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Process Biochemistry 47 (2012) 20722080

Contents lists available at SciVerse ScienceDirect

Process Biochemistry

jo u rn al hom epa ge: www .e lsev ier .com/ locate /procbio

meliorative effect of 20-OH ecdysone on streptozotocin induced oxidativetress and -cell damage in experimental hyperglycemic rats

amalingam Sundarama, Rajendran Naresha, Palanivelu Shanthib, Panchanatham Sachdanandama,

Department of Medical Biochemistry, Institute of Basic Medical Sciences, University of Madras, Taramani Campus, Chennai, Tamil Nadu 600 113, IndiaDepartment of Pathology, Dr. ALM P-G, Institute of Basic Medical Sciences, University of Madras, Taramani Campus, Chennai, Tamil Nadu 600 113, India

r t i c l e i n f o

rticle history:eceived 15 May 2012eceived in revised form 18 July 2012ccepted 18 July 2012vailable online 25 July 2012

eywords:0-OH ecdysoneyperglycemiaxidative stress

a b s t r a c t

The present study was to evaluate the effects of 20-OH ecdysone on hyperglycemia mediated oxida-tive stress in streptozotocin induced diabetic rats. Diabetes was induced in experimental rats by singleintraperitoneal injection of STZ (45 mg/kg b.w.) dissolved in 0.1 mol/L citrate buffer (pH 4.5). Diabeticrats exhibited increased blood glucose with significant decrease in plasma insulin levels. The activi-ties of antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx),glutathione-S-transferase (GST) and the levels of non-enzymic antioxidants vitamin C, vitamin E andreduced glutathione (GSH) were decreased while increases in the levels of LPO markers were observedin liver and kidney tissues of diabetic rats. Moreover, hepatic markers (aspartate aminotransferase andalanine aminotransferase) and renal markers (urea, creatinine) were significantly increased in diabeticntioxidants rats as compared to control rats. Upon treatment with 20-OH ecdysone to diabetic rats showed significantameliorative effects on all the biochemical parameters studied. Biochemical findings were supported byhistological studies. These results indicated that 20-OH ecdysone exerts a protective action on pancreaticbeta cell function and overcomes oxidative stress through its hypoglycemic potential. The effect producedby the 20-OH ecdysone on various parameters was comparable to that of glibenclamide an antidiabeticdrug.. Introduction

Diabetes mellitus (DM) is the most significant chronic diseasend cause of death in modern society. Diabetes is a metabolicisorder characterized by hyperglycemia resulting from defectivensulin secretion, resistance to insulin action or both. DM involvesigh levels of blood glucose, which contributes to an increase inree radical production [1]. Defects in glucose metabolizing machin-ry and consistent efforts of the physiological system to correcthe imbalance in glucose metabolism place an over exertion onhe endocrine system. Continuing deterioration of endocrine con-rol exacerbates the metabolic disturbances and leads primarily toyperglycemia [2]. The elevated levels of blood glucose in diabetesre associated with increased lipid peroxidation (LPO), which mayontribute to long-term tissue damage [3]. Various studies havehown that DM is associated with oxidative stress, leading to anncreased production of reactive oxygen species (ROS), including

uperoxide radical (O2) hydrogen peroxide (H2O2), and hydroxyladical (OH) or reduction of antioxidant defense system [4,5].OS are involved in the pathogenesis of many diseases including

Corresponding author. Tel.: +91 9444060025.E-mail address: psachdanandam2000@yahoo.co.in (P. Sachdanandam).

359-5113/$ see front matter 2012 Elsevier Ltd. All rights reserved.ttp://dx.doi.org/10.1016/j.procbio.2012.07.025 2012 Elsevier Ltd. All rights reserved.

hypoxia, hypercholesterolemia, atherosclerosis, hypertension,ischemia reperfusion injury and heart failure [6].

Streptozotocin (STZ), an antibiotic produced by Streptomycesachromogenes has been widely used for inducing diabetes in theexperimental animals through its toxic effects on pancreatic -cells[7]. The cytotoxic action of STZ is associated with the generation ofROS causing oxidative damage [8]. LPO is a key marker of oxida-tive stress. The increased oxidative stress, as measured by indicesof elevated LPO, depletion of endogenous antioxidant, and antioxi-dant enzymes activities in plasma and tissues, are commonly foundin rats with STZ-induced diabetes, and these alterations may causetissues to be more susceptible to oxidative damage [9]. The signifi-cant extent of LPO byproducts that was measured as thiobarbituricacid reactive substances (TBARS) has been reported in diabetes [10].

