INTRODUCTION

Oxidative stress processes have been suggested to be a contributory factor in complication of diabetes mellitus resulting from increased free radical production or reduced activity of antioxidant defenses or both (Rahimi et al., 2005). During diabetes disease, persistent hyperglycemia in all tissues causes increased production of free radicals, especially Receative Oxygen Molecules (ROM), from glucose auto-oxidation and protein glycosylation. These products have significant influence in the beginning and developing of complications of chronic diabetes (Astaneie et al., 2005). Thus, one of the aims in decreasing of diabetes complication is to use of antioxidant agents (Mohammadirad et al., 2011; Mehri et al., 2010; Momtaz and Abdollahi, 2010).

Selenium as an essential trace element plays a key role in selenoproteins and selenoenzymes structures such as Glutathione Peroxidase (GPx), selenoprotein-P and thioredoxin reductase (Klein, 2004; Zeng and Combs, 2008) that produce antioxidant effects (Miroliaee et al., 2011). The cerium oxide nanoparticles has been recently found helpful in rat diabetes by reducing oxidative stress (Pourkhalili et al., 2011).

In the present study, the effects of cerium oxide nanoparticles was tested and compared with its metal form and the combination form of cerium oxide nanoparticles/sodium selenite on process of oxidative stress in various organs of diabetic rats including brain, heart, lung and kidney.

MATERIALS AND METHODS

Materials: Tris base, 1,1,3,3-tetraethoxypropane (MDA), 5,5’dithiobis-2-nitro benzoic acid (DTNB), methanol (high performance liquid chromatography [HPLC]-grade), trichloroacetic acid (TCA), diethyl ether, tetrabutylammonium hydroxide (TBAHS), n-butanol, 2-thiobarbituric acid (TBA), KH₂PO₄ (analytical grade), 2,4,6-tripyridyl-s-triazine (TPTZ), sodium selenite, ketamin, xylazin and PBS (phosphate buffer solution from Merck (Tehran), 2,7-dichlorodihydrofluorescein diacetate (DCFD) from Sigma-Aldrich (Taufkirchen, Germany), cerium oxide nanoparticles from Navarrean Nenoproducts Technology (Spain), streptozotocine (STZ) from Pharmacia and Upjohn (USA) were used in this study.

Animals: A total of 36 adult male Wistar rats were obtained from Animal House of Faculty of Pharmacy, Tehran University of Medical Science (TUMS). Rats weighing 250-300 g were housed in polypropylene cages under standard conditions of temperature (25°C), relative humidity (50-55%), with free access to drinking water and food and 12 h light/dark cycle. All experiments were performed according to the animal welfare rules approved by TUMS Ethics Committee.

Experimental protocol and groups: Following 12 h of fasting, experimental diabetes was induced by intraperitoneal (ip) injection of dissolved STZ in citrate buffer (0.1 M, pH 4.5) at the single dose of 60 mg kg^-1. Blood glucose of rats were measured by a glucometer three days after injection of STZ and rats with blood glucose of more than (300 mg dL^-1) were selected and randomly divided into five groups with six rats in each group. The animals were treated for two consecutive weeks by ip injection as follows: Group 1 diabetic rats received saline (ip), group 2 diabetic rats received metal form of cerium oxide (60 mg kg^-1), group 3 diabetic rats received cerium oxide nanoparticles (60 mg kg^-1), group 4 diabetic rats received sodium selenite (5 μmol/kg/day), group 5 diabetic rats received combination of cerium oxide nanoparticles (60 mg kg^-1) and sodium selenite (5 μmol/kg/day). Effective doses of compounds were selected from earlier study (Pourkhalili et al., 2011). Moreover, one group including six rats was studied as the vehicle control group that received only ip injection of saline. Treatments were continued up to 2 weeks and animals were weighed daily and their blood glucose was measured using a glucometer. Then animals were anesthetized with ip injection of ketamine (4 mg/100 g) and xylazine (1 mg/100 g) mixture and then immediately, brain, heart, lung and kidney were removed, weighed and stored in -80°C. In the next stage, 100 mg of these tissues were homogenized in PBS (50 mM, pH 7) under ice cooling and then centrifuged at 3000 g for 30 min at 4°C. The supernatant was used to assay oxidative stress markers.

Measurement of reactive oxygen molecules (ROM): 2,7-Dichlorodihydrofluorescein diacetate (DcFD) was used to measure ROM production as previously set up in the lab and described in details (Momtaz et al., 2010). The sample was incubated with 5 μM DCFD at 37°C for 30 min in the dark. Then, fluorescence was read with 488 nm excitation and 525 nm emission using a fluorometer.

