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Effect of antioxidants such as β-carotene, vitamin C and vitamin E on oxidative stress, thermal hyperalgesia and cold allodynia in streptozotocin induced diabetic rats

Manish Sharma M.Pharm
Department of Pharmacology
I.S.F. College of Pharmacy Physical Address

Taruna Katyal M.Pharm
Department of Pharmacology
I.S.F. College of Pharmacy Physical Address

Gagandeep Grewal M.Pharm
Department of Pharmacology
I.S.F. College of Pharmacy Physical Address

Dayanidhi Behera M.Pharm
Department of Pharmacology
School of Pharmacy and Technology Management
NMIMS University Physical Address

Ramji Das Budhiraja PhD
Department of Pharmacology
I.S.F. College of Pharmacy Physical Address

Citation: M. Sharma, T. Katyal, G. Grewal, D. Behera & R. D. Budhiraja : Effect of antioxidants such as β-carotene, vitamin C and vitamin E on oxidative stress, thermal hyperalgesia and cold allodynia in streptozotocin induced diabetic rats. The Internet Journal of Pharmacology. 2009 Volume 6 Number 2

Keywords: β-carotene | vitamin C | vitamin E | oxidative stress | thermal hyperalgesia | cold allodynia

 

Abstract

A study was undertaken to evaluate the effect of antioxidants such as β-carotene, vitamin C and vitamin E on oxidative stress, thermal hyperalgesia and cold allodynia in streptozotocin induced diabetic rats. The hyperalgesia and allodynia was assessed by estimating tail withdrawal latency in warm and cold water immersion method. Furthermore, serum TBARS and plasma GSH levels were estimated to assess the oxidative stress. Treatment with β-carotene (10 mg/kg/day i.p.), vitamin C (10 mg/kg/day i.p.) and vitamin E (40 mg/kg/day i.p.) showed significant ameliorative effects on thermal hyperalgesia and cold allodynia in streptozotocin induced diabetic rats. Moreover, treatment with β-carotene markedly prevented thermal hyperalgesia and cold allodynia in streptozotocin induced diabetic rats.


Address of Research Work

Department of Pharmacology, I.S.F. College of Pharmacy, Moga, Punjab-142 001, India

Introduction

Diabetes is a global health problem and its prevalence is set to increase to 366 million worldwide by the year 2025 1 . Persistent hyperglycemia in diabetic patients, despite, appropriate therapeutic measures leads to several complications including retinopathy, nephropathy and neuropathy. Diabetic neuropathy is the most common complication affecting more than 50% of the diabetic patients. The symptoms of diabetic neuropathy includes pain, parathesia and aberrant, hyperalgesia, allodynia, loss of sensory perception, muscle weakness reduction in motor nerve conduction velocity. 2,3,4 Hyperalgesia is exaggerated response to painful stimuli which is due to reduction in pain threshold and allodynia is nocifensive response to normally innocuous stimuli. 5 Etiology of diabetic neuropathy is complex and multifactorial. Diabetic neuropathy involves complex molecular mechanisms such as auto oxidative glycosylation, formation of glycation products, activation of protein kinase-C, polyol pathway, mitogen activated protein kinases (MAPK), poly (ADP-ribose) polymerase (PARP) and Nicotinamide Adenine Di-nucleotide Phosphate (NADPH) oxidase and induction of oxidative stress. 6,7,8,9 Oxidative stress is a common link between all these pathways and is a major contributor to the development of neuropathy and hyperalgesia in diabetes. 10,11 Oxidative stress occurs in a cellular system due to the imbalance between the generation of reactive oxygen species (ROS) & decrease in amount of antioxidant system to neutralize ROS such as superoxide (O2ˉ), hydrogen peroxide (H2O2), hydroxyl radical (OHˉ), peroxinitrite (ONOOˉ), reactive aldehydes & lipid peroxides. 12,13 Once formed, reactive oxygen species deplete antioxidant defenses (glutathione (GSH) peroxidase, superoxide dismutase and catalase) and render the cells and tissues vulnerable to oxidative damage. 14 Lipids, proteins and DNA are the cellular targets for the oxidative stress and leads to alteration in cellular structure and functions. Oxidative stress activates downstream pathways such as mitogen-activated protein kinases (MAPK), poly (ADP-ribose) polymerase (PARP) and NADPH oxidase. 15 Recent evidences suggest that reactive oxygen species act as second messenger in the regulation of intracellular signaling pathways and ultimately, gene expression. 16 Increased oxidative stress causes vascular impairment leading to endoneurial hypoxia resulting in impaired neural function, reduced nerve conduction velocity and loss of neurotrophic support. 17 Thus, oxidative stress is the one of the potential targets for diabetic neuropathy.