Antioxidant refers to a compound that can delay or inhibit theoxidation of lipids or other molecules by inhibiting the initiation orpropagation of oxidative chain reactions and which can thus pre-vent or repair damage done to the cells by oxygen. They act byone or more of the following mechanisms: reducing activity, freeradical-scavenging, potential complexing of pro-oxidant metals

and quenching of singlet oxygen. The formation of ROS is preventedby an antioxidant system that included non-enzymatic antioxi-dants (vitamin C, vitamin D and glutathione), enzymes regeneratingthe reduced forms of antioxidants and ROS-scavenging enzymes

dx.doi.org/10.1016/j.procbio.2012.07.025http://www.sciencedirect.com/science/journal/13595113http://www.elsevier.com/locate/procbiomailto:psachdanandam2000@yahoo.co.indx.doi.org/10.1016/j.procbio.2012.07.025

R. Sundaram et al. / Process Biochem

OH

OH

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Fig. 1. Chemical structure of 20-OH ecdysone.

uch as superoxide dismutase (SOD), catalase (CAT), glutathioneeroxidase (GPx) and glutathione-S-transferase (GST) [11,12].Recently, plant based therapies gains importance as they have

een shown to regulate the oxidative complications of DM [13].ntioxidants are used as supportive therapy in the treatment of dia-etes [14]. Thus, there is an increasing demand for natural productsith antidiabetic and antioxidant activities to attenuate oxidativetress induced complications. In addition, chemoprevention withatural substances aims to reduce insulin resistance and stimu-ates insulin secretion minimizing the risk of developing DM andts sequels [15].

Vitex negundo (Linn.) belongs to the family Verbanaeceae whichs used as a traditional medicine in different nation and reportedo have variety of pharmacological activities [16,17]. Although, allarts of V. negundo are used as an indigenous system of medicine,he leaves are the most potent for medicinal use due to presence ofumerous phytoconstituents which are therapeutic in nature [18].0-OH ecdysone is one of the core active principles of V. negundoeaves and reported to have antihyperglycemic [19], antihyperlipi-emic [20] and antiosteoporosis [21]. In this view, this plant haseen chosen for isolating an active principle 20-OH ecdysone. In ourrevious studies, we reported that 20-OH ecdysone administrationignificantly improved glucose homeostasis through modificationctivities of key enzymes of carbohydrate metabolism via enhancednsulin secretion in STZ-induced diabetic rats [22]. The presenttudy explores the effect of 20-OH ecdysone on tissue antioxidantnd lipid peroxidation status, histopathological changes of pan-reas, liver and kidney tissues in STZ-induced diabetic rats. Theffect produced by 20-OH ecdysone was compared with gliben-lamide an antidiabetic drug. The chemical structure of 20-OHcdysone was given in Fig. 1.

. Materials and methods

.1. Chemicals

Streptozotocin was purchased from SigmaAldrich, St. Louis, USA. All otherhemicals were of analytical grade obtained from E. Merck and Himedia, Mumbai,ndia.

.2. Animals

Male albino rats of Wistar strain with body weight ranging from 175 to00 g were procured from the Central Animal House Facility University of Madrasaramani Campus, Chennai, Tamil Nadu, India. They were maintained at room tem-erature of 25 2 C and 12/12 h of light/dark cycle. Animals were given standardommercial rat chow and water ad libitum and housed under standard environ-

ental conditions throughout the study. The laboratory animal protocol used in

his study was approved by the Institutional Animal Ethical Committee, Universityf Madras in accordance with the Indian National law on animal care and use (IAECo. 01/013/2009).istry 47 (2012) 20722080 2073

2.3. Induction of diabetes

Overnight (12 h) fasted animals were made diabetic by a single intraperitonealinjection of freshly prepared STZ (45 mg/kg b.w.) dissolved in 0.1 M citrate buffer (pH4.5). STZ injected animals were given 20% glucose solution for 24 h to prevent initialdrug-induced hypoglycemic mortality. Control rats were injected with same volumeof citrate buffer. Plasma glucose was determined at the end of 96 h and those ratswith fasting glucose levels greater than 250 mg/dL were used in the present study.