Measurement of lipid peroxidation (LPO): LPO in brain, heart, lung and kidney samples was determined by the reaction of TBA with lipid peroxides. Brain, heart, lung or kidney samples were mixed with trichloroacetic acid (20%) and the precipitate was dispersed into H₂SO₄ (0.05 M). TBA (0.2% in 2 M sodium sulfate) was added and heated for 30 min in boiling water bath. LPO adducts were extracted by n-butanol and absorbance was measured at 532 nm.

Measurement of total antioxidant power (TAP): TAP was determined by measuring its ability to reduce Fe³⁺ to Fe²⁺ established as the ferric-reducing antioxidant power (FRAP) test. The reagents included 300 mM acetate buffer (pH 3.6) with 16 mL acetic acid per liter of buffer solution, 10 mM TPTZ in 40 mM HCl and 20 mM FeCl₃. Working FRAP reagent was prepared as required by mixing 25 mL acetate buffer, 2.5 mL TPTZ solution and 2.5 mL FeCl₃ solution. Ten μL of H₂O diluted sample was then added to 300 mL freshly prepared reagent warmed at 37°C. The complex between Fe²⁺ and TPTZ gives a blue color with absorbance at 593 nm.

Measurement of total thiol molecules (TTM): A volume of homogenate (0.2 mL) was mixed with 0.6 mL of Tris-EDTA buffer (Tris base [0.25 M], EDTA [20 mM], pH 8.2) in a 10 mL test tube and then mixed with 40 mL of DTNB (10 mM) in methanol. The final volume was made up to 4.0 mL by adding 3.16 mL of methanol. The test tube, after capping, was centrifuged at 3000 g for 10 min at ambient temperature. After 15-20 min the color appeared. The absorbance of the supernatant was measured at 412 nm.

Protein measurements: Protein level was measured in tissues according to Bradford method. Briefly, homogenate samples mixed with Bradford reagent dye and after 5 min, the absorbance were determined at 595 nm using the spectrophotometer. Bovine Serum Albumin (BSA) was used as standard. Stress oxidative markers were presented per milligram protein of tissue homogenate.

Statistical analysis: Data were expressed as Means±SE and compared using one way analysis of variance (ANOVA) followed by Tukey multiple comparisons test. The significance level was tested at p<0.05.

RESULTS

Blood glucose and animal’s tissue weight: As data are shown in Table 1, the weight of diabetic rats (BW) remarkably decreased (p<0.05) and their blood glucose significantly raised as compared with controls (p<0.001). After use of cerium oxide nanoparticles/sodium selenite combination and also cerium oxide nanoparticles alone, an increase in animal’s weight (p<0.01 and p<0.05, respectively) was observed in comparison to diabetic rats. Also, a remarkable decrease in blood glucose (p<0.05) was shown only in the combination of sodium selenite and cerium oxide nanoparticles, when compared to diabetic rats. No significant change in animal weight was observed when using sodium selenite and the metal form of cerium oxide. The body weight/kidney weight index (BW/KW) in diabetic rats remarkably decreased (p<0.01) as compared with control animals.

Table 1:	Effects of various treatments on body weight (BW) and plasma glucose
Data are Mean±SE of six animals in each group, Significantly different from control at aaap<0.001, ap<0.05, Significantly different from diabetic control at bbp<0.01, bp<0.05, Significantly different from cerium oxide nanoparticle/sodium selenite combination at cp<0.05

Table 2:	Effects of various treatments on BW/KW (Body weight/Kidney weight), BW/BRW (Body weight/Brain weight) index
Data are Mean±SE of six animals in each group, Significantly different from control group at aaap<0.001, aap<0.01, ap<0.05, Significantly different from diabetic control group at bbbp<0.001, bbp<0.01, bp<0.05, Significantly different from cerium oxide nanoparticle/sodium selenite at cccp<0.001, ccp<0.01

No significant change in this index was observed by use of metal form of cerium oxide and sodium selenite. Following administration of cerium oxide nanoparticles and a combination of cerium oxide nanoparticles/ sodium selenite, an increase in this index (p<0.001, p<0.001, respectively) was observed in comparison to diabetic rats. Also, BW/BRW in diabetic rats significantly decreased (p<0.01) as compared to control animals. Only, administration of cerium oxide nanoparticle/sodium selenite combination reversed this index (p<0.05) in diabetic rats (Table 2). The similar results were seen in BW/HW. This index in diabetic rats significantly decreased (p<0.001) as compared to control animals. Only, administration of cerium oxide nanoparticle/sodium selenite combination reversed this index (BW/HW) in diabetic rats (p<0.01). No significant change in body weight/lung weight index (BW/LW) was observed in diabetic rats and the other groups as compared to control rats (Table 3).

