . 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 tissues 3,4. 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 tissue damages 5 among which the high level of ROS in pancreatic islets and changes in oxidative stress markers in laboratory animals can be noted 6 . Aerobic cells can be protected against free radicals particularly ROS by antioxidants compounds, such as glutathione, Vitamins E and C, as well as super Oxide Dismutase (SOD), glutathione Peroxidase (GPx), and catalase enzymes 7,8 . On the other hand, studies have also shown a significant decline both in nonenzymatic antioxidants ( including rehabilitated glutathione (GSH) and Vitamin E) and enzymatic antioxidants (such as SOD, catalase, and GPx in diabetic rats) 9,10 . It is also shown that free radicals can cause diabetic damage in different organs like the pancreas and liver by declining SOD, catalase, and antioxidant activities 10,11 . Free radicals can also damage the unsaturated fatty acid in cell membranes 12 . The combination of fatty acids in cell membranes can also affect cell membranerelated phenomena, such as the interaction between insulin and its receptors 13 . In addition, it has been indicated that the fatty acid composition of membrane phospholipids in insulin target tissues like the liver and skeletal muscles, which is an important factor affecting both insulin secretion and its biological activity 14 . 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 hemoglobin 11 . Hence, it is beneficial to use antioxidant compounds (particularly natural antioxidants) and omega-3 fatty acids to prevent oxidative damage.
Vitamin E plus Selenium is one of the important food compounds which not only have high antioxidant properties but it can also affect different biological processes of the body. Shamsi et al. have also shown that Vitamin E decreases blood glucose in diabetic rats and reduces diabetic disorders 15 . It was also reported that Vitamin E declines Malondialdehyde (MDA) and increases GSH and SOD in diabetic rats 16 .
Vitamin E prevents lipid peroxidation and protects cells against peroxide radicals thus it is the most important antioxidant in the biological membrane which can neutralize free radicals 17 . Selenium is the only trace element that enters the genetic code as selenocysteine. This element can be extensively found in selenoproteins, namely GPx enzyme, through which Selenium antioxidant effect is activated 18 . Reports available on the efficacy of Selenium in diabetes have indicated a decline in the effect of streptozotocin (STZ) by Selenium and its positive effect on GPX enzyme activity in laboratory rats 19,20 .
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, antiinflammatory, and antimicrobial) properties 21 .
Although bioactive compounds of Portulaca oleracea can have beneficial effects against diabetes 22 . There is little research addressing the anti-diabetic effects of this plant in previous years 23,24 . Thus, the aim of the 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.

Animals
A total of 96 male Wistar rats aged 2-3 months, with an 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 of dark/light cycle with appropriate ventilation in a particular cage. Out-and-out functions on animals, coincided with the university ethics committee. The rats were divided into four groups with 4 replicates for each group accidentally. The groups contained the control group of diabetic rats, which received the standard ration daily, the second group of diabetic rats was fed the 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 were 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 Kusumaningtyas 25 . The extracts were mixed and condensed with reduced pressure so that their volume reached 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 a dosage of 60 mg/kg which dissolved in buffer citrate 0.1 at pH=4.5. On the second day, blood samples were collected through the 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 the present study.

Data analysis method 2.4.1. 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 a 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 the pathology laboratory, Islamic Azad University, Karaj, Iran for investigating pathological changes.

Statistical analysis
The obtained data from the 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 the 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.

Blood sugar and insulin levels
The blood glucose level in control rats was 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 was significantly lower than the control group on day 28 (p < 0.05, Table 1).
Blood insulin in treated rats increased significantly in comparison with the rats of the 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 the 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 a 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 the control group in the fourth week (p < 0.05, Table 2). The diameter of pancreatic islets in the 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 in the other groups (p < 0.05, Table 2). At the 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).