2.4. Experimental design

In this experiment a total of 30 rats (18 diabetic surviving rats, 12 normal rats)were used. Rats were divided into five groups of six animals in each group. Theywere treated as follows:

Group I: Normal control rats received intra gastrically 0.5 ml of 0.9% saline oncedaily for 30 days.Group II: Normal rats received intra gastrically 20-OH-ecdysone (5 mg/kg b.w.)dissolved in 0.5 ml of 0.9% saline once daily for 30 days.Group III: Diabetic control rats.Group IV: Diabetic rats received intra gastrically 20-OH-ecdysone (5 mg/kg b.w.)dissolved in 0.5 ml of 0.9% saline once daily for 30 days [19].Group V: Diabetic rats received intra gastrically glibenclamide (5 mg/kg b.w.) dis-solved in 0.5 ml of 0.9% saline once daily for 30 days.

At the end of the experimental period (30 days), animals were deprived offood overnight and sacrificed by cervical decapitation under pentobarbitone sodium(60 mg/kg) anaesthesia. Blood was collected in a dry test tube and allowed to coag-ulate at room temperature for 30 min. Serum was separated by centrifugation at2000 rpm for 10 min and used for the estimation of serum ALT, AST, urea and crea-tinine. Liver and kidney were immediately dissected out, washed in ice-cold salineto remove the blood. The tissues were weighed and 10% tissue homogenate wasprepared with 0.01 M TrisHCl buffer, pH 7.4. After centrifugation at 3000 rpm for10 min, the clear supernatant was used to measure thiobarbituric acid reactive sub-stances (TBARS) and hydroperoxides, enzymic and non-enzymic antioxidants.

2.5. Biochemical analysis

Plasma glucose was estimated by the method of Trinder using a reagent kit [23]and plasma insulin was measured by the method of Burgi et al. [24]. The activities ofserum aspartate aminotransferase (AST), alanine aminotransferase (ALT) were esti-mated (by using commercially available kits) by the method of Reitman and Frankel[25]. Serum urea and creatinine were measured by the diacetyl monoxime methodWybenga et al. [26] and Jaffes method [27] respectively. The estimation of proteinwas carried out by the method of Lowry et al. [28]. Lipid peroxidation was estimatedcolorimetrically by measuring thiobarbituric acid reactive substances (TBARS) andhydroperoxides (HP) in liver and kidney tissues by the method of Niehius andSamuelsson [29] and Jiang et al. [30] respectively. SOD was assayed by the methodof Kakkar et al. [31]. The activity of CAT was determined by the method of Sinha[32]. GPx activity was measured by the method described by Rotruck et al. [33]. GSTactivity was measured by the method of Habig et al. [34] Ascorbic acid was estimatedby the method of Omaye et al. [35], -tocopherol was estimated by the method ofBaker and Frank [36].

2.6. Histopathological studies

A portion of the pancreas, liver and kidney tissues of control and experimentalrats were taken and fixed in 10% buffered neutral formal saline for histological stud-ies. After fixation, the tissues were embedded in paraffin; solid sections were cut at5 m and stained with hematoxylineosin. The structural changes of sections wereexamined under a light microscope.

2.7. Statistical analysis

The results are expressed as mean standard deviation (S.D.). Differencesbetween groups were assessed by ANOVA using the SPSS software package forWindows. Post hoc testing was performed for inter-group comparisons using theleast significance difference (L.S.D.) P-values

2074 R. Sundaram et al. / Process Biochem

Table 1Effect of 20-OH ecdysone on plasma glucose, insulin in normal and diabetic rats.

Groups Glucose (mg/dl) Insulin (U/ml)

Normal control 93.5 7.99 17.04 0.84Normal + 20-OH ecdysone 5 mg 92.66 6.91 18.86 1.18Diabetic induced 261.1 16.01b 6.2 0.74bDiabetic + 20-OH ecdysone 5 mg 122.1 8.70c 14.25 1.18cDiabetic + glibenclamide 121.6 7.11 15.35 1.40

Values are given as mean S.D. for six animals in each group.Values are considered significantly different at P < 0.05 with post hoc L.S.D. test.a Control vs. drug control (20-OH ecdysone alone treated rat).

b Control rat vs. diabetic rat.

d

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i

diabetic rats. A fall in the activities of antioxidants enzymes was

TE

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c Diabetic rat vs. 20-OH-ecdysone treated diabetic rat.20-OH-ecdysone treated diabetic rat vs. glibenclamide.

cdysone and glibenclamide compared to diabetic control rats. Nor-al animals treated with 20-OH ecdysone showed no significant

hanges in plasma glucose and insulin levels.

