Toxicology and Applied Pharmacology 244 (2010) 114–129

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Toxicology and Applied Pharmacology

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Streptozotocin induced activation of oxidative stress responsive splenic cell signalingpathways: Protective role of arjunolic acid

Prasenjit Manna, Jyotirmoy Ghosh, Joydeep Das, Parames C. Sil ⁎Division of Molecular Medicine, Bose Institute, P-1/12, CIT Scheme VIIM, Calcutta-700054, West Bengal, India

Abbreviations: AA, arjunolic acid (2,3,23-trihydroxycatalase; DAB, 3,3′-diaminobenzidine tetrahydrochlorideagle's medium; FACS, fluorescence activated cell sorglutathione disulfide; GST, glutathione-S-transferase; GPglutathione reductase; M2VP, 1-methyl-2-vinylpyridiniuMAPKs, mitogen-activated protein kinases; MDA, mfactor kappa B; ROS, reactive oxygen species; RNS, resuperoxide dismutase.⁎ Corresponding author. Fax: +9133 2350 6790.

E-mail addresses: parames@bosemain.boseinst.ac.in(P.C. Sil).

0041-008X/$ – see front matter © 2009 Elsevier Inc. Adoi:10.1016/j.taap.2009.12.024

a b s t r a c t

a r t i c l e i n f o

Article history:Received 23 March 2009Revised 2 December 2009Accepted 17 December 2009Available online 4 January 2010

Keywords:StreptozotocinHyperglycemiaSpleenCytokinesMAPKsApoptosisArjunolic acidChemoprevention

Present study investigates the beneficial role of arjunolic acid (AA) against the alteration in the cytokinelevels and simultaneous activation of oxidative stress responsive signaling pathways in spleen underhyperglycemic condition. Diabetes was induced by injection of streptozotocin (STZ) (at a dose of 70 mg/kgbody weight, injected in the tail vain). STZ administration elevated the levels of IL-2 as well as IFN-γ andattenuated the level of TNF-α in the sera of diabetic animals. In addition, hyperglycemia is also associatedwith the increased production of intracellular reactive intermediates resulting with the elevation in lipidperoxidation, protein carbonylation and reduction in intracellular antioxidant defense. Investigating theoxidative stress responsive cell signaling pathways, increased expressions (immunoreactive concentrations)of phosphorylated p65 as well as its inhibitor protein phospho IκBα and phosphorylated mitogen activatedprotein kinases (MAPKs) have been observed in diabetic spleen tissue. Studies on isolated splenocytesrevealed that hyperglycemia caused disruption of mitochondrial membrane potential, elevation in theconcentration of cytosolic cytochrome c as well as activation of caspase 3 leading to apoptotic cell death.Histological examination revealed that diabetic induction depleted the white pulp scoring which is inagreement with the reduced immunological response. Treatment with AA prevented the hyperglycemia andits associated pathogenesis in spleen tissue. Results suggest that AA might act as an anti-diabetic andimmunomodulatory agent against hyperglycemia.

olean-12-en-28-oic acid); CAT,e; DMEM, Dulbecco's modifiedting; GSH, glutathione; GSSG,x, glutathione peroxidase; GR,m trifluoromethanesulfonate;

alonaldehyde; NF-κB, nuclearactive nitrogen species; SOD,

, parames_95@yahoo.co.in

ll rights reserved.

© 2009 Elsevier Inc. All rights reserved.

Introduction

In modern civilization diabetic mellitus is an increasing healthproblem. It is a group of metabolic diseases characterized byhyperglycemia resulting from defective insulin secretion or resistanceto insulin action or both (Braun et al., 1995). Several lines of earlierinvestigations have examined that oxidative stress plays an importantrole in the etiology of diabetes as well as associated complications(Baynes, 1991; Nogichi, 2007; Oberley, 1988). Various free radicalsand non radical species react with several amino acid residues alteringtheir structures and by extension, the tertiary structures of the protein

molecules. Immune deficiencies, especially cell-mediated immunity,have been suggested as a major contributor to infection and morbidcomplication of diabetes patients (Geerlings and Hoepelman, 1999;Chang, and Shaio, 1999). Spleen is one of the principle sites for theinitiation of most primary immune responses, for B lymphocyteactivation and the production of antibodies. β-Cell destruction in type1 diabetes probably involves cytokine production by spleen cells(Thorvaldson et al., 2003). The balance between cytokines producedby T helper 1 and 2 (Th1 and Th2) lymphocytes has been postulated toinfluence the outcome of diabetes. In type 1 diabetes, Th1 response (acytokine production dominated by a group of cytokines, e.g. TNF-α,IFN-γ, IL-1β and IL-12) has been suggested to be connected with a β-cell destructive insulitis; however, Th2 type response (production ofIL-4 and IL-10) is associated with a non-destructive insulitis (Kolb,1997). The altered cytokine production, in addition, also causedactivation of redox-sensitive transcription factors (TFs), nuclearfactor-κB (NF-κB) and activator protein-1 (AP-1). A variety ofagents which are known to generate ROS, have also been shown toregulate AP-1 activation (Hsu et al., 2000; Klaunig and Kamendulis,2004) and that could be regulated by the activation of mitogenactivated protein kinases (MAPKs). Streptozotocin (STZ), an anti-biotic, produced by Streptomyces achromogenes, is the mostcommonly used agent in experimental diabetes due to its abilityto destruct pancreatic β-islets cells possibly via the formation of

115P. Manna et al. / Toxicology and Applied Pharmacology 244 (2010) 114–129

excessive free radicals (Rakieten et al., 1963). Elsner et al. (2000)showed that the alkylation of DNA plays a major role in STZ inducedcell death. The alkylating activity of STZ is related to its nitrosoureamoiety, especially at the O6 position of guanine. Earlier reports(Geerlings and Hoepelman, 1999; Herold et al., 1996) suggest thatSTZ induced β-cell destruction is mediated via the release ofcytokines, like IFN-γ, IL-2, TNF-α etc. from the spleen.

People suffering from hyperglycemia usually take drugs belongingto the family of sulphonylureas and bisguanidine. These drugs,however, cause various side effects in them. Scientists are, therefore,in search for easily available, inexpensive therapeutics, havingminimum side effects for the better treatment of these patients.Arjunolic acid (AA: 2,3,23-trihydroxyolean-12-en-28-oic acid,Fig. 1A), a natural pentacyclic triterpenoid saponin, is well knownfor various biological functions, including antioxidative (Manna et al.,1999), anti-fungal (Masoko et al., 2008), hepatoprotective (Mannaet al., 2007a, b), anti-bacterial (Djoukeng et al., 2005) and insectgrowth inhibitor activity (Bhakuni et al., 2002). In the present study,the role of AA against STZ induced splenotoxicity under hyper-glycemic condition has been investigated. To achieve the goal, thestatus of the intracellular antioxidant enzymes, GSH/GSSG ratio,cytokine levels, expressions of TFs andMAPKs aswell asmitochondria-dependent cell death pathways have been investigated.

Fig. 1. (A) Chemical structure of arjunolic acid. (B) Mass spectra of the purified arjunolic acid(C) 1H NMR (500 MHz) spectra of arjunolic acid. (D) 13C NMR (125 MHz) spectra of arjun

Materials and methods

Chemicals

DAPI (4′,6-diamidino-2-phenylindole dihydrochloride), DCFDA(2′,7′- dichlorofluorescin diacetate), FBS (fetal bovine serum),Streptozotocin (STZ) [N-(methylnitrosocarbamoyl)-α-D-glucos-amine], proteinase inhibitor PMSF (phenylmethanesulfonyl fluo-ride), phosphatase inhibitor cocktail, anti Caspase 3, anti ERK 1/2,anti phosphorylated ERK 1/2, anti JNK and anti phosphorylated JNKantibodies were purchased from Sigma-Aldrich Chemical Company(St. Louis, USA). Anti p65, anti phosphorylated p65, anti p38 andanti phosphorylated p38 antibodies were purchased from Cellsignaling technology (Beverly, USA). Anti TNF-α, anti IL-2, antiIFN-γ, anti IκBα and anti phosphorylated IκBα were purchased fromSanta Cruz Biotechnology (Santa Cruz, USA). CDNB (1-chloro-2,4-dinitrobenzene), DNPH (2,4-dinitrophenyl hydrazine), DTNB [5,5′-dithiobis(2-nitrobenzoic acid)] [(Ellman's reagent)], MTT [3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide], NBT(nitrotetrazolium blue chloride), PMS (phenazine methosulphate),TCA (trichloroacetic acid), TBA (2-thiobarbituric acid) and othercommon reagents were purchased from Sisco research laboratory,Mumbai, India.

(AA); HRMS (ESI): m/z calculated (C30H48O5Na) 511.3399, found 511.3438 [M+Na]+.olic acid. (E) Reverse phase HPLC analysis of arjunolic acid.

Fig. 1 (continued).

116 P. Manna et al. / Toxicology and Applied Pharmacology 244 (2010) 114–129

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Animals

Swiss albino male rats of weighing approximately 120–130 g werepurchased from M/S Gosh Enterprises, Kolkota, India. They were8 weeks old at the time of study. The animals were acclimatized underlaboratory conditions for 2 weeks prior to the experiments. They weremaintained under standard conditions of temperature (23±2 °C) andhumidity (50±10%) with an alternating 12 h light/dark cycles. Theanimals had free access to tapwater and fed standard pellet diet (AgroCorporation Private Ltd., Bangalore, India). All the experiments withanimals were carried out according to the guidelines of theinstitutional animal ethical committee.

