Contribution of type 1 diabetes to rat liver dysfunction and cellular damage via activation of NOS,...

Free Radical Biology & Medicine 48 (2010) 1465–1484

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Original Contribution

Contribution of type 1 diabetes to rat liver dysfunction and cellular damage viaactivation of NOS, PARP, IκBα/NF-κB, MAPKs, and mitochondria-dependentpathways: Prophylactic role of arjunolic acid

Prasenjit Manna, Joydeep Das, Jyotirmoy Ghosh, Parames C. Sil ⁎Division of Molecular Medicine, Bose Institute, Kolkata 700054, India

Abbreviations: AA, arjunolic acid (2,3,23-trihydroxyadvanced glycation end product; ALT, alanine transaminCAT, catalase; ERK, extracellular signal-regulated kinase; Fsorting; GSH, glutathione; GSSG, glutathione disulfide;GPx, glutathione peroxidase; GR, glutathione reductasesynthase; MAPK, mitogen-activated protein kinase; MDA,factorκB;PARP, poly(ADP-ribose)polymerase; ROS, reactinitrogen species; SOD, superoxide dismutase; STZ, strepto⁎ Corresponding author. Fax: +91 33 2355 3886.

E-mail addresses: [email protected](P.C. Sil).

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a b s t r a c t
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Article history:Received 23 December 2009Revised 15 February 2010Accepted 18 February 2010Available online 25 February 2010

Keywords:HyperglycemiaReactive intermediatesLiver dysfunctionApoptosisArjunolic acidAntioxidantAntidiabeticFree radicals

Diabetic mellitus, a chronic metabolic disorder, is one of the most important health problems in the world,especially in developing countries. Our earlier investigations reported the beneficial action of arjunolicacid (AA) against streptozotocin-mediated type 1 hyperglycemia. We have demonstrated that AA possessesprotective roles against drug- and chemical- (environmental toxins) induced hepatotoxicity. Liver is themain organ of detoxification. The purpose of this study was to explore whether AA plays any protectiverole against hyperglycemic hepatic dysfunctions and, if so, what molecular pathways it utilizes forthe mechanism of its protective action. In experimental rats, type 1 hyperglycemia was induced bystreptozotocin. AA was administered orally at a dose of 20 mg/kg body wt both before and after diabeticinduction. An insulin-treated group was included in the study as a positive control for type 1 diabetes.Hyperglycemia caused a loss in body weight, reduction in serum insulin level, and increased formation ofHbA1C as well as advanced glycation end products (AGEs). Elevated levels of serum ALT and ALP, increasedproduction of ROS and RNS, increased lipid peroxidation, increased 8-OHdG/2-dG ratio, and decreased GSHcontent and cellular antioxidant defense established the hyperglycemic liver dysfunction. Activation of iNOS,IκBα/NF-κB, and MAPK pathways as well as signals from mitochondria were found to be involved ininitiating apoptotic cell death. Hyperglycemia caused overexpression of PARP, reduction in intracellular NADas well as ATP level, and increased DNA fragmentation in the liver tissue of the diabetic animals. Results ofimmunofluorescence (using anti-caspase-3 and anti-Apaf-1 antibodies), DAPI/PI staining, and DNA ladderformation and information obtained from FACS analysis confirmed the apoptotic cell death in diabetic livertissue. Histological studies also support the experimental findings. AA treatment prevented or amelioratedthe diabetic liver complications and apoptotic cell death. The effectiveness of AA in preventing the formationof ROS, RNS, HbA1C, AGEs, and oxidative stress signaling cascades and protecting against PARP-mediatedDNA fragmentation can speak about its potential uses for diabetic patients.

olean-12-en-28-oic acid); AGE,ase; ALP, alkaline phosphatase;ACS, fluorescence-activated cellGST, glutathione S-transferase;; iNOS, inducible nitric oxidemalonaldehyde; NF-κB, nuclearveoxygen species; RNS, reactivezotocin.

