Protective effect of vanadyl sulfate on skin injury in streptozotocin-induced diabetic rats

Abstract

The aim of the present study was to investigate the effect of vanadyl sulfate supplementation on the skin tissues of diabetic and control rats. In this study, 6–6.5 months old male Swiss albino rats were used. The animals were randomly divided into the following four groups: group I, control (nondiabetic intact animals); group II, vanadyl sulfate control; group III, streptozotocin (STZ)-diabetic animals and group IV, STZ-diabetic animals given vanadyl sulfate. The animals were made diabetic by intraperitoneal injection of a single dose of 65 mg/kg STZ in 0.01 M citrate buffer (pH = 4.5). From day 1 to day 60, 100 mg/kg vanadyl sulfate was given daily by gavage technique to one of the control and diabetic groups. Body weights and blood glucose levels were estimated on experimental days 0, 1 and 60. On the 60th day, skin tissue samples were taken, glutathione (GSH), lipid peroxidation (LPO), nonenzymatic glycosylation (NEG) and protein levels, catalase (CAT), superoxide dismutase (SOD) and glutathione-S-transferase (GST) activities were determined. Blood glucose, skin LPO and NEG levels increased, but skin GSH levels and CAT, SOD and GST activities decreased in the STZ group. Treatment with vanadyl sulfate reversed these effects. The present study showed that vanadyl sulfate exerted antioxidant properties and may prevent skin damage caused by diabetes.

Keywords

Oxidative stress skin diabetes mellitus vanadyl sulfate antioxidant enzymes

Introduction

Diabetes mellitus is the most common endocrine disorder, expected to affect 5.4% of the world population by the year 2025. Diabetes mellitus is characterized by high blood glucose levels and by disturbances of carbohydrate and lipid metabolisms and resultant long-term systemic complications.¹ Hyperglycemia or raised blood sugar is a common effect of uncontrolled diabetes and overtime leads to serious damage of several body systems.² The chronic hyperglycemia in diabetes causes oxidative stress.³ It is believed that oxidative stress plays an important role in chronic complications of diabetes.⁴ The generation of reactive oxygen species (ROS) gives rise to oxidative damage, particularly in heart, kidney, eyes, nerves, liver, small and large vessels and gastrointestinal system.⁵ The skin, such as other organs, is affected from the metabolic disturbances caused by diabetes mellitus.⁶ Therefore, alleviation of oxidative stress is essential for preventing or reversing the diabetic complications.⁷

Vanadium and vanadate derivates have been shown to have insulin-like effects on glucose metabolism by Pederson et al.⁸ These effects include stimulation of hexose transport in rat adipocites^9,10 and mouse skeletal muscle,¹¹ stimulation of glycogen synthase in rat adipocytes¹² and stimulation of DNA synthesis in cultured cells.¹³ There are several pharmacological applications of vanadium including treatment of diabetes,¹⁴ cancer therapy,¹⁵ anti-inflammatory activity¹⁶ and hypertension and obesity.¹⁷ The protective effect of vanadyl sulfate on some tissues has been demonstrated previously by our colleagues.^18,19

The aim of this study was to investigate whether vanadyl sulfate has a protective effect on the skin tissue of streptozotocin (STZ)-induced diabetic rats and whether this effect is related with the oxidative/antioxidative system.

Material and methods

Animals

The experiments were reviewed and approved by the Animal Care and Use Institute’s Committee of Istanbul University. In this study, 6–6.5 months old male Swiss albino rats were used. All rats were clinically healthy. The rats were housed in metal cages at room temperature and fed with laboratory pellet chow and given water ad libitum.

Induction of experimental diabetes

Diabetes was induced by intraperitoneal injection of STZ in a single dose of 65 mg/kg body weight. Freshly prepared solution was made by dissolving STZ in cold 0.01 M sodium citrate–hydrochloric acid buffer (pH = 4.5).

Preparation of experimental animals

The animals were randomly divided into the following four groups: group I, control (nondiabetic intact animals; n = 13); group II, vanadyl sulfate control (n = 5); group III, STZ-diabetic animals (n = 11) and group IV, STZ-diabetic animals given vanadyl sulfate (n = 11). Vanadyl sulfate was given by gavage technique to rats in a dose of 100 mg/kg every day for 60 days. The body weight was measured and blood samples were collected from the tail vein of both control and diabetic animals on experimental days 0, 1 and 60.