Extraction of arjunolic acid (AA)

Arjunolic acid (AA) has been extracted from the bark of Terminaliaarjuna following the method of Manna et al. (2007a, b). Briefly, aftercollection, the bark was cut into small pieces, dried, ground intopowder (1 kg) and extracted with petroleum ether (20 h) by usingSoxhlet apparatus to remove greasy non polar materials. Petroleumetherwas removedand the crudematerialwas subsequently extractedwith diethyl ether (40 h). It was filtered off and the mother liquor wasconcentrated under reduced pressure to yield a yellowish whiteamorphous solid which showed two spots on thin layer chroma-tography (TLC). TLC condition was butanol:acetic acid:water=4:1:0.5. This solid material was then subjected to silica gel columnchromatography for the separation of those two compounds. Elutionwith chloroform:methanol (60:40) yielded 250 mg of a compound,later identified as arjunolic acid. Its purity has been confirmed by usingstandard tools [like NMR (1H, 13C), IR, Mass Spectroscopy and opticalrotation studies]. Homogeneous preparation of AA and the absence ofany contaminating substances in its preparation have also beenconfirmed by reverse phase HPLC using a C18 column (8 mm×10 cm).The columnwas elutedwith amobile phase 6:1methanol/water, flowrate 1 mL/min. UV detection was carried out at 206 nm.

Animal treatment

The animals were divided into four groups, consisted of six rats ineach and they were treated as follows.

Group 1 (Normal Control Group)—Animals received vehicle onlyand were sacrificed 9 days after the beginning of the experiment.

Group 2 (AA Treated Group)—AAwas administered orally at a doseof 20 mg/kg body weight for 4 days and the animals were sacrificed24 h after the final dose of AA administration.

Group 3 (STZ Treated Group)—Animals received STZ at a dose of70 mg/kg body weight in the tail vain, left untreated for 5 days andthen sacrificed.

Group 4 (AA Pre-treated Group)—AA was administered orally at adose of 20 mg/kg body weight for 4 days followed by STZ injection inthe tail vain at a dose of 70 mg/kg body weight, left untreated for5 days and then sacrificed.

Before scarification, all animals in each groupwere sacrificed. Fromall the sacrificed animals the spleen tissue were collected and used forthe various experiments as needed.

[The dose schedules of STZ and AA have been selected based on theearlier reports ofDias et al (2005) andManna et al. (2009) respectively].

Preparation of spleen tissue homogenates

Spleen tissues from experimental rats were homogenized usingglass homogenizer in 100mM potassium phosphate buffer containing1 mM EDTA, pH 7.4, PMSF (proteinase inhibitor) and phosphataseinhibitor cocktail. The homogenized mixture was centrifuged at12,000g for 30min at 4 °C. The supernatant was collected and used forthe experiments.

Determination of protein content

The protein contents of the experimental tissue homogenateswere measured by the method of Bradford using crystalline BSA asstandard (Bradford, 1976).

Assessment of the levels of blood glucose

Blood samples collected from puncturing rat heart were keptovernight to clot and then centrifuged at 3000g for 10 min. Bloodglucose levels were estimated by using glucose estimation kit (Spandiagnostic Ltd., India).

Determination of serum IL-2, IFN-γ and TNF-α

IL-2, IFN-γ and TNF-α concentrations in the serum samples wereanalyzed using enzyme linked immune sorbant assay (ELISA). Opticaldensity was measured with a Labsystems integrated EIA ManagementSystem iEMS ELISA and the DeltaSoft3 2.22 EMS software (Dr. EBechthold and BioMetallics).

Measurement of intracellular ROS production

Intracellular ROS production was estimated by using DCFDA as aprobe following themethod of LeBel and Bondy (1990) asmodified byKim et al. (1996). DCF-DA diffuses through the cell membrane whereit is enzymatically deacetylated by intracellular esterases to the morehydrophilic nonfluorescent reduced dye dichlorofluorescin. In thepresence of reactive oxygenmetabolites, nonfluorescent DCFH rapidlyoxidized to highly fluorescent product DCF. Earlier report suggeststhat oxidation of DCFH was unaffected by SOD, catalase or nitric oxide(Zhu et al., 1994). For in vivo study, 100 μL of tissue homogenateswere incubated with the assay media (20 mM Tris–HCl, 130 mM KCl,5 mM MgCl2, 20 mM NaH2PO4, 30 mM glucose and 5 μM DCFDA) at37 °C for 15 min. The formation of DCF was measured at the excitationwavelength of 488 nm and emission wavelength of 610 nm for 10minby using fluorescence spectrometer (HITACHI, Model No F4500)equipped with a FITC filter.

For in vitro study, after treatment, the medium was aspirated andsplenocytes were washed with RPMI 1640 culture medium andincubated in 2 mL of fresh culture medium without FBS. DCFDA wasadded at a final concentration of 10 μmol/L and incubated for 20 min.The cells were then washed twice with RPMI 1640 culture mediumandmaintained in 1mL culture medium. Fluorescence wasmonitoredusing fluorescence spectrometer (the intensity values were calculatedusing the Metamorph software) and also they were also assessedusing a flow cytometer.

Measurement of nitric oxide (NO) levels in terms of nitrite

Nitric oxide synthase (NOS) catalyzes the oxidation of theterminal guanidino nitrogen of the amino acid L-arginine toproduce NO and L-citrulline. NO undergoes a series of reactionswith several molecules present in biological fluids and is eventuallymetabolized to nitrite (NO2

- ) and nitrate (NO3-) (Dezfulian et al.,

2007). Thus, the best index of total NO production is the sum ofboth nitrite and nitrate, commonly quantified in a two-step assay.The first step in the measurement of NO is the conversion of nitrateto nitrite by the use of NADH or NADPH-dependent nitratereductase. Subsequently, the converted nitrite can be quantifiedby the addition of Griess Reagent, which converts nitrite into apurple azo compound. Accurate concentration of nitrite can bedetermined by photometric measurement of the colored azocompound. The serum NO level was indirectly assessed bymeasuring the nitrite levels in plasma using a colorimetric methodbased on the Griess reaction (Beda and Nedospasov, 2005). 20 μL of

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the analyzed sample was added to 100 μL of the Griess reagent(equal volumes of 2% sulfanilamide in 5% HCl and 0.1% N-(1-naphthyl) ethylenediamine dihydrochloride in H2O were mixed justbefore use), NADH and nitrate reductase. 1 mL H2O was added, themixture was stirred, and, after 5 min, the absorbance at 540 nmwas determined.

Estimation of lipid peroxidation and protein carbonyl content

The lipid peroxidation end product in the experimental sampleswasmeasured according to themethods of Esterbauer and Cheeseman(1990). Spleen tissue samples containing1 mg protein were mixedwith 1 mL TCA (20 %), 2 mL TBA (0.67%) and heated for 1 h at100 °C. During heating step 0.01% BHT (butylated hydroxytoluene;2,6-Di-tert-butyl-4-methylphenol) was added to abolish the metalcatalyzes auto-oxidation of lipids. After cooling, the precipitate wasremoved by centrifugation. The pink pigment from all the sampleswas separately extracted with 4 mL of Butanol. The absorbance ofthe sample was measured at 535 nm using a blank containing allthe reagents except the sample. MDA content of the sample wascalculated using the extinction co-efficient of MDA, which is1.56×105 M−1 cm−1.

Protein carbonyl content in the spleen tissue samples wasdetermined following the method as described by Uchida andStadtman (1993). Briefly, the sample was treated with an equalvolume of 0.1% (w/v) DNPH in 2 N HCl, incubated for 1 h at roomtemperature and then treated with 20% TCA. After centrifugation, theprecipitate was washed three times with ethanol/ethyl acetate anddissolved in 8 M guanidine hydrochloride in 133 mM tris solutioncontaining 13mMEDTA. The absorbance was recorded at 365 nm. Theresults were expressed as nmol of DNPH incorporated/mg proteinbased on the molar extinction coefficient of 22000 M−1 cm−1 foraliphatic hydrazones.

Assay of antioxidant enzymes

Determination of SOD activity. In case of SOD assay spleen tissuehomogenates were centrifuged at 600 g for 8 min at 4 °C.Supernatants were decanted and re-centrifuged at 5,500 g for15 min to form mitochondrial pellets. The supernatant was kept as“cytosolic fraction”. The pellets were resuspended in MSH buffer(210 mM mannitol, 70 mM sucrose, 5 mM HEPES, pH 7.5) withoutEDTA and termed as “mitochondrial fraction”. The activities of Mn-SOD were measured in the mitochondrial and Cu/Zn-SOD in thecytosolic fractions following the method of Nishikimi et al. (1972) andKakkar et al. (1984). The addition of cyanide ion (in mitochondrialfraction) to a final concentration of 2 mM inhibits Cu/Zn-SOD activityby over 90%. Mn-SOD is unaffected by cyanide. For bothmeasurements, about 5 μg protein were mixed with sodiumpyrophosphate buffer, PMS and NBT. The reaction was started bythe addition of NADH. Then the reaction mixture was incubated at30 °C for 90 s and stopped by the addition of 1mL of glacial acetic acid.The absorbance of the chromogen formed was measured at 560 nm.One unit of SOD activity is defined as the enzyme concentrationrequired to inhibit chromogen production by 50% in 1 min under theassay condition.

Determination of CAT activity. The enzyme CAT converts H2O2

formed via the action of SOD on superoxide radical, into water. TheCAT activity was measured by the method of Bonaventura et al.(1972). About 5 μg protein from spleen homogenate was mixed with2.1 mL of 7.5 mM H2O2 and a time scan was performed at 240 nmspectrophotometrically for 10 min at 25 °C. The disappearance ofperoxide depending on the CAT activity was observed. One unit of CATactivity is defined as the amount of enzyme, which reduces 1 μmol ofH2O2 per minute.