β-carotene, vitamin C and vitamin E are natural antioxidants normally found in diet that scavenges reactive oxygen species. In the present study, we have investigated the effect of β-carotene, vitamin C and vitamin E on hyperalgesia and allodynia in streptozotocin induced diabetic rats by assessing nociception, intracellular antioxidants and oxidative stress.

Materials and Methods

Materials

Streptozotocin and β-carotene were purchased from Sigma Chemicals (Missouri, USA), Vitamin C and Vitamin E were purchased from S.D. Fine Chemicals (Mumbai, India). Blood glucose kit was purchased from Vital Diagnostics Private LTD. (Mumbai, India). All other chemicals used in the study were of analytical grade and purchased from commercial suppliers.

Animals

Wistar albino rats (150-250 gm) bred in-house at I.S.F. College of Pharmacy, Moga, Punjab, India, were maintained in a temperature and humidity-controlled room with a 12-h light/dark cycle with free access to standard diet and water. All animals used in this study were mature and healthy and were not subjected to any form of treatment/medication. Guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India were followed and the in-house animal ethical committee approved all experimental procedures.

Streptozotocin-induced experimental diabetes

The rats were rendered diabetic by a single intra peritoneal (i.p.) injection of streptozotocin (STZ) (50 mg/kg) dissolved in 0.1 M citrate buffer (pH 4.5). 18 Blood samples were collected by retro-orbital puncture after 48 hours of injection of STZ for the glucose estimation and the animals having blood glucose level more than 200 mg/dl 19 were selected for the study. The treatment with β-carotene (10 mg/kg i.p.), vitamin C (10 mg/kg i.p.) and vitamin E (40 mg/kg i.p.) were started from day 1 of STZ injection and continued till the end of study. 20,21,22

Assessment of diabetes by estimating blood glucose

Blood glucose was estimated by glucose oxidase/peroxidase method using commercially available enzymatic kit obtained from Vital Diagnostic Private Ltd., Mumbai, India. In this method, 1000 l working glucose reagent was added to 10 l of serum, 10 l of standard glucose (100 mg/dl) and 10 l of purified water to prepare test, standard and blank sample respectively. All the test tubes were incubated at room temperature for 30 min. To each test tube, 1000 l of purified water was added. The absorbance of test and standard samples were measured against blank at 505 nm spectrophotometrically (Beckman DU 640B, Nyon, Switzerland). The concentration of glucose was calculated using the following formulae:

Optical density of test

Concentration of glucose (mg/dl) = x 100

Optical density of standard

Assessment of Oxidative stress

The oxidative stress was assessed by estimating serum thiobarbituric acid reactive substances (TBARS) and reduced GSH concentrations in plasma.

Estimation of serum TBARS concentration

The lipid peroxidation was assessed by measuring malondialdehyde (MDA) concentration, which was assessed by TBARS. 23 In this method, 1000 µl of 20% trichloroacetic acid was added to 100 µl serum in a test tube to which 1000 µl of 1 % TBARS reagent (mixture of equal volume of 1% TBA aqueous solution and glacial acetic acid) was added, mixed, and incubated at 100ºC for 30 min. After cooling on ice, samples were centrifuged at 1000 x g for 20 min. The absorbance was recorded spectrophotometrically at 532 nm against suitably prepared blank solution. A standard curve using 1,1,3,3-tetraethoxypropane was plotted to calculate the concentration of TBARS.

Estimation of reduced GSH concentration

GSH was estimated using previously reported method of Ellman et al., 1959. In this method, 0.02 ml of fresh or citrated blood was added to 9.0 ml of distilled water to which 1.0 ml of phosphate buffer (pH 8.0) was added. 3 ml of this solution was placed into each of two Beckman 1-cm cells, using one to adjust the absorbance to zero. To the other, 0.02 ml, 5,5’-dithiobis [2-nitrobenzoic acid] (DTNB) absorbance was determined at 420 nm after 30 min. Results were expressed as mmoles (SH)/l blood. The concentration of reduced glutathione was calculated using the following formulae:

Co = 36.8 X Absorbance

Where, Co - Original concentration

A - Absorbance at 420 nm

Assessment of Hyperalgesia

Hyperalgesia was assessed by tail (warm water) immersion test (50°C). In this method, the tip of the tail of each rat was dipped in warm water (50° C) and the tail withdrawal latency was taken. 25 A cut off latency time was fixed at 15 sec.

