RUTIN RESTORE BIOCHEMICAL CHANGES, OXIDATIVE STRESS AND BETATROPHIN LEVEL IN STZ-INDUCED DIABETIC RATS

Objective: Diabetes mellitus (DM) is associated with long-term damage, dysfunction, of various organs. Study aims to assessrole of rutin on experimentally induced diabetes. Methods: 50 adult male albino rats divided into 5 groups. Group I (control group, rats were orally administered with 1 ml saline daily). Group II (DMSO group, rats were orally administered with 0.2 % DMSO for 60 d orally). Group III (positive control, animals were injected intraperitoneally with 60 mg/kg b. wtstreptozotocin followed by intraperitoneal injection with 120 mg/kg b. wt of Nicotinamide after 15 min). Group IV (therapeutic group, diabetic rats treated with 100 mg/kg b. wt of rutin for 60 d orally). Group V (standard group, diabetic animals treated with 100 mg/kg b. wt of metformin for 60 d orally). At the end of the experimental period blood serum and plasma, liver, kidney and pancreatic tissues were collected. Results: Diabetic rats showed a significant increase in plasma glucose, serum urea, creatinine, cholesterol and triglyceride. Also, induced oxidative stress as pointed out an increase in MDA level, decrease in GSH level, GST and CAT activities in compared to control group. Also, showed an increase in plasma and tissues levels of betatrophin. Oral administration of rutin cause decrease in elevated biochemical and oxidative stress parameters. Also, decrease betatrophin level when compared with diabetic rats. Our results were confirmed by histopathological examination of different tissues. Conclusion: This study suggests that rutin exihibit santihyperglycemic and antioxidant activity in streptozotocin-induced diabetic rats.


INTRODUCTION
Diabetes mellitus (DM) is one of the most important health problems in all world. It is the seventh cause of death all over the world [1]. It was documented that 25% of the world population having DM.
DM is a metabolic disease characterized by chronic high blood glucose with an imbalance in metabolism of carbohydrates, proteins and fat caused by impair in insulin secretion, insulin action, or both. It is symptoms were thirst, urination, impair vision, and decrease in weight.

Streptozotocin (STZ) is a deoxy-s [((methyl-nitrosoamino) carbonyl)
-amino]-D glucopyranose molecule that caused toxic action on β cells and induced DM in most laboratory animals [2]. STZ enters the pancreatic β-cells through a glucose transporter-GLUT2 and induces alkylation of genetic material DNA. In addition to, STZ caused activation of poly adenosine diphosphate (ADP) ribosylation and nitric oxide (NO) release. Finally of STZ action caused pancreatic βcells destroy by necrosis. The real mechanism of its toxicity is still unclear, it is proposed that site of action is at nuclear DNA. Through the STZ metabolism, highly reactive carbonium ions (CH+ 5 ) are produced, that induce alkylation of DNA bases and also STZ may damage the membrane of β cell and break the DNA strand which causes activation of poly (ADP-ribose) synthetase and NAD decrease, which leads to cell death [3].
Rutin is considered to be a very good antioxidant because of its ability to bind free radicals and metal ions [6]. Rutin is capable of chelating iron ions (II and III valence), which can initiate oxygen free-radical formation [7]. The aglycone of rutin has a protective effect by binding to free radicals during reperfusion injury of ischemic tissues [8]. Rutin can also be used as an anti-inflammatory agent because of binding of free radicals that prevents the induction of inflammatory cytokine transcription factors [9]. Thus, rutinis important in treatment chronic inflammatory diseases [10].

Animal management
Adult male albino rats, weighing 180-200 g, were obtained from the Experimental Animal Care Center from Cairo university and were keeped in cages at experimental animal house of faculty of Science, Zagazig University under regulated environmental conditions (25 °C and a 12 h light/dark cycle) 7 d before starting the experiment.

Diabetic model
Diabetes was induced by STZ, purchased from Sigma Chemical Co. (St. Louis, MO, USA).
Induction of Type 2 diabetes (T2DM) by single intraperitoneal (i. P) injection of STZ at a dose of 60 mg/kg b. wt in fasting rats followed by the i. P injection of nicotinamide (NIC) at a dose of 120 mg/kg b. wt after 15 min. STZ was dissolved in freshly prepared cold citrate buffer (100 mmol, pH= 4.5) to immediate use through five minutes. While nicotinamide was dissolved in 0.9% saline of sodium chloride [11]. Blood glucose levels in all animals were measured after 72 h of drug administration and rats of fasting blood glucose levels higher than 250 mg/dl were considered to be diabetic and used for the further study [12].