Determination of GST activity. The GST activity was measured bythe method of Habig et al. (1974). The reaction mixture containedsuitable amount of enzyme (25 μg of protein in homogenates),potassium phosphate buffer, EDTA, CDNB and GSH. The reaction wascarried out at 37 °C and monitored spectrophotometrically by theincrease in absorbance of the conjugate of GSH and CDNB at 340 nm. Ablank was run in absence of the enzyme. One unit of GST activity is1 μmol product formation per minute.

Determination of GR activity. GR activity was determined accordingto the method of Smith et al. (1988). The increase in absorbance at412 nmwas monitored spectrophotometrically for 3 min at 24 °C. Theenzyme activity was calculated using molar extinction coefficient of13,600 M−1 cm−1. One unit of enzyme activity is defined as theamount of enzyme, which catalyzes the oxidation of 1 μmol NADPHper minute.

Determination of GPx activity. GPx activity in the experimentalsamples was measured following the method of Flohe and Gunzler(1984) using H2O2 and NADPH as substrates. The conversion ofNADPH to NADP+ was observed by recording the changes inabsorption intensity at 340 nm and one unit of enzyme activity isdefined as the amount of enzyme that catalyzes the oxidation of1 μmol NADPH per minute.

Assay of cellular metabolites

Cellular redox environment in spleen of normal and experimentalrats was assessed by measuring the glutathione (GSH) and glutathi-one disulfide (GSSG) levels.

Determination of GSH. GSH level was measured by the method ofEllman (1959). Spleen tissue homogenate (720 μL) was diluted to1440 μL by using Tris-EDTA buffer and 5% TCA was added to it toprecipitate the protein content of the homogenate. After centrifuga-tion (10,000g for 5 min) the supernatant was taken, DTNB solution(Ellman's reagent) was added to it and the absorbance was measuredat 417 nm. A standard graph was drawn using different con-centrations of GSH solution (1 mg/mL) and fixed concentration ofDTNB. With the help of this graph GSH content of tissue homogenateswere calculated. The method describes here basically determines thetotal non-protein sulfhydryls (NPSH) in the experimental samples asproteins are precipitated initially by TCA. However, GSH is the mostabundant NPSH in the tissue. Thus, the aforementioned method canbe regarded as the GSH estimation procedure.

Determination of GSSG. Glutathione disulfide (GSSG) was assessedby measuring its level using a kit from Calbiochem, USA following themethod of Finck et al. (2003). In this method, GSSGwas enzymaticallyreduced to GSH (in presence of NADPH and GR as given below) whichreacts with DTNB to produce the yellow coloured TNB.

GSSG + NADPH + Hþ→GR 2GSH + NADP

GSH + DTNB→TNB Yellow colourð ÞBriefly, spleen tissue samples were homogenized in ice cold m-phosphoric acid (100 g/L, Fluka). This treatment resulted in theprecipitation of the proteins. After allowing the mixture to stand for5 min at room temperature, homogenates were centrifuged (10,000g;10 min at 4 °C). Supernatants were frozen at −80 °C until furtheranalysis. Prior to the estimation of GSSG, neutralization was achievedby adding 5 μL of triethanolamine (4 mmol/L, Sigma Chemicals) to a100 μL supernatant. For GSSG assay, the thiol scavenging reagent1-methyl-2-vinylpyridinium trifluoromethanesulfonate (M2VP) wasused to mask GSH very rapidly. 10 μL of M2VP was added to100 μL of neutralized supernatant. Then 100 μL of the recycling

Table 1Effect of streptozotocin (STZ) and arjunolic acid (AA) on body weight (BW) and bloodglucose.

Parameters Normal Control AA treated STZ treated AA+STZ

Body weight(BW) (g)

135.84±6.82a 133.88±6.72a 88.24±4.45b 115.24±5.75c

Blood glucose(mg/dL)

65.12±3.31a 71.54±3.61a 275.98±8.85b 125.94±6.34c

Normal Control: normal animals; AA Treated: animals treated with AA; STZ Treated:animals treated with STZ and AA+STZ: animals treated with AA followed by STZexposure. Data are mean±SD, for 6 animals per group and were analyzed by one-wayANOVA, with Student–Newman–Keuls post hoc tests. Differences were attributed atpb0.05, and homogeneous subgroups share common superscripted letters.

Table 2Effect of streptozotocin (STZ) and arjunolic acid (AA) on the cytokine levels, like IL-2,IFN-γ and TNF-α.

Parameters Normal control AA treated STZ treated AA+STZ

IL-2 (ng/mL) 0.29±0.01a 0.30±0.02a 0.22±0.02a 0.27±0.02a

IFN-γ (ng/mL) 5.12±0.49a 4.98±0.36a 2.85±0.25b 4.65±0.44a

NF-α (ng/mL) 2.15±0.15a 2.28±0.12a 3.75±0.10b 2.55±0.16a

Normal Control: normal animals; AA Treated: animals treated with AA; STZ Treated:animals treated with STZ and AA+STZ: animals treated with AA followed by STZexposure. Data are mean±SD, for 6 animals per group and were analyzed by one-wayANOVA, with Student–Newman–Keuls post hoc tests. Differences were attributed atpb0.05, and homogeneous subgroups share common superscripted letters.

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reagent, which contained NADPH (0.30 mM), DTNB (0.225 mM),and GR (1.6 units/mL) in a 100mMphosphate/1mMEDTA buffer (pH7.4), was added. The change of absorbance was monitored at 412 nmfor 3 min with a spectrophotometer. Standards and test samples wererun in triplicate for each assay and the measurements were repeatedthree times. The reaction rate and calibration curves were used tocalculate concentrations of GSSG.

Determination of total thiol. Total thiol (total protein bound as wellas non-protein bound sulfhydryl groups) content was measuredaccording to the method of Sedlak and Lindsay (1958) with somemodifications. About 50 μL of the spleen tissue homogenatewasmixedwith 0.6mL of Tris-EDTA buffer and 40 μL of 10mMDTNB inmethanol.The final volume was made up to 1 mL by adding methanol. Thereaction mixture was incubated at room temperature for 20 min andthe absorbance was measured at 412 nm. The content of total thiolswas calculated usingmolar extinction coefficient of 13,600M−1 cm−1.

Immunoblotting

Samples containing 50 μg proteins were subjected to 10% SDS-PAGE and transferred to a nitrocellulose membrane. Membranes wereblocked at room temperature for 2 h in blocking buffer containing 5%non-fat dry milk to prevent non specific binding and then incubatedwith anti phosphorylated p65, anti unphosphorylated p65 (1:250dilution), anti phosphorylated IκBα (1:500), anti unphosphorylatedIκBα (1:1000 dilution), anti phosphorylated p38 (1:1000), antiunphosphorylated p38 (1:1000 dilution), anti phosphorylatedERK1/2 (1:1000), anti unphosphorylated ERK1/2 (1:1000 dilution),anti phosphorylated JNK (1:1000), anti unphosphorylated JNK(1:1000 dilution) and anti caspase 3 (1:1000 dilution) primaryantibodies at 4 °C overnight. The membranes were washed in TBST(50 mmol/L Tris-HCl, pH 7.6, 150 mmol/L NaCl, 0.1% Tween 20) for30 min and incubated with appropriate HRP conjugated secondaryantibody (1:2000 dilution ) for 2 h at room temperature anddeveloped by the HRP substrate 3,3′-diaminobenzidine tetrahydro-chloride (DAB) system (Bangalore, India).

Isolation of splenocytes

Splenocytes were isolated from the spleen tissue of normalanimals (rats) following the method of Wang et al. (2008). A portionof the spleen tissue was passed through a stainless steel mesh in RPMI1640 culture medium. The cell suspension was centrifuged at 1000gfor 5 min at 4 °C. The cell pellet was resuspended in 6 mL of Hank'ssolution and laid on to 6 mL of Histopaque-1083 (Sigma, St. Louis,MO). After centrifugation at 400 g for 30 min, the interface (contain-ing splenocytes) was transferred into a fresh tube and washed twicewith RPMI 1640 without serum. The cell pellet was suspended inRPMI 1640 medium supplemented with 2 mM glutamine, 50 μg/mLgentamycin and 10% heat-inactivated FBS, and total splenocytes werecounted.

Cell viability assessment

About 2×106 cells were incubated with STZ either alone or incombination with the AA in varying concentrations. After treatmentwith STZ orwith the AA and STZ combination, themedia was removedand the splenocytes were washed twice with phosphate bufferedsaline. Cell viability assessment has been measured by MTT reductionassay following the method of Edmondson et al. (1988).

Experimental setup with isolated splenocytes

All experiments were performed with different sets of splenocytescontaining 1 mL of suspension (∼2×106 cells) in each. After isolation,

cells were incubated in a CO2 incubator at 37 °C. The splenocytes keptin culture medium only was served as normal control. To investigatethe effect of AA alone, 1 mL of splenocytes suspension was incubatedwith AA (100 μg/mL) for 1 h. The toxin control was prepared byincubating the splenocytes with STZ (2 mM) for 1 hr. The effect of AAagainst STZ induced toxicity has been investigated by incubating thesplenocytes with AA (100 μg/mL) prior to STZ addition. All theincubationswere performed at 37 °Cwith gentle shaking. At the end ofincubation period all splenocyte suspensions were separated intothree different sets. Cell viability assaywasmeasured using the first setof splenocyte suspension. The second set was used for investigatingthe ROS production assay. The third set was used for the isolation ofmitochondria and measuring the mitochondrial membrane potential.