The activities of serum AST, ALT and the levels of urea, creatininen normal and diabetic rats were shown in Table 2. The activities

able 2ffect of 20-OH ecdysone on activities of serum AST, ALT, urea and creatinine in normal a

Groups AST (IU/L) AL

Normal control 70.63 6.09 38Normal + 20-OH ecdysone 5 mg 71.43 7.47 37Diabetic induced 133.18 8.96b 71Diabetic + 20-OH ecdysone 5 mg 77.01 6.56c 45Diabetic + glibenclamide 5 mg 76.57 7.60 43alues are given as mean S.D. for six animals in each group.alues are considered significantly different at P < 0.05 with post hoc L.S.D. test.Control vs. drug control (20-OH ecdysone alone treated rat).b Control rat vs. diabetic rat.c Diabetic rat vs. 20-OH-ecdysone treated diabetic rat.20-OH-ecdysone treated diabetic rat vs. glibenclamide treated diabetic rats.

able 3ffect of 20-OH ecdysone in the levels of TBARS and hydroperoxides in tissues of normal

Groups TBARS (mM/100 g tissues)

Liver K

Normal control 0.87 0.06 1Normal + 20-OH ecdysone 5 mg 0.85 0.07 0Diabetic control 2.57 0.23b 2Diabetic + 20-OH ecdysone 5 mg 1.45 0.10c 1Diabetic + glibenclamide 5 mg 1.41 0.10 1alues are given as mean S.D. for six animals in each group. Values are considered signiControl vs. drug control (normal rats treated with 20-OH ecdysone alone).b Control rat vs. diabetic rat.c Diabetic rat vs. 20-OH-ecdysone treated diabetic rat.20-OH-ecdysone treated diabetic rat vs. glibenclamide treated diabetic rats.

able 4ffect of 20-OH ecdysone on activities of superoxide dismutase, catalase, glutathione per

Groups Superoxide dismutase Catalase

Liver Kidney Liver

Normal control 8.82 0.51 16.54 1.52 81.17 6.07 Normal + 20-OH ecdysone 5 mg 8.91 0.57 17.26 1.62 83.56 6.23 Diabetic control 5.09 0.47b 8.37 0.71b 46.71 4.23bDiabetic + 20-OH ecdysone 5 mg 7.47 0.60c 12.56 1.20c 71.14 6.09cDiabetic + glibenclamide 5 mg 7.76 0.59 13.16 1.13 71.81 5.43 he activities of enzymes are expressed as follows: SOD one unit of activity was taken asn 1 min/mg protein; CAT mol of H2O2 consumed/minute; GPx g of glutathioneonjugate formed/minute/mg protein.alues are given as mean S.D. for six animals in each group. Values are considered signiControl vs. drug control (normal rats treated with 20-OH ecdysone alone).b Control rat vs. diabetic rat.c Diabetic rat vs. 20-OH-ecdysone treated diabetic rat.20-OH-ecdysone treated diabetic rat vs. glibenclamide treated diabetic rats.istry 47 (2012) 20722080

of serum hepatic marker enzymes AST and ALT and levels of renalfunction markers, urea and creatinine were significantly higher inthe diabetic rats when compared to control rats. Administrationof 20-OH ecdysone and glibenclamide to diabetic rats significantlydecreased the activities of AST and ALT and the levels of urea andcreatinine to near normal when compared to diabetic untreatedrats.

Table 3 shows the levels of TBARS and HP in liver and kidney tis-sues of normal and diabetic rats. Diabetic rats exhibited increasedlevels of TBARS and HP when compared to normal control. Intra-gastric administration of 20-OH ecdysone and glibenclamide todiabetic rats significantly decreased lipid peroxidation markersin liver and kidney tissues when compared to diabetic untreatedrats.

Table 4 represents the activities of antioxidant enzymes (SOD,CAT, GPx and GST) in hepatic and renal tissues of normal andobserved in diabetic rats when compared to normal control. 20-OH-ecdysone and glibenclamide administration to diabetic ratssignificantly improved the activities of the above enzymes.

nd diabetic rats.