Levels of ROM in tissues: There was a significant increase (p<0.001) in ROM in all of the diabetic rat tissues as compared with the normal rat tissues (Table 4). Treatment of diabetic animals with cerium oxide nanoparticles decreased ROM in kidney and lung (p<0.01 and p<0.05, respectively) as compared to diabetic group. Administration of sodium selenite alone decreased ROM in kidney (p<0.05). Combination of cerium oxide nanoparticles/sodium selenite significantly decreased ROM in all tissues in comparison to diabetic group (p<0.001 in kidney and lung; p<0.01 in heart and brain) and reached close to normal values in all tissues expect the lung. No significant change was detected by administration of metal form of cerium oxide.

Table 3:	Effects of various treatments on BW/HW (Body weight/Heart weight) and BW/LW (Body weight/Lung weight) index
Data are Mean±SE of six animals in each group, Significantly different from control group at aaap<0.001, aap<0.01, ap<0.05, Significantly different from diabetic control at bbp<0.01, Significantly different from cerium oxide nanoparticle/sodium selenite combination at cccp<0.001, cp<0.05

Table 4:	Effects of various treatments on production of ROM in kidney, heart, brain and lung
Data are mean±SE of six animals in each group, Significantly different from control group at aaap<0.001, aap<0.01, ap<0.05, Significantly different from diabetic control at bbbp<0.001, bbp<0.01, bp<0.05, Significantly different from cerium oxide nanoparticle/sodium selenite at cccp<0.001, ccp<0.01, cp<0.05

Table 5:	Effects of various treatments on LPO in kidney, heart, brain and lung
Data are Mean±SE of six animals in each group, Significantly different from control group at aaap<0.001, aap< 0.01, Significantly different from diabetic control at bbbp<0.001, bbp<0.01, bp<0.05, Significantly different from cerium oxide nanoparticle/sodium selenite at cccp<0.001, ccp<0.01, cp<0.05

Levels of LPO in tissues: There was a significant elevation (p<0.001) in LPO in all diabetic rat tissues as compared with normal rats. Treatment of diabetic animals with cerium oxide nanoparticle decreased LPO in kidney, heart and brain (p<0.01, p<0.05 and p<0.01, respectively) as compared to diabetic group. Administration of sodium selenite alone decreased LPO in heart and brain (p<0.01 and p<0.01, respectively). Combination of cerium oxide nanoparticles and sodium selenite significantly (p<0.001) decreased LPO in all tissues as compared with diabetic rats. No significant change was detected by administration of metal form of cerium oxide (Table 5).

Levels of TAP in tissues: There was a significant decrease (p<0.001) in TAP in all diabetic rat tissues as compared with normal group.

Table 6:	Effects of various treatments on TAP in kidney, heart, brain and lung
Data are Means±SE of six animals in each group, Significantly different from control group at aaap<0.001, aap<0.01, Significantly different from diabetic control at bbbp<0.001, bbp<0.01, bp<0.05, Significantly different from cerium oxide nanoparticle/sodium selenite combination at cccp<0.001, ccp<0.01, cp<0.05

Table 7:	Effects of various treatments on TTM in kidney, heart, brain and lung
Data are Mean±SE of six animals in each group, Significantly different from control group at aaap<0.001, aap<0.01, ap<0.05, Significantly different from diabetic control at bbp<0.01, bp<0.05, Significantly different from cerium oxide nanoparticle/sodium selenite at ccp<0.01, cp<0.05

Treatment of diabetic animals with cerium oxide nanoparticle increased TAP in heart and kidney as compared to that of diabetic group (p<0.01 and p<0.05, respectively) (Table 6). Administration of cerium oxide nanoparticle/sodium selenite combination significantly increased (p<0.001) TAP in all tissues expect the lung. No significant change was observed by administration of sodium selenite and metal form of cerium oxide in diabetic rats.

Levels of TTM in tissues: As data are shown in Table 7, the TTM level in all tissues considerably decreased in diabetic group when compared to normal group (p<0.001 in kidney and brain; p<0.01 in heart and lung).

Combination of cerium oxide nanoparticle/sodium selenite increased TTM in all tissues expect the lung as compared with diabetic group (p<0.01 in kidney and brain; p<0.05 in heart). No significant change was observed by administration of cerium oxide nanoparticles, sodium selenite, and metal form of cerium oxide.