Determination of mitochondrial membrane potential

Mitochondria from splenocytes were isolated following themethod of Hodarnau et al. (1973). Mitochondrial membrane potentialwas estimated on the basis of cell retention of the fluorescent cationicprobe rhodamine 123 (Mingatto et al., 2002). Briefly, the mitochon-drial suspension was incubated with 1 μM rhodamine 123 for 10 min,centrifuged at 50g for 5min at 4 °C,washed and resuspended in 1mLof0.1% TritonX-100. After centrifugation at 2000g for 5min,fluorescenceof rhodamine 123 was determined using BD-LSR flow cytometer. Celldebris, characterized by a low FSC/SSC was excluded from analysis.The data was analyzed by Cell Quest software. In this assay carbonylcyanide m-chlorophenyl hydrazone (CCCP) was used as a positivecontrol to prove that membrane potential could be destroyed by drugexposure. CCCP directly interferes with mitochondrial function andinduces apoptosis. The results are expressed as percentage of thefluorescence values for control (untreated).

Assay of the concentration of cytosolic cytochrome c

The concentration of cytosolic cytochrome c was measuredfollowing the method as described by Adrain et al. (2001) with thecytochrome c enzyme immunometric assay kit (Minneapolis, USA).The kit uses a monoclonal antibody to cytochrome c immobilized on amicrotiter plate to bind the cytochrome c in the standards or sample

Table 3Effect of streptozotocin (STZ) and arjunolic acid (AA) on the levels of ROS as well as NO production (measured by nitrite level), lipid peroxidation (measured by MDA content) andprotein carbonylation.

Parameters Normal control AA treated STZ treated AA+STZ

Rate of intracellular ROS production (nmol of DCF/min/mg protein) 36.88±1.92a 41.79±2.11a 109.84±5.55b 75.98±3.82c

Nitrite level (mM) 62.37±3.15a 65.97±3.33a 125.49±6.25b 81.94±4.09c

MDA (nmol/mg protein) 5.24±0.26a 5.31±0.31a 15.47±0.88b 8.74±0.45c

Protein Carbonylation (nmol/mg protein) 3.75±0.22a 3.82±0.24a 10.97±0.83b 4.47±0.25a

Normal Control: normal animals; AA Treated: animals treated with AA; STZ Treated: animals treated with STZ and AA+STZ: animals treated with AA followed by STZ exposure. Dataare mean±SD, for 6 animals per group and were analyzed by one-way ANOVA, with Student–Newman–Keuls post hoc tests. Differences were attributed at pb0.05, andhomogeneous subgroups share common superscripted letters.

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which was detected by adding streptavidin conjugated to alkalinephosphatase. The optical density of each well was measured at405 nm using a microplate reader. The concentration of cytochrome cwas calibrated from a standard curve based on reference standards.

Flow cytometry analysis

The flow cytometric analysis was done on BD-LSR flow cytometer.Cell debris, characterized by a low FSC/SSC was excluded fromanalysis. The data was analyzed by Cell Quest software.

Assessment of apoptotic and necrotic cells

The apoptotic and necrotic cell distribution was analyzed byAnnexin V binding and PI uptake. Positioning of quadrants on AnnexinV/PI dot plots was performed and living cells (Annexin V−/PI−), earlyapoptotic/primary apoptotic cells (Annexin V+/PI−), late apoptotic/secondary apoptotic cells (Annexin V+/PI+) and necrotic cells(Annexin V−/PI+) were distinguished (Vermes et al., 1995).Therefore, the total apoptotic proportion included the percentage ofcells with fluorescence Annexin V+/PI− and Annexin V+/PI+. Thecells were suspended in 1mL binding buffer (1×). An aliquot of 100 μLwas incubated with 5 μL Annexin V-FITC and 10 μL PI for 15 min indark at room temperature and 400 μL binding buffer (1×) was addedto each sample. The FITC and PI fluorescence were measured throughFL-1 filter (530 nm) and FL-2 filter (585 nm), respectively, and 10,000events were acquired.

Histological studies

Spleens from the normal and experimental animals were fixed in10% buffered formalin and were processed for paraffin sectioning.Sections of about 5 μm thickness were stained with haematoxylinand eosin to evaluate under light microscope. For semi-quantitativeanalysis, a scheme described in a previous report (Xiao et al., 2001)was used. In analyzing histopathology of the spleen, a subjectiveimpression of none, minimal, mild, moderate, and severe, wasgraded as 0, 1, 2, 3, and 4, respectively. The parameters assessedwere white pulp depletion and red pulp activation. The samples

Table 4Effect of streptozotocin (STZ) and arjunolic acid (AA) on the activities of the antioxidant en

Name of the antioxidant enzymes Normal control

Cu/Zn-SOD (Unit/mg protein) 38.21±1.97a

Mn-SOD (Unit/mg protein) 40.14±2.02a

CAT (μmol/min/mg protein) 78.14±4.22a

GST (μmol/min/mg protein) 0.55±0.035a

GR (nmol/min/mg protein) 35.87±1.82a

GPx. (nmol/min/mg protein) 72.34±3.65a

Normal Control: normal animals; AA Treated: animals treated with AA; STZ Treated: animalsare mean±SD, for 6 animals per group and were analyzed by one-way ANOVA, withhomogeneous subgroups share common superscripted letters.

were scored with no knowledge of treatment group by the personscoring the slides.

Statistical analysis

All the values are expressed as mean±S.D. (n=6). Significantdifferences between the groups were determined with SPSS 10.0software (SPSS Inc., Chicago, IL, USA) for Windows using one-wayanalysis of variance (ANOVA) and the groupmeanswere compared byStudent–Newman–Keuls post hoc tests. A difference was consideredsignificant at the pb0.05 level.

Results

Chemical structure of arjunalic acid (AA) has been presented inFig. 1A. It possesses three hydroxyl (one primary and two secondary)and one carboxylic acid groups. In addition, it has one double bond.

Fig. 1B represents the mass spectra of AA. For MS analysis,0.005 mg compound was dissolved in 1 mL methanol (concentration5 ng/μL) and this solution was analyzed using a Mass Spectrometer(WATERS, Model No. Qtof Micro YA263). Electron Spray Ionization(ESI) mode was used for generating ions needed for the analysis.

Result of 1H NMR spectra of AA has been represented in Fig. 1C. 1HNMR (500 MHz, DMSO-d6) δ (ppm) 11.98 (s, 1H, -COOH), 5.15 (s,1H), 4.38 (app. s,1H), 4.20 (s, 1H), 4.13 (s, 1 H), 3.46 (m,1H), 3.32 (m,1H), 3.16 (d, J=9.1 Hz, 1H), 3.02 (d, J=9.7 Hz, 1H), 2.72 (dd,J1=13.6 Hz, J2=3.6 Hz, 1H), 2.0–0.80 (terpenpoid H's, 20H), 1.13 (s,3H), 0.89 (s, 3H), 0.86 (s, 6H), 0.70 (s, 3H), 0.52 (s, 3H).

Fig. 1D shows the 13C NMR spectra of AA. 13C-NMR (125 MHz,DMSO-d6) δ (ppm) 179.4, 144.8, 122.3, 76.4, 68.3, 64.8, 47.9, 47.5,46.9, 46.5, 46.3, 43.4, 42.2, 41.7, 39.7, 38.2, 33.7, 32.9, 32.7, 31.3, 28.0,26.5, 24.2, 23.9, 23.4, 21.9, 18.3, 17.8, 17.7, 14.5.

Fig. 1E represents the reverse phase HPLC chromatograph of AA. Inthis figure a single peak of arjunolic acid appeared with the retentiontime 4.792 min confirming its homogeneous preparation and absenceof any contaminating substances.

From optical rotation study [α]D29.5 of AA was found to be +60.53.This typical value as well as sign (dextrorotatary) is specific for thiscompound.

zymes in spleen tissue of the normal and experimental animals.

AA treated STZ treated AA+STZ

35.15±1.79a 14.14±0.77b 29.75±1.52a

39.55±1.99a 20.47±1.05b 37.32±1.89a

75.91±3.82a 45.39±3.31b 68.57±3.51a

0.52±0.031a 0.22±0.015b 0.45±0.025a

32.91±1.72a 15.87±0.88b 29.87±1.51a

69.57±3.49a 29.87±1.53b 55.47±2.81a

treated with STZ and AA+STZ: animals treated with AA followed by STZ exposure. DataStudent–Newman–Keuls post hoc tests. Differences were attributed at pb0.05, and

Table 5Status of the thiol based antioxidant in the spleen tissue of the streptozotocin (STZ) and arjunolic acid (AA) treated animals.

Parameters Normal control AA treated STZ treated AA+STZ

GSH (nmol/mg protein) 19.81±1.02a 17.33±0.92a 9.47±0.55b 15.98±0.83a

GSSG (nmol/mg protein) 0.35±0.021a 0.31±0.018a 0.91±0.044b 0.41±0.022c

Total thiols (nmol/mg protein) 161.37±8.33a 155.15±7.81a 85.78±4.32b 139.61±7.01a

Normal Control: normal animals; AA Treated: animals treated with AA; STZ Treated: animals treated with STZ and AA+STZ: animals treated with AA followed by STZ exposure. Dataare mean±SD, for 6 animals per group and were analyzed by one-way ANOVA, with Student–Newman–Keuls post hoc tests. Differences were attributed at pb0.05, andhomogeneous subgroups share common superscripted letters.

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Effects on body weight and blood glucose level

Reduction in body weight along with an increase in blood glucoselevel is a marker for the development of diabetes. This pathophysio-logy has been observed in STZ treated animals (Table 1) suggestingtheir diabetic nature. AA supplements, however, significantly loweredthe blood glucose level and prevented the loss in body weightcompared to the diabetic control group.