Rutin dose selection and treatment
Rutin was obtained from Sigma Chemical Co (St. Louis, MO, USA).
The rats were orally administrated with Rutin at a dose of 100 mg/kg each day dissolved in 0.2 % DMSO [13].

Experimental design
To accomplish the ultimate goal of this study, after the acclimatization period of 7 d with a standard basal diet, a total of 50 adult male albino rats were classified into five groups with 10 animals in each group.
Group I (control Group): Rats were administrated orally with 1 ml single saline dose.
Group II (DMSO group): Rats were administrated by gavaging 1 ml of 0.2% DMSO for 60 d.
Group III (Positive control): Rats received STZ (60 mg/kg b. wt) followed by the i. p administration of Nicotinamide (120 mg/kg b. wt) after 15 min.
Group IV (Therapeutic Group):Rats were induced for DM. After 1 w of DM induction, animals were post-treated with Rutin (100 mg/kg daily for 60 d orally) [13].
GroupV (standard therapeutic): were induced for DM. After 1 w of DM induction, animals were post-treated with metformin (100 mg/kg daily for 60 d orally [14].
Doses of rutin and metformin were adjusted every week according to any change in body weight to maintain the same dose per each kg body weight of rat during the entire period of study for each group.

Collection and sampling of blood
At the end of the study and after last treatment, rats were fasted for 12 h; blood samples were collected from the retro-orbital venous plexus under light ether anesthesia. Where blood samples were collected in three different tubes, first tube containing sodium fluoride for blood glucose estimation, second tube containing EDTA to obtain plasma and third empty tube to obtain serum by centrifugation at 4000 rpm for 20 min. Serum and plasma were transferred into eppendorff tubes and stored frozen at-20 °C til an analysis of different biochemical measurements (glucose, liver, kidney lipid function tests and betatrophin).

Tissue sample
After blood collection, animals were killed by cervical decapitation and different tissues (liver, kidney, and pancrease) were excised from animals, rinsed in with ice-cold phosphate-buffered saline (pH 7.4) to flush out any blood.
The first part of different tissue samples was homogenized with icecold phosphate-buffered saline (pH 7.4) to prepare a 10% (w/v) tissue homogenate for determination MDA and GSH levels. Also, CAT and GST activities and estimation of level of betatrophin.
Second part of different tissue samples was used for histopathological study.

• Estimation of biochemical parameters
Plasma glucose was performed by glucose oxidase peroxidase activity using commercial kit derived from Elitech clinical system, france [15]. Serum urea was measured by Berthelot emzymatic colorimetric method [16] and creatinine was measured by Buffered kinetic jaffè reaction without deproteinization [17]. Also serum cholesterol concentration was determinated by CHOD-POD enzymatic colorimetric method [18] while serum triglyceride concentration was measured by GPO-PAP enzymatic colorimetric method [19].

Histopathological examination
Different tissues were then immersed with molted paraffin wax, then embedded and blocked out. Paraffin sections (4-5 um) were stained with hematoxylin and eosin then examined through light electric microscope [25].

Statistical analysis
All results were analyzed by SPSS software (SPSS, ver.14.00, USA). Data were expressed as mean±SEM. Comparison of mean values of studied variables among different groups was done using ANOVA test. P<0.05 was considered to be significant [26].

Effect of rutin on body weight
The initial and final body weight was given in (table 1). There was significant decrease (p<0.001) in finial body weight of diabetic induced group (positive group) which amounted to-31.29 % when compared to control group, while groups treated with rutin and metformin showed slight decrease in the final body weight which was statistically non-significant in compared to control group which amounted to-6.77 % and-2.9 % respectively (P˃0.05).

Effect of rutinon biochemical parameter
Result presented in (

Effect of rutin on levels of betatrophin in plasma and different tissues
Result in (table 6) indicated significant increase of levels of betatrophin in plasma (13.76 %, p>0.01), tissues of liver (23.6 %, P= 0.001), kidney (72.23 %, p>0.001) and pancrease (139.2 %, p>0.001) in positive group when compared to control group. but this elevation was reduced in treatment with rutin (therapeutic group) and metformin (standard group).