Assessment of Allodynia

Allodynia was estimated through tail (cold water) immersion test (5°C) in which the tail of the rat was dipped in cold water (5°C) and the tail withdrawal latency was noted. 26 A cut off latency was fixed at 15 sec.

Experimental Protocol

Five groups were employed in the present study and each group comprises of six animals.

Control Group

The non diabetic rats were included in this group and citrate buffer was administered. All parameters were assessed weekly from 0 day to 35 days.

Diabetic Control Group

The animals were made diabetic by administration of streptozotocin (50 mg/kg, i.p, once) in citrate buffer. All parameters were assessed weekly from 0 day to 35 days.

β-carotene Treated Group

The treatment with β-carotene (10 mg/kg/day i.p.) was started in diabetic animals on the same day of administration of streptozotocin and continued till 35 th day. All parameters were assessed weekly from 0 day to 35 days.

Vitamin C Treated Group

The treatment with vitamin C (10 mg/kg/day i.p.) was started in diabetic animals on the same day of administration of streptozotocin and continued till 35 th day. All parameters were assessed weekly from 0 day to 35 days.

Vitamin E Treated Group

The treatment with vitamin E (40 mg/kg/day i.p.) was started in diabetic animals on the the same day of administration of streptozotocin and continued till 35 th day. All parameters were assessed weekly from 0 day to 35 days.

Statistical analysis

Statistical analysis was performed using SigmaStat2 statistical software. All the results are expressed in mean ± SD. The data obtained from various groups were statistically analyzed using one way ANOVA followed by post hoc Tukey’s multiple range test. The P < 0.05 was considered to be statistically significant.

Results

Effect of Antioxidants on blood glucose

The significant increase in blood glucose level was noted in diabetic control rats from 7 th day to 35 th day of protocol when compared with the control group rats. The marked increase in blood glucose level was noted in diabetic control rats at 21 st , 28 th and 35 th day of protocol. However, treatment with various antioxidants such as β-carotene (10 mg/kg/day i.p.), vitamin C (10 mg/kg/day i.p.) and vitamin E (40 mg/kg/day i.p.) showed a significant reduction in blood glucose levels at 21 st , 28 th and 35 th day of treatment when compared with the diabetic control rats. Moreover, β-carotene treatment markedly attenuated the streptozotocin-induced increase in blood glucose levels at 21 st , 28 th and 35 th day of treatment when compared with vitamin C and vitamin E treatment groups (Table 1).

Thumbnail: Table 1: Effect of Pharmacological Interventions on Blood Glucose.
Table 1: Effect of Pharmacological Interventions on Blood Glucose.

Effect of Antioxidants on reduced GSH

The significant reduction in GSH level was noted in diabetic control rats from 7 th day to 35 th day of protocol when compared with the control group rats. The marked reduction in GSH level was noted at 28 th and 35 th day of protocol. However, treatment with β-carotene (10 mg/kg/day i.p.), vitamin C (10 mg/kg/day i.p.) and vitamin E (40 mg/kg/day i.p.) showed a significant increase in diabetes induced decrease in GSH levels at 21 st , 28 th and 35 th day of treatment when compared with the diabetic control rats. Moreover, β-carotene treatment markedly prevented the decrease in GSH levels at 28 th and 35 th day of treatment when compared with vitamin C and vitamin E treatment groups (Table 2).

Thumbnail: Table 2: Effect of Pharmacological Interventions on Reduced Glutathione (GSH) {mmoles (SH)/l blood}.
Table 2: Effect of Pharmacological Interventions on Reduced Glutathione (GSH) {mmoles (SH)/l blood}.

Effect of Antioxidants on serum TBARS

The significant increase in TBARS level was noted in diabetic control rats from 7 th day to 35 th day of protocol when compared with the control group rats. The marked increase in TBARS level was noted at 28 th and 35 th day of protocol. However, treatment with β-carotene (10 mg/kg/day i.p.), vitamin C (10 mg/kg/day i.p.) and vitamin E (40 mg/kg/day i.p.) showed a significant reduction in diabetes induced increase in TBARS level at 21 st , 28 th and 35 th day of treatment when compared with the diabetic control rats. Moreover, β-carotene treatment markedly attenuated the high TBARS level at 28 th and 35 th day of treatment when compared with vitamin C and vitamin E treatment groups (Table 3).