Fig. 2. Western blot analysis for the expression of p65 and IκBα (both phosphorylated aphosphorylated p65, panel B: unphosphorylated p65; panel C: phosphorylated IκBα andimmunoreactive concentrations have been plotted below the picture of western blotting. CAA+STZ: animals treated with AA followed by STZ exposure. The relative intensities of barbitrary value of 1. Data are mean±SD, for 6 animals per group and were analyzed by oneat pb0.05, and homogeneous subgroups share common superscripted letters.

Effect on the levels of cytokines in serum

Cytokines are thought to be involved in the type1 diabetes. Table 2represents the levels of the cytokines measured from the serumsample of the experimental animals. STZ exposure decreased thelevels of IL-2 as well as IFN-γ and increased the level of TNF-α in thesera of the diabetic animals. Treatment with AA could, however,prevent the STZ induced alteration in the levels of the cytokines.

nd unphosphorylated) in the spleen tissue of STZ and AA treated animals. Panel A:panel D: unphosphorylated IκBα In each figures the relative band intensities i.e. theont: normal animals; AA: animals treated with AA; STZ: animals treated with STZ andands were determined using NIH-image software and the control band was given an-way ANOVA, with Student–Newman–Keuls post hoc tests. Differences were attributed

Fig. 3.Western blot analysis for the expression of different mitogen activated protein kinases (MAPKs) (both phosphorylated and unphosphorylated) in the spleen tissue of STZ andAA treated animals. Panel A: phosphorylated p38, panel B: unphosphorylated p38, panel C: phosphorylated ERK 1/2, panel D: unphosphorylated ERK 1/2, panel E: phosphorylatedJNK and panel F: unphosphorylated JNK. In each figures the relative band intensities i.e. the immunoreactive concentrations have been plotted below the picture of western blotting.Cont: normal animals; AA: animals treated with AA; STZ: animals treated with STZ and AA+STZ: animals treated with AA followed by STZ exposure. The relative intensities of bandswere determined using NIH-image software and the control band was given an arbitrary value of 1. Data are mean±SD, for 6 animals per group and were analyzed by one-wayANOVA, with Student–Newman–Keuls post hoc tests. Differences were attributed at pb0.05, and homogeneous subgroups share common superscripted letters.

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Fig. 4. (A) Dose dependent effect of STZ on the reduction in cell viability of primarysplenocytes. Cont: normal splenocytes; “STZ-0.,5” “STZ-1.0,” “STZ-1.5,” “STZ-2.0,” “STZ-2.5” and “STZ-3.0”: splenocytes treated with STZ at a dose of 0.5, 1, 1.5, 2, 2.5 and 3 mMfor 1 h respectively. Data are mean±SD, for 6 animals per group and were analyzed byone-way ANOVA, with Student–Newman–Keuls post hoc tests. Differences wereattributed at pb0.05, and homogeneous subgroups share common superscriptedletters. (B) Dose dependent effect of the AA on STZ induced loss of cell viability for 1 h.Cont: normal splenocytes; STZ: splenocytes treated with STZ at a dose of 2 mM for 1 h;AA-20+STZ, AA-40+STZ, AA-60+STZ, AA-80+STZ, AA-100+STZ, AA-120+STZ:splenocytes treated with AA at a dose of 20, 40, 60, 80, 100 and 120 μg/mL respectivelyfor 1 h followed by STZ exposure (2 mM, 1 h) individually. Data are mean±SD, for 6animals per group and were analyzed by one-way ANOVA, with Student–Newman–Keuls post hoc tests. Differences were attributed at pb0.05, and homogeneoussubgroups share common superscripted letters.

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Effects on intracellular ROS and NO production

Several investigations support the fact that increased productionof ROS as well as RNS induces organ pathology under STZ inducedhyperglycemic condition. STZ exposure caused alteration in the levelsof intracellular ROS as well as NO production (measured by nitritelevel). Under hyperglycemic condition, STZ intoxication increased theproduction of ROS as well as nitrite level (Table 3). AA administrationat a dose of 20 mg/kg body weight for 4 days, however, could preventthe STZ induced increased production of ROS as well as nitrite level.

Estimation of lipid peroxidation and protein carbonyl content

Lipid peroxidation and protein carbonyl content arewidely used asthe marker of cell membrane damage and oxidative modification ofproteins. In the present study the lipid peroxidation has beenmeasured by estimating the concentration of MDA (lipid peroxidationend product). STZ intoxication increased the levels of MDA andprotein carbonylation in the spleen tissue of the experimental animals(Table 3). Treatment with AA prior to the STZ exposure has beenfound to be effective in preventing the STZ induced splenotoxicityunder hyperglycemic condition.

Activities of antioxidant enzymes

To investigate whether the radical scavenging activity of AA wasmediated by the activities of antioxidant enzymes, we have measuredthe activities of intracellular antioxidant enzymes, like, SOD (Mn-SODand Cu/Zn-SOD), CAT, GST, GR and GPx. Mn-SOD activity has beenmeasured in the mitochondrial fraction and Cu/Zn-SOD activity hasbeen measured in the cytosolic fraction of the spleen tissue homo-genates. Table 4 represents the activities of the antioxidant enzymes inspleen tissue of the experimental animals. Our study reveals that STZexposure significantly attenuated the activities of the antioxidantenzymes. Results suggest that AA treatment could be able to preventthe STZ induced decreased activities of the antioxidant enzymes.

Levels of the cellular metabolites

Table 5 depicts the effects of AA and STZ on the levels of cellularmetabolites. STZ exposure decreased the levels of total thiols as wellas GSH and increased the level of GSSG. Streptozotocin is a NO donor.During metabolism, it releases NO which may cause S-nitrosation ofprotein bound thiol groups. In addition the cystein residue of proteinacts as a nucleophile and they are very susceptible towardsglycosylation. Thus under STZ induced diabetic milieu, S-nitrosationas well as S-glycosylation cause substantial reduction of the level oftotal thiol. AA pretreatment, however, could prevent the alteration inintracellular thiol status via its radical scavenging activities.

Effect on the expression of p65 and IκBα

p65 is the vital component of the activated NF-κB whichtranslocates to the nucleus. Transcriptional activity of p65 could beregulated by IκB. IκB causes translocation of p65 into the nucleuswhere it binds with the DNA and induces gene expression. Westernimmunoblotting was used to determine whether p65 and IκBα areinvolved in the STZ induced splenotoxicity and its prevention by AA.We observed that STZ exposure elevated the immunoreactiveconcentration of phosphorylated p65 and phosphorylated IκBα(Fig. 2). Treatment with AA prior to the STZ exposure prevented theSTZ induced alteration in the immunoreactive concentration of p65and IκBα. No significant difference has been observed in theexpression of phosphorylated p65 and phosphorylated IκBα betweennormal and AA treated groups. STZ exposure also lowered theimmmunoreactive concentration of unphosphorylated IκBα. However,

there is no significant difference from control levels in the other twogroups (AA and AA+STZ). No significant differences have also beenobserved between the immunoreactive concentrations of the unpho-sphorylated p65 of different groups.

Effect on the expressions of MAPKs

MAPKs are the upstream critical signaling proteins. To asses theeffect of STZ and AA exposure on the expressions ofMAPK subfamilies,

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the spleen tissue homogenates were analyzed for both unpho-sphorylated and phosphorylated forms of ERK (1/2), JNK and p38by immunoblotting. Fig. 3 revealed that STZ exposure increased theimmunoreactive concentrations of all three subfamilies of MAPKs intheir phosphorylated levels leaving unchanged in the immuno-reactive concentrations of unphosphorylated ERK, JNK and p38.Treatment with AA prior to STZ exposure prevented the STZ inducedincreased immunoreactive concentrations of phosphorylated ERK,JNK and p38.

Effect of STZ and AA on cell viability (splenocytes)

STZ caused a progressive loss in cell viability in splenic cells(Fig. 4). In order to determine whether this loss could be prevented bythe AA treatment, we performed MTT assay. Fig. 4A shows that thereduction in cell viability increasedwith increasing STZ concentration.At a concentration of nearly 2mM, optimum reduction in viability wasobserved. Thereafter, the loss in cell viability remains constant evenwith increasing STZ concentration. Hence, this particular concentra-tion of STZ has been used throughout the study. Fig. 4B shows that STZexposure reduced the cell viability of splenocytes to 45%. Treatmentwith AA at a dose of 20 μg/mL and 40 μg/mL could not prevent thereduction in cell viability against STZ exposure. However treatmentwith AA at a dose of 60 μg/mL to120 μg/mL gradually increased the

Fig. 5. The intracellular ROS production was detected by DCF-DAmethod. Panel A: Representafluorescence measured using a flow cytometer with FL-1 filter. Panel C: show average fluoressplenocytes treated with AA; STZ: splenocytes treated with STZ and AA+STZ: splenocytes tare mean±SD, for 6 animals per group and were analyzed by one-way ANOVA, withhomogeneous subgroups share common superscripted letters.

cell viability against STZ exposure. Optimum increase in viabilityoccurred with 100 μg/mL of AA, and this concentration has been usedin all the subsequent studies.

Effect on the ROS production in splenocytes

ROS plays amajor role in the STZ induced organ pathology. In orderto asses the changes in the splenocyte morphology under oxidativestress, STZ induced increased production of ROS in the splenocytes hasalso been included in our study. Fig. 5 shows the effect of STZ inducedROS production in isolated splenocytes and its reversal by AAtreatment. Here, we observed (Fig. 5) that STZ exposure increasedthe level of ROS production, (as revealed by the increased no of greenfluorocent cells) with simultaneous alteration in the splenocytemorphology. FACS analysis also supported this result. Treatmentwith AA prior to the STZ addition prevented the STZ induced ROSproduction in splenocytes. However, no significant difference has beenobserved between the normal cell and AA treated cell.