Histopathological examination
Histology ofliver, kidney and pancreatic tissues were studied.
The normal pancrease section of control and DMSO group showed healthy n parenchyma, normal pancreatic acini and islets. In positive group showed congestion of the blood vessels with thickened wall with fibrous connective tissue proliferation. Treapeutic and standard group showed healthy parenchyma, normal acini and islets ( fig. 2).
The normal histological liver section in control group and DMSO showed healty liver parenchyma, normal hepatocytes and blood sinusoids. In positive group it showed change in the portal tracts and congested hepatoportal blood vessel. In therapeutic and standard groups showed healthy liver parenchyma, normal hepatocytes and blood sinusoids (fig. 3).
The kidney section of control and DMSO group showed healthy renal parenchyma, normal glomeruli and renal tubules. Positive group showed congestion in interstitial blood vessel with thickened wall together with degeneration changes in both glomeruli and renal tubules. Therapeutic and standard group showed healthy renal parenchyma normal glomeruli and renal tubules ( fig. 4).

DISCUSSION
Diabetes mellitus is a heterogeneous metabolic disease described by high blood glucose caused by defect in insulin secretion, resistance to insulin action or both [27]. Streptozotocin (STZ) is agent which affects insulin secretion it decrease it and causes a condition of insulin dependent diabetes mellitus. The selective pancreatic β-cell toxicity of STZ related to the glucose moiety in its chemical structure that enables STZ for entering the β-cell via the low affinity GLUT2 glucose transporter in the plasma membrane [28].
The present work investigate the role of rutin in treatment of type 2 diabetes induced by STZ in rats.
Our data illustrated that significant decrease (p<0.001) in finial body weight of diabetic induced group (positive group) when compared with control group. While group treated with rutin and metformin showed non-significant difference in final body weight in treated groups in compared to control group.
Our result in agreement with who showed significant decrease in body weight of diabetic rats in compared to control group [29].
The decrease in weight of body is related to the increase of blood glucose with decrease of insulin level, decrease of tissue proteins, and enhancement of muscle wasting in STZ diabetic animals [30]. DM is accompanied by increased glycogen breakdown, lipolysis, and gluconeogenesis and these metabolic procedures bring about muscles squandering and loss of tissue protein. The body depends on insulin as a major anabolic hormone. The inhibition of insulin secretion caused metabolic disorders of glucose and also lipids and protein. The decline and inadequacy of insulin changed over anabolism to catabolism of proteins and lipids. Building of glucose relies upon proteolysis and gluconeogenic amino acids by liver. Induction of negative nitrogen equalization credited to the catabolism of proteins and lipids; along these lines the hunger and polyphagia were expanded [31].
Rutin administration to diabetic rats improved body weight and this could be because of a superior control of the hyperglycaemic state in the diabetic rats and it is synergistic anti-inflammatory and against oxidative Properties [32]. Diminished levels of blood glucose could improve body weight in streptozotocin-diabetic rats [32].
Streptozotocin administration to rats indicated critical (P˂ 0.001) expanded blood glucose. Rutin treated group exhibited a decline in plasma glucose when compared with control group.
Our outcome in accordance with who found that diabetic animals that administrated rutin had a decrease of 20.5% in the glucose levels when contrasted and beginning treatment (p<0.05) [33] Renal illness is one of the most widely recognized and extreme difficulties of diabetes [34]. Our data found that the positive control group showed high elevation (p>0.001) in serum urea, creatinine, cholesterol and triglyceride (p>0.001) when compared to control group. On the other hand, treated group with rutin (therapeutic) and metformin (standard) groups showed significant decrease in comparison with positive group.
Rutin diminishes the levels of Serum Urea and Serum Creatinine in diabetes mellitus by uprightness of its antioxidant property [37].
Serum lipid levels are commonly elevated in diabetes mellitus and such an elevation speaks to a hazard factor for coronary heart disease [38].
Previous studies investigated that, there was a high increase in serum non-esterified cholesterol, triglycerides and phospholipids in STZ-induced diabetic rats, accompanied by a decrease in highdensity lipoprotein (HDL)-cholesterol [39].
Our outcomes are in accordance with who announced that total cholesterol and LDL-c levels in serum of the diabetic rats treated with Rutin were fundamentally lower in the diabetic rats (p<0.05 and p<0.01, respectively). These values were considerably more fundamentally diminished when contrasted with diabetic rats treated. Moreover showed the administration of rutin encourages lipid metabolism in diabetic rats so decrease levels of triglyceride and cholesterol [33].