Biochemical assays

On day 60 of the experiment, animals were fasted overnight, and the following day, they were killed under ether anesthesia. Blood and skin tissue samples were taken and skin samples were frozen until needed for study. In all samples, 18 h-fasting blood glucose levels were determined by o-toluidine method.²⁰ Skin tissues were homogenized in cold saline using a glass homogenizer to make 10% (w/v) homogenate. The homogenates were centrifuged and the clear supernatants were used for glutathione (GSH), lipid peroxidation (LPO) and nonenzymatic glycosylation (NEG). All enzymatic activities of antioxidant enzymes, including catalase (CAT), superoxide dismutase (SOD), glutathione-S-transferase (GST) and protein analysis, were determined using skin tissue homogenates in appropriate dilutions.

The skin tissue GSH, LPO and NEG levels were determined by Beutler,²¹ Ledwozyw et al.²² and thiobarbutiric acid methods, respectively.²³ CAT activity was determined according to Aebi,²⁴ SOD activity according to Mylorie et al.²⁵ and GST activity according to Habig and Jacoby.²⁶ Protein levels were measured by the method of Lowry using serum albumin as standard.²⁷

Statistical analysis

Biochemical results were evaluated using an unpaired t test and analysis of variance (ANOVA) using the NCSS statistical computer package. The values were expressed as mean ± SD. p < 0.05 was considered as significant.

Biochemical results

Changes in the body weight in control and experimental groups are shown in Table 1. There is a significant difference in the body weight between the four groups on days 1 and 60 (p _ANOVA = 0.011 and p _ANOVA = 0.0001), respectively. Body weight in the diabetic group compared with control group showed a notable decrease on days 1 and 60 (p < 0.01 and p < 0.0001).

Table 1.

Levels of weight parameters for all the (g)

Groups	Day 0	Day 1	Day 60
Control	246.64 ± 44.43	243.35 ± 39.50	288.27 ± 22.10
Control + Va	224.17 ± 16.13	221.03 ± 12.58	251.91 ± 20.42
Diabetic	231.67 ± 37.71	197.02 ± 37.47^c	171.70 ± 34.67^c
Diabetic + Va	229.66 ± 32.15	203.93 ± 30.57	199.16 ± 36.20
p _ANOVA	0.571	0.011	0.0001

ANOVA: analysis of variance; Va: vanadyl sulfate.

^aMean ± SD.

^b p < 0.01 compared with the control group.

^c p < 0.0001 compared with the control group.

Alterations in the blood glucose levels on the treatment of diabetic rats with vanadyl sulfate are given in Table 2. The blood glucose levels were significantly increased in STZ-diabetic rats compared with the control rats on 1st and 60th days (p < 0.0001). Oral administration of vanadyl sulfate for 60 days significantly decreased the blood glucose levels in diabetic rats (p < 0.005; Table 2).

Table 2.

Levels of blood glucose for all the (mg/dl)

Groups	Day 0	Day 1	Day 60
Control	74.52 ± 14.69	78.20 ± 12.74	69.05 ± 9.16
Control + Va	72.92 ± 9.40	85.05 ± 19.31	86.40 ± 11.03
Diabetic	65.52 ± 5.77	202.30 ± 31.21^b	323.39 ± 131.29^b
Diabetic + Va	71.88 ± 13.78	253.93 ± 38.63^c	131.68 ± 72.30^d
p _ANOVA	0.324	0.0001	0.0001

ANOVA: analysis of variance; Va: vanadyl sulfate.

^aMean ± SD.

^b p < 0.0001 compared with the control group.

^c p > 0.05 compared with the diabetic group.

^d p < 0.005 compared with the diabetic group.

Table 3 gives the levels of GSH, LPO and NEG in the skin of control and diabetic groups of rats. In diabetic rats, a significant decrease in skin GSH levels was observed when compared with control groups (p < 0.0001). The administration of vanadyl sulfate significantly increased the skin GSH levels in diabetic rats (p < 0.0001). A significant difference in the skin GSH levels was observed in all the groups (p _ANOVA = 0.0001; Table 3). In diabetic groups, LPO levels were higher than those in the other groups (p _ANOVA = 0.003). There was a significant increase in the levels of LPO in the skin of diabetic rats when compared with control group (p < 0.005). Vanadyl sulfate given to the diabetic rats lowered the skin LPO levels in a significant manner when compared with diabetic rats (p < 0.05). NEG levels were significantly increased in diabetic rats when compared with the control rats (p < 0.0001). In the diabetic rats treated with vanadyl sulfate, the skin NEG levels significantly decreased when compared with the diabetic group (p < 0.005). The NEG levels in skin were significantly increased in the diabetic animals as compared to the other groups (p _ANOVA = 0.0001).