Discussion
Although the pathogenesis of diabetes induced by chemicals and the inflammation of the pancreatic islets has not been fully identified 22 , streptozotocin causes damage to DNA molecules and beta cells by alkylating basal and alloxan when producing free radicals 26,27 . The penetration of mononuclear inflammatory cells, especially lymphocytes, can form an autoimmune reaction leading to the loss of cells in the pancreatic Islets, especially beta cells 28,29 .However, the inflammation process is a complementary component for an autoimmune response that is performed against beta cells in some animal models of diabetes 26 .
The results of the present study revealed a decrease in inflammation of pancreatic islets as well as a significant increase in insulin levels and hypoglycemia in the treated rats. Moreover, histopathological findings indicated the regeneration of pancreatic islets that were destroyed by STZ. Furthermore, changes in pancreatic tissue in untreated diabetic rats were similar to those of patients with type 1 (Insulin-dependent) diabetes mellitus.
Since there has been no report of suitable medication for the treatment of some acute and chronic pain conditions, especially in the state of diabetes mellitus with no side effects, researchers have focused on the medicinal plants and their effective extracted substances 30 .
Portulaca oleracea extract contains pharmacological active agents, such as alkaloids, glycosides, terpenoids, sterols, and flavonoids. It may be stated that some of these compounds can reduce the severity of autoimmune reactions and the inflammation process to some extent that leads to the destruction of beta cells. Consequently, the destruction of the remaining cells is prevented which provides ample opportunity for the proliferation of these cells and the regeneration of the pancreatic islets. It was found that the consumption of Portulaca oleracea extract caused the regeneration of pancreatic islets in diabetic rats with STZ due to the presence of flavonoids, such as quercetin, existing in the aerial parts of the plant which can release insulin by changes in Ca ++ metabolism 31 . These compounds have insulin-like properties and may cause beta cell regeneration 31 . The results of a study addressing the effect of Portulaca oleracea extracts on alloxan-induced diabetic rats have indicated a significant decrease in the Hemoglobin A1C (Hb A1C), serum levels of glucose, Tumor Necrosis Factor-alpha (TNF-α), and Interleukin 6 ( IL-6) of Portulaca oleracea pre-treated diabetic rats confirming that Portulaca oleracea is a general tissue-protective and regenerative agent 32 and also demonstrated the anti-diabetic effect of hydroethanolic extract Portulaca oleracea seeds on diabetic animals.
In an investigation by Dehghan et al., it was indicated that 16 weeks of aerobic training or/and Portulaca oleracea seed consumption were effective in the regulation of diabetic parameters and biomarkers associated with atherosclerosis in women with Type 2 Diabetes (T2D) 33 . Another relevant study revealed that diabetes significantly impaired brain abilities and swimming training and Portulaca oleracea synergistically reversed and ameliorated neurobehavioral dysfunction in type 2 diabetic rats 34 .
It should be noted that no significant toxic marks have yet been reported about Portulaca oleracea 35 . water, minerals, pectin, protein, carbohydrates, fatty acids, especially omega-3 unsaturated fatty acids, antioxidants, and numerous minerals (such as ferritin, copper, manganese, potassium, calcium, and cessation) are found in different parts of this plant 36,37 . Portulaca oleracea is the richest plant source of omega-3 fatty acids 36 . Its antioxidant compounds include Olefatoferol, Ascorbic acid, and Glutathione 4 . In addition, the antioxidant properties of the plant extract have been confirmed in laboratory studies 12 . In a study, it was found that aqueous and ethanolic extracts of Portulaca oleracea leaves can produce various antioxidants 38 . Moreover, quinoline as the main alkaloid of Portulaca oleracea had a significant stimulating effect on insulin secretion and improving glucose uptake 39 .
There are reports on the effectiveness of Selenium in diabetes as well as its significant role in reducing the effect of STZ, and enhancing the activity of GPx enzyme in rats 19,20 . However, Selenium has a narrow therapeutic index, and the increase in consumption can have toxic effects 40 .
Vitamin E is known as the most important anti-oxidant of biological membranes that can neutralize free radicals 17 . Jamilian and Ravanbakhsh 41 reported that Vitamin E plus omega-3 fatty acid supplementation in gestational diabetes mellitus (GDM) women had beneficial effects on biomarkers of inflammation and oxidative stress. Baburao Jain and Anand Jain 42 demonstrated that Vitamin E supplementation has an important role in delaying the onset of diabetic complications as well as slowing down the progression of the complications. Another meta-analysis indicated that the supplementation of Vitamin E may be a valuable strategy for controlling diabetes complications and enhancing antioxidant capacity 43 .
The essential fatty acids are reported to have a low level in different tissues of diabetic patients. Therefore, the use of omega-3 fatty acids can be effective in reducing the effects of fatty deficiencies 44 . The causes of low levels of essential fatty acids in diabetic patients are unclear, but some researchers assert that diabetics have less ability to convert linoleic acid into Eicosa Pentaenoic Acid and Docosa Hexaenoic Acid. In one of the latest studies on the antidiabetic effects of Vitamins C, A, and E as well as omega-3 fatty acids, it was found that lipid peroxidation and malondialdehyde levels were reduced due to the decreased production of free radicals or inhibition of oxidative damage 45 .

Conclusion
In treated diabetic rats with Portulaca oleracea, regenerated pancreatic islet levels were more than two groups of omega-3 and Vitamin E + Selenium. Moreover, inflammatory cells decrease in one pancreatic islet were better than omega-3 group. Minimum islets diameter in the omega-3 group on day 14 was more than other groups but it significantly increased at the end of the study, compared to the control group. Beta cells percentage in two groups of omega-3 and Portulaca oleracea was more than Vitamin E + Selenium group.
At the end of the study, blood glucose levels in the omega-3 group significantly decreased, compared to Portulaca oleracea extract and Vitamin E + Selenium groups. At the end of the study, blood insulin levels increased significantly in Portulaca oleracea extract group, compared to other groups.

Declarations
Arman Moshaveri and Pouria Ahmadi Simab wrote the draft of the manuscript. All authors check the final proof of the article and the statistical results.

Availability of data and materials
All data and related findings of the thesis are prepared for publishing in the present journal.