DM is related to oxidative stress occurring as an outcome of expanded development of free radicals, such as superoxide (O2-) and hydroxyl (OH) radicals, and lower activity of antioxidant defense systems [40]. Reactive oxygen species (ROS) can negativelly influence different cell biomolecules as protein, RNA and DNA making damage to tissues and organs, so to prevent cellular damage induced by ROS, the organism has a lot of antoxidative defense system, such as the non-enzymatic (mainly GSH) and enzymatic antioxidant defenses for example (GST, CAT, SOD, GR, and GPx) which consider the key enzymes in elimination of free radicals.
Our present study approved oxidative stress in liver, kidney and pancrease and showed that oxidative stress as indicated by increased production of MDA, accompanying with decreased activities of antioxidants including GST, CAT and GSH level in positive control group when compared with control group (table 3,4,5).
Our data are in agreement with previous findings that showed streptozotocin (STZ)-induced diabetic rats caused increased MDA level that reflect the levels of tissue lipid peroxidation and decreased SOD activity that scavenge superoxide products in kidney tissue [36]. Glucose is oxidized to reactive ketoaldehyde and superoxide radicals. On the off chance that it isn't decayed by CAT or GSH peroxidase it causes generation of responsive hydroxyl radicals. Excess amounts of free radicals damage cellular proteins and nucleic acids by attaching to them [41].
The GST catalyzes the conjugation of glutathione to a wide range of electrophiles and support a protective mechanism against hyperglycemia mediated oxidative stress. The GST is critical in the protection of cells from reactive species because they utilize a wide variety of products of oxidative stress as substrates [42].
Rutin scavenges free radicals and decreases superoxide radical formation in addition to enhance the activity of antioxidant enzymes, glutathione peroxidase and reductase to maintain the levels of the reduced glutathione, which is a biological antioxidant [43].
Previous data showed that oral treatment with rutin to diabetic rats for a period of 6 w showed significant ameliorative effects on GSH, CAT, SOD, GPx values in liver tissue. The elevated activities of antioxidant enzymes may act as an added additional remuneration system to keep up the cell integrity and protection against free radical damage. This showed that free radical scavenging ability of rutin could exert a beneficial action against pathogenic alterations caused O2 and OH [32].
Betatrophin is a hormone gotten from liver and white adipose tissue that was found to advance beta cell multiplication in a mouse model of insulin resistance induced by S961, an antagonist of the insulin receptor [44]. In our result, the betatrophin level increased in positive control group in both plasma and tissues (liver, kidney and pancrease) when compared with control group. These result in agreement with who showed that serum betatrophin was significantly higher in type 2 diabetes mellitus patients as compared to control group [45]. Also who stated that serum betatrophin levels were significantly increased in type 2 diabetic patients compared to non-diabetic subjects [46] and [47]. This correlates with the results of our study. Also, who found that high circulating betatrophin levels were discovered uniquely in patients with T2DM however not in prediabetic subjects who as of now have insulin resistance [48].
Opposed with who expressed that no connections among betatrophin and glycemic control indices for example FBG and HbA1c.
Recommending that betatrophin probably won't assume a significant relation in controlling glucose homeostasis. Also, showed positive relationship of circulating betratophin levels with blood lipids levels in subjects with impaired glucose tolerance (IGT) or type 2 diabetes, and in healthy subjects. Since it is recommended that betatrophin is related with circulating low-density lipoprotein cholesterol (LDL-C), highdensity lipoprotein cholesterol (HDL-C), and triglyceride (TG) probably via the respectively different mechanisms [49].
The formation of betatrophin in liver is highly produced under insulin resistance caused by uptake of an insulin receptor blocker such as was transporter 2 (SGLT2) inhibitors [50].
Our histopathological examination showed severe alterations of liver, kidney and pancreatic tissues were observed in untreated diabetic rats. Also, rutin attenuated the histopathological changes in STZ diabetic rats.
Our results are in accordance with who stated that pancreatic tissues of diabetic control rats showed a decrease of Langerhans islet size and multiple degeneration and injuries. In addition to, the number of β-cells was reduced, and some necrosis and destruction were found [51].
Also, who stated that the structure of renal tissue was damaged in STZ diabetic rats [36].
Generally, the present obtained findings confirm that the influences of rutin is attributed to the antioxidant properties.

CONCLUSION
Natural food and medicinal plants and supplements have the potential to become valuable complementary therapy in the treatment of DM and its complications. The present study evaluated the hypoglycemic activitiy of rutin on diabetic male rats. Based on the present experimental data, it can be concluded that this rutin improve the physiological and histological changes induced by STZ in the experimental animals.