Effect of AA against STZ induced mitochondrion-dependent celldeath pathway

Disruption of mitochondrial membrane potential and increasingconcentration of cytosolic cytochrome c are the novel biomarker of

tive images under fluorescencemicroscope (original magnification ×400), Panel B: DCFcence intensity as calculated using Metamorph software. Cont: normal splenocytes; AA:reated with AA prior to STZ exposure. The measurements were made in six times. DataStudent–Newman–Keuls post hoc tests. Differences were attributed at pb0.05, and

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oxidative stress induced cell damage and subsequent activation ofcaspase 3 represent a key step in the mitochondrion dependentapoptotic cell death pathway. To determine whether AA exerts itsanti-apoptotic action against STZ-induced apoptotic death via thismechanism, we measured (Fig. 6) the mitochondrial transmembranepotential, concentration of cytosolic cytochrome c and immunoreac-tive concentration of caspase 3 in the spleen tissue of experimentalanimals. STZ administration significantly attenuated the mitochon-drial transmembrane potential, elevated the concentration of cyto-solic cytochrome c and simultaneously increased the immunoreactiveconcentration of caspase 3. Treatment with AA prior to the STZ admin-istration could, however, significantly inhibit STZ induced alterationsof these effects.

Effect of STZ and AA on cell death pathway

To understand the nature of cell death due to STZ toxicity, doublelabeling techniques has been utilized using Annexin V /PI todistinguish between apoptotic and necrotic cells. Flowcytometricdata (Fig. 7) revealed that, in comparison with control untreated

Fig. 6. (A) Study on the mitochondrial membrane potential in the splenocytes by flow cytomplot. Results expressed as dot plot representing as one of the six independent experiments.and D: splenocytes treated with AA prior to STZ exposure. The measurements were made in ssplenocytes; AA: splenocytes treated with AA; STZ: splenocytes treated with STZ and AA+STper group and were analyzed by one-way ANOVA, with Student–Newman–Keuls post hoc tesuperscripted letters. (C) Western blot analysis for the expression of caspase 3. The relativepicture of western blotting. Cont: normal splenocytes; AA: splenocytes treated with AA; STZexposure. Data are mean±SD, for 6 animals per group and were analyzed by one-way ANOand homogeneous subgroups share common superscripted letters.

splenocytes, STZ intoxicated splenocytes showed maximum AnnexinV staining but very little PI binding, indicating majority of cells deathvia apoptotic pathway. Treatment with AA prior to the STZ additionshows less number of apoptotic cells, indicating that AA treatmentprotected the apoptotic cell death under hyperglycemic condition.

Histological assessment

Spleen is the largest lymphoid organ in the body. It acts as a part ofthe immune system and as a filter. In spleen, there are two distinctcomponents, the white pulp and the red pulp. The white pulp consistsof aggregates of lymphoid tissue (T cells, B cells and accessory cells).Antibodies are made inside the white pulp and the purpose of thewhite pulp is to mount an immunological response to antigens withinthe blood. The red pulp consists of large numbers of sinuses andsinusoid filled with blood and is responsible for the filtration functionof the spleen. Histological studies showed that (Fig. 8) diabeticinduction caused white pulp depletion and the red pulp activation. Inagreement with the alteration in the cytokines levels, this histologicalobservation suggests that diabetic milieu weakened the immuno-

etry analysis. Right figure represents histogram plot and the left one represents the dotA: normal splenocytes; B: splenocytes treated with AA; C: splenocytes treated with STZix times. (B) Study on the release of cytochrome c from the mitochondria. Cont: normalZ: splenocytes treated with AA prior to STZ exposure. Data are mean±SD, for 6 animalssts. Differences were attributed at pb0.05, and homogeneous subgroups share commonband intensities i.e. the immunoreactive concentrations have been plotted below the: splenocytes treated with STZ and AA+STZ: splenocytes treated with AA prior to STZVA, with Student–Newman–Keuls post hoc tests. Differences were attributed at pb0.05,

Fig. 7. Effect of STZ and AA on percent distribution of apoptotic and necrotic cells. Celldistribution analyzed using Annexin V binding and PI uptake. The FITC and PIfluorescence measured using flow cytometer with FL-1 and FL-2 filters, respectively.Results expressed as dot plot representing as one of the six independent experiments.Cont: normal splenocytes; AA: splenocytes treated with AA; STZ: splenocytes treatedwith STZ and STZ+AA: splenocytes treated with AA prior to STZ exposure.

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logical response of our body. Treatment with AA prior to STZadministration reduced the STZ-induced pathological lesion andthat is in agreement with the results of the other parameters.

Discussion

Present study demonstrates the prophylactic role of AA againstSTZ-induced spleno-toxicity developed under hyperglycemia. STZexposure damages β cells and reduces insulin secretion. Glycogenesisis thus inhibited and blood glucose level goes up. AA treatment priorto STZ administration enhances glycogenesis and prevents glucoseaccumulation in blood. The weight loss detected in the STZ treatedanimals is most likely due to the induction of insulitis and/or the lackof insulin (Kim et al., 2009). No weight loss was registered in theanimals treated with AA prior to STZ, probably those animals were notas hyperglycemic as that of STZ treated group.

Increasing evidence suggests that proinflammatory cytokines,released from spleen, are critically involved in the pathogenesis oftype 1 diabetes (Rabinovitch, 1998). Mandrup-Poulsen et al. (1985)reported that IL-2, IFN-γ and TNF-α synergistically impaired β-cellfunction and cause β-cell death. STZ exposure leads to the activationof TFs causing phosphorylation of upstream critical signaling proteins[IKKα/β and MAPKs (ERK1/2, JNK1/2 and p38)] and results in theupregulation of pro-inflammatory and pro-fibrogenic cytokines in thespleen (Lau et al., 2006). IL-2, a potent immunomodulatory cytokine,is effective in conferring protection against viral, bacterial andintracellular parasitic infections (Doolan and Hoffman, 1999). Inaddition, IL-2 also affects humoral immunity by inducing isotypeswitching through both IFN-γ dependent and independent mechan-isms (Trinchieri, 1995; Crutcher et al., 1995). IFN-γ plays a protectiverole against microbial infection (Young and Hardy, 1995) althoughseveral studies have subsequently established that the primary role ofIFN-γ is associated with autoimmunity (Gottenberg and Chiocchia,2007). Jang et al. (2009) reported that cytokines IFN-γ and TNF-α areprimarily involved in the immunologic destruction of pancreatic β-cells in STZ induced type 1 diabetes mellitus. In agreement with theearlier findings, we also observed the reduced levels of IL-2 and IFN-γas well as increased level of TNF-α in the serum of the STZ treatedanimals. This effect might be due to the STZ induced splenotoxicity viasuppressing the T cell proliferative response. Treatment with AAenhanced the levels of IL-2 as well as IFN-γ and decreased the level ofTNF-α compared to the diabetic control group.

During intracellular metabolization, STZ itself liberates nitricoxide, NO (Turk et al., 1993; Kroncke et al., 1995) and this processinduces the increased ROS production as well as increased expressionof proinflammatory cytokines (Tabatabaie et al., 2003). In our study,we also observed that STZ administration increased the serum nitritelevel as well as the production of intracellular ROS in spleen tissue. AAtreatment, however, could prevent the alterations of STZ inducedintracellular RNS and ROS formation in spleen tissue. STZ inducedoxidative as well as nitrosative stress altered both enzymatic as wellas non-enzymatic antioxidant defense system. In this context, weassayed the activities of the antioxidant enzymes SOD, CAT, GST, GR,GPx and G6PD and the levels of the non-enzymatic antioxidantmolecules GSH and its metabolite GSSG. We observed that STZintoxication decreased the activities of the antioxidant enzymes, theGSH/GSSG ratio and increased lipid peroxidation as well as proteincarbonylation. Treatment with AA, prior to STZ intoxication, aug-mented the intracellular antioxidant defense.

Oxidative stress disturbs cellular redox status, causing oxidativedamage to cellular molecules and altering gene expression possiblythrough post-translational modification of redox sensitive TFs (Yanand Hales, 2005). Phosphorylation at multiple serine sites of p65increases the transcriptional activity of p65 in the nucleus (Vermeulenet al., 2003). Activation of phosphorylated p65 is connected tocarcinogenesis via regulation of genes involved in cell transformation,proliferation and angiogenesis (Baldwin, 1996). In our study, weobserved that STZ exposure increased the immunoreactive concen-tration of phosphorylated p65 leaving unchanged the immuno-reactive concentration of unphosphorylated p65 compared tonormal control. Thus STZ induced early activation of phosphorylatedp65 could regulate a number of genes leading to inflammatory,fibrogenic and carcinogenic responses in the spleen. Transcriptionalactivity of p65 could be regulated by the phosphorylation of inhibitor-κB (IκB) (Sakurai et al., 2003). Removal of IκB induces translocation ofp65 into the nucleus where it binds to the DNA and induces geneexpression (Akira and Kishimoto, 1997). In the present study, STZinduced activation of phosphorylated p65 is linked with the increasedimmunoreactive concentration phosphorylated IκBα as well asdecreased concentration of unphosphorylated IκBα. AA treatment,however, could prevented the STZ induced alteration in the expressionsof phosphorylated p65, phosphorylated IκBα as well as its unpho-sphorylated form.