Thumbnail: Table 3: Effect of Pharmacological Interventions on Serum TBARS (nmol/ml).
Table 3: Effect of Pharmacological Interventions on Serum TBARS (nmol/ml).

Effect of Antioxidants on Hyperalgesia and Allodynia

Hyperalgesia and allodynia was assessed by tail (warm water) immersion method and tail (cold water) immersion method respectively.

Effect of antioxidants on Tail (Warm Water) Immersion Method

The diabetic control group showed a significant reduction in tail withdrawal latency from 7 th day to 35 th day of protocol when compared with the control group. The marked decrease in tail withdrawal latency was noted at 28 th and 35 th day of protocol. However, treatment with β-carotene (10 mg/kg/day i.p.), vitamin C (10 mg/kg/day i.p.) and vitamin E (40 mg/kg/day i.p.) showed a significant improvement in diabetes induced decrease in tail withdrawal latency at 21 st , 28 th and 35 th day of treatment when compared with the diabetic control rats. Moreover, β-carotene treatment markedly increased the tail withdrawal latency at 28 th and 35 th day of treatment when compared with vitamin C and vitamin E treatment groups (Table 4).

Thumbnail: Table 4: Effect of Pharmacological Interventions on Tail Withdrawal Latency by Tail (Warm Water) Immersion Method (seconds).
Table 4: Effect of Pharmacological Interventions on Tail Withdrawal Latency by Tail (Warm Water) Immersion Method (seconds).

Effect of antioxidants on Tail (Cold Water) Immersion Method

The diabetic control group animals showed a significant reduction in tail withdrawal latency from 7 th day to 35 th days of protocol when compared with the control group rats. The marked decrease in tail withdrawal latency was noted at 28 th and 35 th day of protocol. However, treatment with β-carotene (10 mg/kg/day i.p.), vitamin C (10 mg/kg/day i.p.) and vitamin E (40 mg/kg/day i.p.) showed a significant improvement in diabetes induced decrease in tail withdrawal latency at 14 th , 21 st , 28 th and 35 th day of treatment when compared with the diabetic control rats. Moreover, β-carotene treatment markedly increased the tail withdrawal latency at 21 st , 28 th and 35 th day of treatment when compared with vitamin C and vitamin E treatment groups (Table 5).

Thumbnail: Table 5: Effect of Pharmacological Interventions on Tail Withdrawal Latency by Tail (Cold Water) Immersion Method (seconds).
Table 5: Effect of Pharmacological Interventions on Tail Withdrawal Latency by Tail (Cold Water) Immersion Method (seconds).