T (IU/L) Urea (mg/dL) Creatinine (mg/dL)

.24 3.19 33.94 3.50 0.67 0.06

.58 3.81 32.91 3.09 0.66 0.05

.90 6.71b 60.05 6.09b 1.73 0.07b

.11 4.04c 35.15 3.42c 0.70 0.09c

.77 5.12 34.12 3.91 0.69 0.06

and diabetic rats.

Hydroperoxides (mM/100 g tissues)

idney Liver Kidney

.11 0.10 78.90 6.12 45.52 3.63

.98 0.07 75.76 5.23 42.29 3.71

.34 0.13b 126.4 6.41b 71.20 5.03b

.47 0.12c 86.81 6.27c 58.07 5.89c

.45 0.15 81.28 5.64 54.49 5.31ficantly different at P < 0.05 with post hoc L.S.D. test.

oxidase and glutathione-S-transferase in tissues of normal and diabetic rats.

Glutathione peroxide Glutathione S transferase

Kidney Liver Kidney Liver Kidney

37.90 3.47 9.42 0.50 7.30 0.59 8.32 0.53 5.45 0.4339.37 3.22 9.60 0.61 7.58 0.63 8.54 0.58 5.76 0.5022.75 1.84b 5.16 0.47b 3.71 0.34b 4.63 0.35b 3.52 0.31b31.98 2.92c 7.32 0.61c 6.25 0.55c 7.42 0.50c 4.81 0.43c32.86 2.77 7.75 0.55 6.64 0.47 7.76 0.42 4.93 0.44

the enzyme quantity, which gave 50% inhibition of nitroblue tetrazolium reduction consumed/minute/mg protein; GST mol of 1-chloro 2,4-dinitrobenzene-GSH

ficantly different at P < 0.05 with post hoc L.S.D. test.

R. Sundaram et al. / Process Biochemistry 47 (2012) 20722080 2075

Table 5Effect of 20-OH ecdysone on levels of vitamin C, vitamin E and reduced glutathione in tissues of normal and diabetic rats.

Groups Vitamin C (M/mg tissue) Vitamin E (M/mg tissue) Reduced glutathione (M/mg tissue)

Liver Kidney Liver Kidney Liver Kidney

Normal control 1.46 0.18 1.23 0.08 0.90 0.08 0.49 0.03 76.29 5.38 60.57 5.47Normal + 20-OH ecdysone 5 mg 1.60 0.15 1.31 0.08 0.96 0.07 0.51 0.04 79.33 6.67 61.22 5.50Diabetic control 0.79 0.06b 0.60 0.05b 0.36 0.02b 0.25 0.02b 42.38 4.15b 37.96 3.32bDiabetic + 20-OH ecdysone 5 mg 1.12 0.13c 0.96 0.08c 0.68 0.05c 0.39 0.03c 66.09 5.90c 49.68 4.24cDiabetic + glibenclamide 5 mg 1.20 0.14 1.05 0.10 0.74 0.06 0.42 0.03 68.39 5.88 50.75 5.06

Values are given as mean S.D. for six animals in each group. Values are considered significantly different at P < 0.05 with post hoc L.S.D. test.a Control vs. drug control (normal rats treated with 20-OH ecdysone alone).

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Control rat vs. diabetic rat.c Diabetic rat vs. 20-OH-ecdysone treated diabetic rat.20-OH-ecdysone treated diabetic rat vs. glibenclamide treated diabetic rats.

Table 5 represents the levels of Vitamin C, -tocopherol andeduced glutathione in liver and kidney tissues of normal and dia-etic rats. Diabetic rats exhibited a significant fall in the levels ofitamin C, -tocopherol and reduced glutathione when comparedo normal control. However, treatment with 20-OH-ecdysone andlibenclamide brought back these parameters to near normalcy.