AP-1, a redox sensitive early response TF, plays a pivotal role in theregulation of a variety of down stream target genes includinginflammatory, fibrogenic and cell proliferation genes (Filosto et al.,2003). Activation of AP-1 is regulated by the activation of MAPKs(involving three major pathways: ERKs, JNKs and p38). Earlierinvestigation reported that all three MAPK signaling pathways havebeen implicated in the activation of. phosphorylated p65 through thephosphorylation of its inhibitor, IκBα (Castrillo et al., 2001; Kapahiet al., 2000). Kefaloyianni et al. (2006) reported that MAPKs could alsophosphorylate p65. STZ exposure, in our study, led to increase in theimmunoreactive concentrations of phosphorylated ERK, phosphory-lated JNK as well as phosphorylated p38 and that could be reduced bythe AA treatment. However, treatment of the animals with AA alonedid not exert any effect on the expression of phosphorylated MAPKs.

Apoptosis is known to be a delicately controlled programmed celldeath pathway (Singh and Anand, 1994). Several lines of earlierinvestigations suggest that the change in mitochondrial transmem-brane potential is able to switch the committed cells to apoptoticdeath with oxidative stress as the mediator (Keeble and Gilmore,2007). To investigate the mitochondria dependent cell death path-ways in hyperglycemia, we used spleen cells in vitro because of thedifficulty of isolating the immune cells from spleen tissue of the STZtreated animals (the islet mass is reduced and the islet structure isdisrupted). We found that STZ administration increased the intra-cellular ROS production along with decreased the mitochondrial

Fig. 8. (A) Haematoxylin and eosin stained spleen section of (Cont) normal rats spleen (×100); (AA) spleen from animals treated with AA; (STZ) STZ treated group, showing whitepulp (marked with solid arrow) diminished in size and the red pulp (marked with dotted arrow) tended to enlarge (×100); (AA+STZ) AA pretreated spleen section before STZadministration (×100) showing almost normal morphology. (B) Histological scoring system used to quantitate spleen pathology. Cont: spleen section from normal animals; AA:spleen section from AA alone treated animals; STZ: spleen section from diabetic animals and AA+STZ: spleen section from the animals treated with AA prior to diabetic exposure.Data are mean±SD (n=6) and were analyzed by one-way ANOVA, with Student–Newman–Keuls post hoc tests. Differences were attributed at pb0.05, and homogeneoussubgroups share common superscripted letters.

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transmembrane potential in splenocytes. This event caused increasedconcentration of cytosolic cytochrome c, activation of caspase 3(immunoreactive concentration of caspase 3 has been measured)followed by the cell death mostly via the apoptotic pathway. AAtreatment prior to the STZ addition could prevent the STZ inducedalterations in the mitochondria dependent apoptotic cell death.

Pathological changes by STZ administration and its reversal by AAtreatment have been observed in the spleen tissue of the experimen-tal animals. White pulp and red pulp are the two important

parenchymal arrangement of the spleen. Among them, white pulp isresponsible for the immunological function of the spleen. Earlierreport (Konrad et al., 1986) showed that diabetic mellitus causeddepression of cellular immunoresponsiveness. In our study we foundthat diabetic milieu as a result of STZ administration depleted thesplenic white pulp scoring and thus suppressed the cellular immune-response. Treatment with AA alone showed normal histology and AAtreatment prior to the STZ administration prevented the severity ofthe lesions.

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In conclusion, results of the present study demonstrate that STZinduced oxidative and nitrosative stress, stimulated the phosphory-lation of p65 as well as the upstream signaling molecules; IκBα andMAPKs. Upregulation of fibrogenic cytokines in spleen tissue couldcontribute to initiating or developing a fibrogenic response in the STZtreated spleen. These early signaling events reduced mitochondrialtransmembrane potential and initiates mitochondria-dependentapoptotic cell death pathways. Results also suggest that AA ispotentially useful for preventing STZ induced splenotoxicity andcell death.

Conflict of interest statementThe authors have declared that no conflict of interest exists.

Acknowledgments

The authors are grateful to Mr. Prasanta Pal for excellent technicalassistance for the study.

References

Adrain, C., Creagh, E.M., Martin, S.J., 2001. Apoptosis-associated release of Smac/DIABLO from mitochondria requires active caspases and is blocked by Bcl-2. EMBOJ. 20, 6627–6636.

Akira, S., Kishimoto, T., 1997. NF-IL6 and NF-kB in cytokine gene regulation. Adv.Immunol. 65, 1–46.

Baldwin, A.S., 1996. The NF-kappa B and I kappa B proteins: new discoveries andinsights. Annu. Rev. Immunol. 14, 649–683.

Baynes, J.W., 1991. Role of oxidative stress in development of complications in diabetes.Diabetes 40, 405–412.

Beda, N., Nedospasov, A., 2005. A spectrophotometric assay for nitrate in an excess ofnitrite. Nitric Oxide 13, 93–97.

Bhakuni, R.S., Shukla, Y.N., Tripathi, A.K., Prajapati, V., Kumar, S., 2002. Insect growthinhibitor activity of arjunolic acid isolated from Cornus capitata. Phytother. Res. 16,68–70.

Bonaventura, J., Schroeder, W.A., Fang, S., 1972. Human erythrocyte catalase: animproved method of isolation and a revaluation of reported properties. Arch.Biochem. Biophys. 150, 606–617.

Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgramquantities of protein utilizing the principle of protein-dye binding. Anal. Biochem.72, 248–254.

Braun, B., Zimmermann, M.B., Kretchmer, N., 1995. Effects of exercise intensity oninsulin sensitivity in womenwith non-insulin-dependent diabetes mellitus. J. Appl.Physiol. 78, 300–306.

Castrillo, A., de Las Heras, B., Hortelano, S., Rodriguez, B., Villar, A., Bosca, L., 2001.Inhibition of the nuclear factor kappa B (NF-kappa B) pathway by tetracyclickaurene diterpenes inmacrophages. Specific effects on NF-kappa B-inducing kinaseactivity and on the coordinate activation of ERK and p38 MAPK. J. Biol. Chem. 276,15854–15860.

Chang, F.Y., Shaio, M.F., 1999. Decreased cell-mediated immunity in patients withnoninsulin dependent diabetes mellitus. Diabetes Res. Clin. Pract. 528, 137–146.

Crutcher, J.M., Stevenson, M.M., Sedegah, M., Hoffman, S.L., 1995. Interleukin-12 andmalaria. Res. Immunol. 146, 552–559.

Dezfulian, C., Raat, N., Shiva, S., Gladwin, M.T., 2007. Role of the anion nitrite inischemia–reperfusion cytoprotection and therapeutics. Cardiovas. Res. 75,327–338.

Dias, A.S., Porawski, M., Alonso, M., Marroni, N., Collado, P.S., Gonzalez-Gallego, J., 2005.Quercetin decreases oxidative stress, NF-κB activation, and iNOS: overexpression inliver of streptozotocin-induced diabetic rats. J. Nutr. 135, 2299–2304.

Djoukeng, J.D., Abou-Mansour, E., Tabacchi, R., Tapondjou, A.L., Bouda, H., Lontsi, D., 2005.Antibacterial triterpenes from Syzygium guineense (Myrtaceae). J. Ethnopharmacol.101, 283–286.

Doolan, D.S., Hoffman, S.L., 1999. IL-12 and NK cells are required for antigen-specificadaptive immunity against malaria initiated by CD8+ T cells in the Plasmodiumyoelii model. J. Immunol. 163, 844–892.

Edmondson, J.M., Armstrong, S.L., Martinez, A.O., 1988. A rapid and simple MTT basedspectrophotometric assay for determining drug sensitivity in monolayer cultures.Methods Cell Sci. 11, 15–17.

Ellman, G.L., 1959. Tissue sulfhydryl group. Arch. Biochem. Biophys. 82, 70–77.Elsner, M., Guldbakke, B., Tiedge, M., Mundy, R., Lenzen, S., 2000. Relative importance of

transport and alkylation for pancreatic beta-cell toxicity of streptozotocin.Diabetologia 43, 1528–1533.

Esterbauer, H., Cheeseman, K.H., 1990. Determination of aldehydic lipid peroxidationproducts: malonaldehyde and 4-hydroxynonenal. Methods Enzymol. 186,407–421.

Filosto, M., Tonin, P., Vattemi, G., Savio, C., Rizzuto, N., Tomelleri, G., 2003. Transcriptionfactors c-Jun/activator protein-1 and nuclear factor-kappa B in oxidative stressresponse in mitochondrial diseases. Neuropathol. Appl. Neurobiol. 29, 52–59.

Finck,B.N.,Han,X., Courtois,M.,Aimond, F., Nerbonne, J.M., Kovacs, A.,Gross, R.W., Kelly,D.P.,2003. A critical role for PPARalpha-mediated lipotoxicity in the pathogenesis of dia-

betic cardiomyopathy:modulationbydietary fat content. Proc.Natl. Acad Sci. U.S.A. 100,1226–1231.

Flohe, L., Gunzler, W.A., 1984. Assay of glutathione peroxidase. Methods Enzymol. 105,114–121.

Geerlings, S.E., Hoepelman, A.I., 1999. Immune dysfunction in patients with diabetesmellitus (DM). FEMS Immunol. Med. Microbiol. 26, 259–265.

Gottenberg, J.E., Chiocchia, G., 2007. Dendritic cells and interferon-mediated autoim-munity. Biochimie 89, 856–871.

Habig, W.H., Pabst, M.J., Jakoby, W.B., 1974. Glutathione-S-transferases. The firstenzymatic step in mercapturic acid formation. J. Biol. Chem. 249, 7130–7139.

Herold, K.C., Vezys, V., Sun, Q., Viktora, D., Seung, E., Reiner, S., Brown, D.R., 1996.Regulation of cytokine production during development of autoimmune diabetesinduced with multiple low doses of streptozotocin. J. Immunol. 156, 3521–3527.