Table 3.

Effect of vanadyl sulfate on the levels of GSH, LPO and NEG in the skin tissue of all the groups.^a

Groups	GSH (nmol GSH/mg protein)	LPO (nmol MDA/mg protein)	NEG (nmol fructose/mg protein)
Control	9.29 ± 0.87	0.43 ± 0.21	6.58 ± 1.66
Control + Va	7.02 ± 0.26	0.53 ± 0.08	5.60 ± 0.67
Diabetic	6.39 ± 0.65^b	1.09 ± 0.47^d	26.04 ± 5.67^b
Diabetic + Va	9.39 ± 1.00^c	0.44 ± 0.37^e	15.26 ± 2.46^f
p _ANOVA	0.0001	0.003	0.0001

GSH: glutathione; LPO: lipid peroxidation; NEG: nonenzymatic glycosylation; ANOVA: analysis of variance; Va: vanadyl sulfate.

^aMean ± SD.

^b p < 0.0001 compared with the control group.

^c p < 0.0001 compared with the diabetic group.

^d p < 0.005 compared with the control group.

^e p < 0.05 compared with the diabetic group.

^f p < 0.005 compared with the diabetic group.

The changes in the activities of skin CAT, SOD and GST are shown in Table 4. A considerable increase was observed in skin CAT activity in diabetic rats (p < 0.005). In the diabetic rats treated with vanadyl sulfate, the skin CAT activity increased when compared with the diabetic group. SOD activity was significantly decreased in diabetic group (p < 0.05) and administration of vanadyl sulfate restored this decrease significantly (p < 0.05). A significant decrease was also seen in GST activities in the diabetic group (p < 0.005), whereas activity was significantly higher in the vanadium-treated diabetic group (p < 0.05).

Table 4.

Effect of vanadyl sulfate on the activities of CAT, SOD and GST in the skin tissue of all the groups.^a

Groups	CAT (U/mg protein)	SOD (U/mg protein)	GST (U/g protein)
Control	40.52 ± 5.90	6.60 ± 0.77	3.79 ± 0.76
Control + Va	38.84 ± 2.93	6.30 ± 0.50	2.69 ± 0.45
Diabetic	30.19 ± 2.72^b	5.11 ± 0.63^c	2.58 ± 0.47^b
Diabetic + Va	32.30 ± 2.40	6.87 ± 0.78^d	4.97 ± 0.61^e
p _ANOVA	0.001	0.014	0.0001

CAT: catalase; SOD: superoxide dismutase; GST: glutathione-S-transferase; ANOVA: analysis of variance; Va: vanadyl sulfate.

^aMean ± SD.

^b p < 0.005 compared with the control group.

^c p < 0.05 compared with the control group.

^d p < 0.05 compared with the diabetic group.

^e p < 0.0001 compared with the diabetic group.

Discussion

Diabetes mellitus is a disease that results in chronic hyperglycemia due to an absolute or relative lack of insulin and/or insulin resistance that in turn impairs the glucose, protein and lipid metabolisms, and finally, results in characteristic secondary complications.²⁸ STZ-induced diabetes is characterized by severe loss in body weight. The body weight decrease in diabetic rats suggests that the loss or degradation of structural proteins may be due to an unavailability of carbohydrates for utilization as an energy source in diabetes, whereas structural proteins are known to contribute to reduction in body weight.²⁹ The loss in the body weight, which was also seen in the present study, was partly prevented by vanadyl sulfate (Table 1) and thus could be attributed to the antidiabetic role of the vanadyl sulfate.³⁰

Several investigators have reported that vanadyl sulfate in STZ-induced diabetic rats alleviates some signs of diabetes, the most marked one being a normalization of blood glucose level.^31–33 The administration of vanadyl sulfate to STZ-diabetic rats reduced the blood glucose levels, in accordance with our previous studies. These effects may attributed to the insulin-mimicking effect of vanadyl sulfate.³⁰

GSH is one of the essential compounds for maintaining cell integrity against ROS, as it scavenges free radicals and reduces hydrogen peroxide (H₂O₂).³⁴ Yanardag et al. have shown that the tissue concentration GSH of STZ-induced diabetic rats is significantly lower when compared with the control rats.³⁵ Decreased levels of GSH in the skin tissue of diabetic rats may increase susceptibility to oxidative damage. In the present study, the elevation of GSH levels in the skin tissue was observed in the vanadyl sulfate-treated diabetic rats. This indicates that the vanadyl sulfate can increase the biosynthesis of GSH and reduce the oxidative stress.