.1. Histological study

.1.1. PancreasThe histopathological examination revealed extensive alter-

tions in the pancreas of STZ-induced diabetic rats. Pancreaticection from normal and drug control rats showed normal archi-ecture of the islets of langerhans and well-defined granulated darkcells (Fig. 2A and B). The pancreatic tissue of diabetic rats showed

he islets of langerhans with imperfect atrophied cells (Fig. 2C)ancreatic section from diabetic rats after treatment with 20-OH-cdysone and glibenclamide showed the islets of langerhans withell-organized, granulated cells (Fig. 2D and E)

.1.2. LiverHepatic section from normal and drug control rats showed nor-

al arrangement of hepatocytes and no fibrosis (Fig. 3A and B).epatic section from diabetic rats was showed the distortion inhe arrangement of cells around the central vein of the liver. Theapillaries were enlarged and the walls of veins and capillariesere thickened in the diabetic liver (Fig. 3C). Hepatic section fromiabetic rats treated with 20-OH-ecdysone and glibenclamide pre-ented the cellular changes such as disturbed arrangement of theepatocytes in the liver tissue of the diabetic rats (Fig. 3D and E).

.1.3. KidneyRenal section from normal and drug control rats showed no

tructural changes in the glomeruli, proximal convoluted tubules,istal convoluted tubules and collecting duct (Fig. 4A and B).enal tissue section from diabetic control rats showed expandedlomerulus and also thickening walls of renal tubules filling withumen. The glomerular lesions characteristic of the long-term dia-etic nephropathy were not visible in the kidney of 4 weeks diabeticats. The collecting tubules showed dilation in the cortex regionay be under the pressure of increased urine flow (Fig. 4C). Renalection from 20-OH-ecdysone and glibenclamide treated diabeticats reversed the cellular changes towards near normal (Fig. 4D and).

. DiscussionSTZ-induced diabetes is a well-characterized experimentalodel for type 1 diabetes due to its ability to selectively destroyancreatic -cells leading insulin deficiency and hyperglycemia37]. Diabetogenic effect of STZ is mainly attributed to the excessproduction of ROS leading to toxicity in pancreatic beta cells whichreduces the synthesis and the release of insulin and affecting organssuch as liver, kidney and hematopoietic system [37].

In the present study, STZ-induced diabetic rats showed signifi-cant increase in plasma glucose and decrease in insulin levels whencompared to normal rats. The increased levels of plasma glucoseand decreased levels of insulin were brought back to near nor-mal by the administration of 20-OH-ecdysone and glibenclamidewhich is an essential trigger for the liver and kidney to revert backto its normal homeostasis during experimental diabetes. Because,persistent hyperglycemia increases the generation of free radicalsand exhausts the antioxidant defenses thus leading to the disrup-tion of cellular functions, oxidative damage to membranes andenhanced susceptibility to LPO [38]. The hypoglycemic action of20-OH ecdysone may either be due to enhanced production of theinsulin secretion from remnant pancreatic -cells or which protectthe intact functional -cells from further deterioration, so that theyremain active and continue to produce insulin as it is evidenced bythe significant increase in the level of insulin in diabetic treated ratswhich is supported by the improved histopathology of pancreas.

Recently, we reported that 20-OH-ecdysone administration sig-nificantly improved glucose homeostasis through modification ofactivities of key enzymes of carbohydrate metabolism via enhancedinsulin secretion in STZ-induced diabetic rats [22]. Moreover, sev-eral studies have proved that 20-OH-ecdysone treatment reducedthe blood glucose by increasing insulin in alloxan induced diabeticrats [19,39]. Further, Muthuraman and Srikumar [40] have reportedthat oral administration of oxysterol which is structurally homol-ogous to 20-OH ecdysone decreased the blood glucose levels inSTZ-induced diabetic rats.

Lipid peroxidation is considered a hallmark of oxidative stressin which ROS interact with polyunsaturated fatty acids and leadsto the formation of lipid products such as MDA and 4-HNE(4-hydroxynonenal) which then causing damages to the mem-brane components of the cell, cell necrosis and inflammation [41].Extensive evidence has demonstrated that the increase of lipid per-oxidation plays an important role in the progression of diabetesby altering the transbilayer fluidity gradient which could hamperthe activities of membrane-bound enzymes and receptors [42]. Inour study, there were significant increases in lipid peroxidation inliver and kidney of diabetic rats as measured by lipid peroxidationmarkers such as thiobarbituric acid reactive substances (TBARS),hydroperoxides (HP). These results are in agreement with severalstudies that have reported an increase in TBARS and HP levels inkidney, liver, serum and erythrocytes of animal with experimen-tal and clinical diabetes [10,4346]. Taken together these findingssuggest that the exposure to high glucose levels may elevate the

generation of ROS through the non-enzymatic glycation of proteinand glucose autooxidation. As a consequence, it provokes damagein structural and functional integrity of hepatic and renal tissues,as evidenced by an increase in the oxidative deterioration of the