Discussion

Diabetes mellitus can be chemically induced in animals by administration of either streptozotocin or alloxan. 27,28 Streptozotocin, a glucosamine-nitrosourea compound obtained from Streptomyces achromogenes, is used to induce insulin-dependent diabetes mellitus (IDDM) in rodents. Streptozotocin produces cytotoxic effect on β-cells of islets of langerhans of pancreas by interfering with glucose transporter GLUT-2 and causes DNA damage either by alkylation or peroxynitrite formation. 29,30,31 . The DNA strands breakage by streptozotocin activates poly (ADP-ribose) polymerase (PARP) and causes ATP depletion, which leads to cell death. 32,33,34 Streptozotocin-induced diabetic animals exhibit two phases of thermal pain sensitivity, an initial phase of hyperalgesia and a late phase of hypoalgesia and also induces TRPV1 protein expression by involving ROS-p38 MAPK pathway which plays an important role in both diabetic and inflammatory hyperalgesia. 35,36 Hyperglycemia was confirmed by estimating blood glucose levels in the animals after 48 hours of streptozotocin administration (50 mg/kg/day i.p.). It is well documented that hyperglycemia-induced oxidative stress in diabetes could be the major cause of development and progression of diabetic microvascular complications such as neuropathy. 37,38 The decrease in reduced glutathione and increase in serum TBARS are taken as the markers of oxidative stress. 23,24 It is reported that hyperglycemia-induced increase in oxidative stress produces lipid peroxidation and consequent generation of MDA. 24 The decrease in reduced glutathione (GSH) level has been observed during reduced intracellular antioxidant defense system. 39 Hence, these parameters have been used in the present study to assess the degree of oxidative stress. In the present study, the increase in serum TBARS, which is an index of lipid peroxidation was noted in diabetic rats. Moreover, reduced glutathione level was noted to be decreased in diabetic rats. These results suggest the development of oxidative stress in diabetic rats. In the present study, the administration of β-carotene, vitamin C and vitamin E significantly attenuated the STZ-induced rise in blood glucose level. The β-carotene markedly attenuated the STZ-induced increase in blood glucose level as compared to vitamin C and vitamin E. This observation is consistent with a recent study in which β-carotene has been reported to ameliorate STZ-induced rise in blood glucose level. 40 The treatment with β-carotene, vitamin C and vitamin E significantly attenuated the diabetes-induced increase in TBARS level and decrease in reduced glutathione level. The β-carotene markedly reduced the high TBARS level and increased the level of reduced glutathione in diabetic rats as compared to vitamin C and vitamin E, which may be due to its potent antioxidant property. β-carotene, apart from its potent antioxidant property, has inhibitory effect on the formation of advanced glycation end products by inhibiting the Maillard reaction. 41 Thus, it may be suggested that the reduction in blood glucose level with β-carotene, vitamin C and vitamin E may be due to their antioxidant properties and reduction in oxidative stress in β-cells of islets of langerhans. This contention is supported by the fact that quercetin, an antioxidant flavanoid, showed protection on pancreatic β-cells by decreasing the oxidative stress in STZ-induced diabetic rats. 42 Moreover, the result obtained in the present study is further supported by the recent report in which rutin, an antioxidant bioflavanoid has been shown to scavenge free radicals, inhibit lipid peroxidation and protect β-cells of islets of langerhans, resulting in increased insulin secretion and decreased blood glucose levels. 43

The significant decrease in nociceptive threshold in streptozotocin induced diabetic rats as compared to control rats indicates the development of significant hyperalgesia and allodynia in diabetes. 44,45 Diabetic neuropathy is one of the most frequent neuropathies associated with hyperalgesia and hyperaesthesia. The development of diabetic neuropathy in STZ-induced diabetic rats can be ascertained from the alteration in motor nerve conduction velocity and nerve blood flow and nociception. Several mechanisms such as tissue injury due to ischemia, sensitization of peripheral receptors, ectopic activity in sprouting fibers, alteration in dorsal root ganglia cells are reported to contribute to nociception. 46,47 In the present study, tail flick latencies measured by tail warm water and cold water immersion test were noted to be decreased in diabetic rats which suggest the development of hyperalgesia and allodynia. However, treatment with β-carotene, vitamin C and vitamin E have significantly attenuated streptozotocin-induced hyperalgesia and allodynia in diabetic rats. This observation is consistent with other reports where antioxidants like GSH and α-lipoic acid significantly prevented thermal and mechanical hyperalgesia. 48 Moreover, β-carotene treatment markedly attenuated STZ-induced decrease in tail-flick latencies because of its potent antioxidant property. The activation of protein kinase-C (PKC), polyol pathway, mitogen activated protein kinases (MAPK), poly (ADP-ribose) polymerase (PARP) and NADPH oxidase have been shown to play a key role in the pathogenesis of diabetic hyperalgesia and neuropathy. 6,7,8,9 It has been reported that diabetes-induced oxidative stress involves activation of various signaling pathways such as polyol, PKC, AGE and hexosamine. In conclusion, the ameliorative effects of antioxidants such as β-carotene, vitamin C and vitamin E on hyperalgesia and allodynia in diabetic rats may be due to their antioxidant properties and consequent inhibition of polyol, PKC, Advanced Glycation End-products (AGE) and hexosamine pathways. The β-carotene was found to be more potent in preventing hyperalgesia and allodynia in diabetic rats than vitamin C and vitamin E which may be due to its potent antioxidant property.

Acknowledgements

The authors wish to thank Chairman, Shri. Parveen Garg and Dr. Balakumar P, I.S.F. College of Pharmacy, Moga, Punjab, India for providing facilities to carry out this project.

Corresponding Author

Dayanidhi Behera, M.Pharm
Department of Pharmacology, School of Pharmacy and Technology Management, NMIMS University, Vile-Parle-400 056, Mumbai, India
Contact: (M)- +91- 9324025458
Email- [dayaxworld@gmail.com]
[dayaxworld@yahoo.com]

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