Oxidative stress leads to an increased production of ROS, and finally, cellular LPO has been found to play an important role in the development of diabetes.³⁶ In the present study, STZ treatment significantly increased LPO products and decreased enzymatic and nonenzymatic antioxidant levels in the skin tissue of rats. LPO elevated leads to decrease the level of reduced GSH in STZ-induced diabetes. A potent endogenous antioxidant LPO-mediated damage has been observed in the development of types I and II diabetes mellitus. The increased LPO during diabetes, as found in the present study, may be due to the inefficient antioxidant system prevalent in diabetes.³⁷ Administration of vanadyl sulfate to diabetic rats significantly decreased the levels of LPO. Vanadyl sulfate acts as an antioxidant by scavenging free radicals that result in decreased LPO in diabetic rats.

Diabetes mellitus is a common metabolic disease, representing a serious risk factor for the development of cardiovascular, gastrointestinal, retinal, peripheral and central nervous, kidney, liver and urinary bladder disfunctions.³⁸ Increasing evidence indicates that hyperglycemia is the initiating cause of tissue damage in diabetes mellitus either through repeated acute changes in cellular glucose metabolism or through long-term accumulation of glycated biomolecules and advanced glycation end products (AGEs) of Maillard reaction.³⁹ These are causing the complications of diabetes and skin aging, primarily via adventitious and cross-linking of proteins. Long-lived proteins such as structural collagen are particularly implicated as pathogenic targets as AGEs process. NEG was found to be increased in diabetes mellitus, and the amount of increase is directly proportional to the fasting blood glucose level.⁴⁰ In this study, the levels of NEG were found to be increased in the STZ-diabetic groups with respect to untreated controls. In this study, the increase in skin tissue NEG levels provoked by diabetes was significantly lowered by vanadyl sulfate. This suggests that amelioration of oxidative stress is due to hyperglycemia by vanadyl sulfate administration.

Diabetes mellitus is associated with increased formation of free radicals and decrease in antioxidant potential. Antioxidants constitute the foremost defense system that limit the toxicity associated with free radicals. Oxidative stress in diabetes is coupled with decrease in the antioxidant status, which can increase the deleterious effects of free radicals.⁴¹ Antioxidant enzymes including SOD, CAT, GPx and GST form the first line of defense against ROS in the organism, which play an important role in scavenging the toxic intermediate of incomplete oxidation. Due to these events, the balance normally present in cells between radical formation and protection against them is disturbed.⁴² In the current study, CAT and SOD showed lower activities in skin tissue during diabetes. The decreased activities of CAT and SOD may be in response to increased production of H₂O₂ and O₂ ^−· by the auto-oxidation of the excess of glucose and nonenzymatic glycation of proteins.⁴³ The decreased activity of CAT and SOD could also be due to their decreased protein expression levels in the diabetic condition.⁴⁴ Treatment with vanadyl sulfate increased the activities of SOD and CAT in diabetic rats when compared with untreated diabetic rats. In fact, the reactivation in SOD activity promoted by vanadyl sulfate may accelerate the dismutation of O₂ ^−· to H₂O₂, which is quickly removed by CAT. GST is an important antioxidant enzyme, whose activity was significantly decreased in diabetic liver tissues, indicating impaired scavenging of H₂O₂ and lipid hydroperoxides. It also plays an important role in detoxification of xenobiotic compounds thereby protecting the cell from peroxidative damage.⁴⁵ Decreased activity of GST in diabetic rats could be directly explained by the low content of GSH found in these rats since GSH is the substrate and cofactor of GST.⁴⁶ Administration of vanadyl sulfate to STZ-induced diabetic rats resulted in increased activity of skin GST compared to diabetic rats, indicating the protective effect of vanadyl sulfate against oxidative damage observed in STZ-induced diabetes.

As a result, we can say that vanadyl sulfate has antioxidant and protective effects on the damage caused by diabetes on the skin tissue.

Footnotes

Funding

This work was supported by Scientific Research Projects Coordination Unit of Istanbul University (project number: YADOP-4408).

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