2 iochem

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076 R. Sundaram et al. / Process B

ipids of cellular membrane in diabetic state. Peroxidative regu-ation can be achieved by intervention with lipid, enzymic andon-enzymic antioxidants. Moreover, following administration of0-OH-ecdysone and glibenclamide to diabetic rats prevented theipid peroxidation markers to near normal levels which could be as

result of improved glycemic control and antioxidants status.Tissues are endowed with antioxidant defense mechanisms,

hich include the concerted action of both enzymic and non-nzymic antioxidants. An alteration in the activities of antioxidantnzymes SOD, CAT, GST, GPx and in glutathione metabolism resultsn an imbalance of oxidant/antioxidant defense systems leading tohe accumulation of highly reactive free radicals [41]. In the present

tudy, we observed a fall in antioxidant enzymes and low molec-lar weight antioxidant levels with elevation of lipid peroxidationarkers in liver and kidney tissues of diabetic rats.

Fig. 2. Histopathological section of pancreas istry 47 (2012) 20722080

Free radicals are continuously generated as a result of nor-mal metabolic processes and on interaction with environmentalstimuli. ROS are the unavoidable evils of oxygen metabolism andsuperoxide anion is the primary radical produced in vivo whichlead to secondary radicals. Chronic hyperglycemia, the hallmark ofdiabetes mellitus leads to an overdrive of mitochondrial respira-tory chain resulting in increased production of superoxide anions[47]. Antioxidant enzymes form the first line of defense againstROS in the organism include the enzymes SOD, CAT GST and GPx,which play an important role in scavenging the toxic intermediateof incomplete oxidation. CAT and SOD are the two major enzymesthat remove ROS in vivo. SOD catalyzes the dismutation of superox-

ide anion into hydrogen peroxide (H2O2), which is then degraded toH2O by CAT or by glutathione peroxidase. A decrease in the activ-ity of these antioxidants may lead to an excess of availability of

of control and experimental rats (20).

R. Sundaram et al. / Process Biochemistry 47 (2012) 20722080 2077

ver of

si[fnpg[mc

i

Fig. 3. Histopathological section of li

uperoxide anion and H2O2 which in turn generates hydroxyl rad-cals, resulting in initiation and propagation of lipid peroxidation41,4850]. In line with this, in the present study, we observed aall in both enzymic and non-enzymic antioxidant in liver and kid-ey of diabetic rats associated with a concomitant increase in lipideroxidation markers in these tissues. In diabetic state abnormallycation of SOD impairs its activity resulting in oxidative stress51]. Reduced SOD activity is responsible for the activation of all

ajor pathways underlying the different components of vascularomplications [41].

Antioxidants enzymes CAT and GPx are involved in the elim-nation of H2O2 at high and low concentrations respectively. Incontrol and experimental rats (20).

diabetes, the activities of CAT and GPx are significantly decreasedby superoxide radical and by glycation reactions. Glutathione, atripeptide, functions as a direct scavenger of free radicals andalso a co-substrate for GPx. As NADPH necessary for GSH regen-eration is utilized by the polyol pathway which is prominent inhyperglycemic conditions, there occurs a depletion of GSH result-ing in lowered GPx activity [52]. Low GPx activity is associatedwith thrombosis and cardiovascular complications in diabetes [53].

Reduced activities of enzymic antioxidants have been observedduring diabetes and this may result in number of deleterious effectsdue to the accumulation of free radicals [54,55]. The activities ofthe above enzymes influence the susceptibility of various tissues

2078 R. Sundaram et al. / Process Biochemistry 47 (2012) 20722080

ney o

tika

dwgscmfab

Fig. 4. Histopathological section of kid

o oxidative stress. In the present study we also observed a signif-cant decrease in the activities of antioxidant enzymes in liver andidney tissues of diabetic rats when compared to normal controlnimals.Tocopherol is the primary lipophilic chain breaking antioxi-

ant and is a component of total peroxyl radical trapping systemhich reacts directly with peroxyl, superoxide and singlet oxy-en and protects membranes from lipid peroxidation [56]. It isuggested to be a beneficial antioxidant for treatment of diabeticomplication in humans as well as in STZ-treated rats [57,58]. Vita-

in C works in synergy with tocopherol against different types of

ree radicals and inhibits hydroperoxide formation. We observed significant decrease in non-enzymic antioxidant levels in dia-etic control rats and those levels were elevated upon treatmentf control and experimental rats (20).