Hodarnau, A., Dancea, S., Barzu, O., 1973. Isolation of highly purified mitochondria fromrat pancreas. J. Cell. Biol. 59, 222–227.

Hsu, T.C., Young, M.R., Cmarik, J., Colburn, N.H., 2000. Activator protein 1 (AP-1)- andnuclear factor kappaB (NF-kappaB)-dependent transcriptional events in carcino-genesis. Free Rad. Biol. Med. 28, 1338–1348.

Jang, S.M., Yee, S.T., Choi, J., Choi, M.S., Do, G.M., Jeon, S.M., Yeo, J., Kim, M.J., Seo, K.I., Lee,M.K., 2009. Ursolic acid enhances the cellular immune system and pancreatic â-cellfunction in streptozotocin-induced diabetic mice fed a high-fat diet. Int.Immunopharmacol. 9, 113–119.

Kakkar, P., Das, B., Viswanathan, P.N., 1984. A modified spectrophotometric assay ofsuperoxide dismutase. Ind. J. Biochem. Biophys. 21, 130–132.

Kapahi, P., Takahashi, T., Natoli, G., Adams, S.R., Chen, Y., Tsien, R.Y., Karin, M., 2000.Inhibition of NF-kappa B activation by arsenite through reaction with a criticalcysteine in the activation loop of Ikappa B kinase. J. Biol. Chem. 275, 36062–36066.

Keeble, J.A., Gilmore, A.P., 2007. Apoptosis commitment—translating survival signalsinto decisions on mitochondria. Cell Res. 17, 976–984.

Kefaloyianni, E., Gaitanaki, C., Beis, I., 2006. ERK1/2 and p38-MAPK signaling pathways,through MSK1, are involved in NF-κB transactivation during oxidative stress inskeletal myoblasts. Cell Signal 18, 2238–2251.

Kim, J.D., McCarter, R.J.M., Yu, B.P., 1996. Influence of age, exercise and dietaryrestriction on oxidative stress in rats. Aging. Clin. Exp. Res. 8, 123–129.

Kim, J.O., Lee, G.D., Kwon, J.H., Kim, K.S., 2009. Anti-diabetic effects of newherbal formulain neonatally streptozotocin-induced diabetic rats. Biol. Pharm. Bull. 32, 421–426.

Klaunig, J.E., Kamendulis, L.M., 2004. The role of oxidative stress in carcinogenesis.Annu. Rev. Pharmacol. Toxicol. 44, 239–267.

Kolb, H., 1997. Benign versus destructive insulitis. Diabetes Metab. Rev. 13, 139–146.Konrad, P., Forsgren, A., Husberg, B.S., Nilsson, L., 1986. The immunosuppressive effect

of streptozotocin-induced diabetes in rats. Res. Exp. Med. 186, 455–462.Kroncke, K.D., Fehsel, K., Sommer, A., Rodriguez, M.L., Kolb-Bachofen, V., 1995. Nitric

oxide generation during cellular metabolization of the diabetogenic N-methyl-N-nitroso-urea streptozotocin contributes to islet cell DNA damage. Biol. Chem.Hoppe-Seyler. 376, 179–185.

Lau, J., Börjesson, A., Holstad, M., Sandler, S., 2006. Prolactin regulation of the expressionof TNF-alpha, IFN-gamma and IL-10 by splenocytes in murine multiple low dosestreptozotocin diabetes. Immunol. Lett. 102, 25–30.

LeBel, C.P., Bondy, S.C., 1990. Sensitive and rapid quantitation of oxygen reactive speciesformation in rat synaptosomes. Neurochem. Int. 17, 435–440.

Mandrup-Poulsen, T., Bendtzen, K., Nielsen, J.H., Bendixen, G., Nerup, J., 1985. Cytokines causefunctional and structural damage to isolated islets of Langerhans. Allergy 40, 424–429.

Manna, P., Sinha, M., Sil, P.C., 2007a. Arjunolic acid, a triterpenoid saponin, amelioratesarsenic-induced cyto-toxicity in hepatocytes. Chem. Biol. Interact. 170, 187–200.

Manna, P., Sinha, M., Sil, P.C., 2007b. Protection of arsenic-induced hepatic disorder byarjunolic acid. Basic Clin. Pharmacol. Toxicol. 101, 333–338.

Manna, P., Sinha, M., Sil, P.C., 2009. Protective role of arjunolic acid in response tostreptozotocin-induced type-I diabetes via the mitochondrial dependent andindependent pathways. Toxicology 257, 53–63.

Masoko, P., Mdee, L.K., Mampuru, L.J., Eloff, J.N., 2008. Biological activity of two relatedtriterpenes isolated from Combretum nelsonii (Combretaceae) leaves. Nat. Prod.Res. 22, 1074–1084.

Mingatto, F.E., Rodrigues, T., Pigoso, A.A., Uyemura, S.A., Curti, C., Santos, A.C., 2002. Thecritical role of mitochondrial energetic impairment in the toxicity of nimesulide tohepatocytes. J. Pharmacol. Exp. Ther. 303, 601–607.

Nishikimi, M., Rao, N.A., Yagi, K., 1972. The occurrence of superoxide anion in thereaction of reduced phenazine methosulfate and molecular oxygen. Biochem.Biophys. Res. Commun. 46, 849–854.

Nogichi, H., 2007. Stem cells for the treatment of diabetes. Endocr. J. 54, 7–16.Oberley, L.W., 1988. Free radicals and diabetes. Free Rad. Biol. Med. 5, 113–124.Rabinovitch, A., 1998. An update on cytokines in the pathogenesis of insulin dependent

diabetes mellitus. Diabetes Metab. Rev. 14, 129–151.Rakieten, N., Rakieten, M.L., Nadkarni, M.V., 1963. Studies on the diabetogenic action of

streptozotocin (NSC-37917). Cancer Chemother. Rep. 29, 91–98.Sakurai, H., Suzuki, S., Kawasaki, N., Nakano, H., Okazaki, T., Chino, A., Doi, T., Saiki, I.,

2003. Tumor necrosis factor-alpha-induced IKK phosphorylation of NF-kappaB p65on serine 536 is mediated through the TRAF2, TRAF5, and TAK1 signaling pathway.J. Biol. Chem. 278, 36916–36923.

Sedlak, J., Lindsay, R.H., 1958. Estimation of total, protein-bound, and non proteinsulfhydryl groups in tissue with Ellman's reagent. Anal. Biochem. 24/25, 192–205.

Singh, N., Anand, S., 1994. Cell death by apoptosis. Indian J. Exp. Biol. 32, 843–847.Smith, I.K., Vierheller, T.L., Thorne, C.A., 1988. Assay of glutathione reductase in crude tissue

homogenatesusing 5,5′-dithiobis (2-nitrobenzoic acid). Anal. Biochem. 175, 408–413.Tabatabaie, T., Vasquez-Weldon, A., Moore, D.R., Kotake, Y., 2003. Free radicals and the

pathogenesis of type 1 diabetes: β-cell cytokine-mediated free radical generationvia cyclooxygenase-2. Diabetes 52, 1994–1999.

129P. Manna et al. / Toxicology and Applied Pharmacology 244 (2010) 114–129

Thorvaldson, L., Holstad, M., Sandler, S., 2003. Cytokine release by mueine spleen cellsfollowing multiple low dose streptozotocin-induced diabetes and treatment with aTNFa transcriptional inhibitor. Int. Immunopharmacol. 3, 1609–1617.

Trinchieri, G., 1995. Interleukin-12: a proinflammatory cytokine with immunoregula-tory functions that bridge innate resistance and antigen-specific adaptiveimmunity. Annu. Rev. Immunol. 3, 251–276.

Turk, J., Corbett, J.A., Ramanandham, S., Bohrer, A., McDaniel, M.L., 1993. Biochemicalevidence for nitric oxide formation from streptozotocin in isolated pancreatic islets.Biochem. Biophys. Res. Commun. 197, 1458–1464.

Uchida, K., Stadtman, E.R., 1993. Covalent attachment of 4-hydroxynonenal toglyceraldehydes-3-phosphate dehydrogenase. J. Biol. Chem. 268, 6388–6393.

Vermes, I., Haanen, C., Steffens-Nakken, H., Reutelingsperger, C., 1995. A novel assay forapoptosis, flow cytometric detection of phosphatidylserine expression on earlyapoptotic cells using fluorescein-labeled Annexin V. J. Immunol. Methods 184,39–45.

Vermeulen, L., De Wilde, G., Van Damme, P., Vanden Berghe, W., Haegernan, G., 2003.Transcriptional activation of the NF-kappaB p65 subunit by mitogen- and stressactivated protein kinase-1 (MSK1). EMBO J. 22, 1313–1324.

Wang, J., Wang, G., Ansari, G.A.S., Khan, M.F., 2008. Activation of oxidative stress-responsive signaling pathways in early splenotoxic response of aniline. Toxicol.Appl. Pharmacol. 230, 227–234.

Xiao, SY, Zhang, H, Guzman, H, Tesh, R, 2001. Experimental yellow fever virus infectionin the golden hamster( Mesocricetus auratus): II. Pathology. J. Infect. Dis. 183,1437–1444.

Yan, J., Hales, B.F., 2005. Activator protein-1 (AP-1) DNA binding activity is induced byhydroxyurea in organogenesis stage mouse embryos. Toxicol. Sci. 85, 1013–1023.

Young, H.A., Hardy, K.J., 1995. Role of interferon-gamma in immune cell regulation.J. Leukoc. Biol. 58, 373–381.

Zhu, H., Bannenberg, G.L., Moldéus, P., Shertzer, H.G., 1994. Oxidation pathways for theintracellular probe 2′,7′-dichlorofluorescein. Arch. Toxicol. 68, 582–587.