with 20-OH-ecdysone and glibenclamide. These results suggestthat 20-OH-ecdysone and glibenclamide significantly improvedantioxidant status and reduced lipid peroxidation in liver and kid-ney tissues of diabetic treated rats through their hypoglycemicpotential which might directly be corrected the above antioxidantsenzymes and protected the tissues damage from oxidative stress.

Another important aspect to be discussed in this study is serumaminotransferases activities, which have long been considered assensitive indicators of hepatic damage [59]. Injury to the hepato-cytes alters their transport function and membrane permeability,

leading to leakage of enzymes from the cells [60]. Therefore, themarked increase of AST and ALT in the plasma by leakage ofthese enzymes from liver cytosol into the blood stream. Thus,increased activities of AST and ALT and found in this study may be

R. Sundaram et al. / Process Biochemistry 47 (2012) 20722080 2079

STZ

-cells damage

Insulin secre on

Hepac & R enal markers(AST, ALT & Urea, Crea nine)

Alter naon of carboh ydrat e Blood Glu cose meta bolizing enzymes

HbA1c

OPL SOR Anox idan t

Glyc oly c enzymes Glucon eogenic enzymes

Periph eral ssue glucose uptak e

Body weight

Abno rmality in pa rameter s due inducon of diab etes

Correction/ pha rmac olog ical ef fect of 20-OH ec dysone and glibenc lamide

s and

imi[deSl

rvaaetavbper2ts

Fig. 5. Schematic diagram of the streptozotocin induced diabete

nterpreted as a result of the liver cell destruction or changes in theembrane permeability indicating severe hepatocellular damage

s induced by diabetes which is in accordance with other reports6062]. Administration of 20-OH-ecdysone and glibenclamide toiabetic rats showed significant decrease in the activities of thesenzymes and consequently prevented liver damage caused byTZ-induced diabetes as evidenced by improved histopathology ofiver.

In addition to the hepatic damage, in the current study, diabeticats also presented renal damages that were evidenced by the ele-ation in serum urea and creatinine levels which are considereds significant markers of renal dysfunction [63]. 20-OH-ecdysonend glibenclamide treatment prevented the increase in the lev-ls of urea and creatinine in diabetic rats. These findings suggesthat 20-OH-ecdysone and glibenclamide possess the potential tottenuate renal injury caused by hyperglycemic state and pre-ents the further progression of renal damage and is evidencedy improved histopathology of kidney which supports to thesearameters. Based on these results, we can suggest that 20-OH-cdysone and glibenclamide inhibits the progression of hepatic and

enal dysfunction in STZ-induced diabetic rats. In previous reports,0-OH-ecdysones containing rich extract of Ajuga iva has showno improve hepatic and renal markers in diabetic rats which areimilar to that of our results [64]. its prevention/curation by 20-OH-ecdysone and glibenclamide.

5. Conclusion

The findings of the present study demonstrated that 20-OH-ecdysone treatment significantly improved glycemic, hepatic, renalmarkers and antioxidant status in diabetic rats by enhancing insulinsecretion from remnant beta cells of pancreas in STZ-induced dia-betic rats. (The impacts of STZ on pancreatic -cells and curativepotential of 20-OH ecdysone and glibenclamide are shown in Fig. 5.)The effect produced by 20-OH ecdysone on various parameters wascomparable to that of glibenclamide.

Conflict of interest statement

The authors declare that there are no conflicts of interest.

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Ameliorative effect of 20-OH ecdysone on streptozotocin induced oxidative stress and -cell damage in experimental hypergl...1 Introduction2 Materials and methods2.1 Chemicals2.2 Animals2.3 Induction of diabetes2.4 Experimental design2.5 Biochemical analysis2.6 Histopathological studies2.7 Statistical analysis

3 Results3.1 Histological study3.1.1 Pancreas3.1.2 Liver3.1.3 Kidney

4 Discussion5 ConclusionConflict of interest statementReferences