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In modern civilization diabetic mellitus is one of themost commonendocrine metabolic disorders. Worldwide, the number of diabeticpatients is rapidly increasing in the general population, especiallyamong the obese and the elderly. A large body of earlier investigationshas examined the role of oxidative stress in insulin resistance in type 1

and type 2 diabetes and its associated complications [1]. In addition,nonenzymatic glycation of protein and glucose autoxidation are alsoclosely associated with the development of hyperglycemia [2]. Hyper-glycemia also favors the increased expression of inducible nitric oxidesynthase (iNOS)1 via the activation of NF-κB, and this phenomenon isaccompanied by increased generation of nitric oxide [3]. Interactionbetween nitric oxide and superoxide generates the strong oxidantperoxynitrite, which causes increased lipid peroxidation, proteinnitration, and low-density lipoprotein oxidation, affecting manysignal transduction pathways [4]. Stadler et al. [5] reported thatbefore the evolution of late diabetic complications, nitric oxide, ROS,and peroxynitrite play a complex role in the development of earlydiabetes tissue injury. Antioxidants protect against glycation-derivedfree radicals and may have therapeutic potential. In addition, recentstudies have shown that compounds with combined antioxidant andantiglycation properties are more effective in treating diabetesmellitus [6]. Thus, the screening and development of new compounds

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1466 P. Manna et al. / Free Radical Biology & Medicine 48 (2010) 1465–1484

with combined antioxidant and antiglycation properties would bebeneficial in the treatment of diabetes mellitus.

Scientists are in search of easily available, inexpensive therapeu-tics having minimum side effects for the better treatment of thesediabetic patients. India has a well-known plethora of medicinal plants.Since ancient times herbal medicines have been used in the treatmentof diabetic mellitus and its complications. There are many compoundsisolated from traditional Indian medicinal plants with antidiabeticactivity [7], of which triterpenoid saponins are promising candidateswith the potential to be developed into new drugs for diabetes [8]. Inmany studies, these compounds showed potent inhibitory activityon the increased serum glucose levels [9]. Shimizu et al. [10] reportedthat triterpenoid saponins suppress the elevated blood glucoselevel by inhibiting the rate of glucose absorption. Arjunolic acid(AA; 2,3,23-trihydroxyolean-12-en-28-oic acid, Fig. 1A), a naturalpentacyclic triterpenoid saponin (isolated from the bark of Terminalia

Fig. 1. (A) Chemical structure of arjunolic acid. (B–D) Free radical scavenging activities of A(C) superoxide and hydroxide radical scavenging activities, and (D) hydrogen peroxide an(E) Inhibition of glycation by AA at various concentrations. Values represent the means of

arjuna), is well known for various biological functions, includingantioxidative [11], antifungal [12], hepatoprotective [13], antibacterial[14], and insect growth inhibitory activity [15].

In our earlier investigation, we found that AA plays a beneficialrole in reducing streptozotocin (STZ; a commonly used agent inexperimental diabetes)-induced type 1 diabetes via mitochondria-dependent as well as independent pathways [16]. In addition, we alsoinvestigated the prophylactic role of AA in response to STZ-mediateddiabetic renal injury via the activation of the polyol pathway andoxidative stress-responsive signaling cascades [17]. As the liver is themain organ of detoxification in the body, very recently we haveinvestigated the beneficial role of AA against acetaminophen (APAP)-induced liver injury via the inhibition of APAP bioactivation and c-JunN-terminal kinase (JNK)-mediated mitochondrial protection [18].In disease conditions, at an early stage the biomarkers of oxidativestress are elevated rapidly in the liver. It has been reported that under

A in a cell-free system. (B) Peroxynitrite and nitric oxide radical scavenging activities,d singlet oxygen scavenging activities. Values represent the means of six replications.six replications.

1467P. Manna et al. / Free Radical Biology & Medicine 48 (2010) 1465–1484

hyperglycemia the liver is subjected to ROS-mediated injury [19]. Inthis study, we therefore planned to investigate the role of AA againstSTZ-induced hepatotoxicity in an experimental model of chronichyperglycemia. To achieve this goal, we investigated the status of thephysiological indices related to hyperglycemic liver disease, intracel-lular antioxidant enzymes, GSH/GSSG ratio, and expression of tran-scription factors and MAPKs (mitogen-activated protein kinases) aswell as mitochondria-dependent cell death pathways.

Materials and methods

Chemicals

STZ [N-(methylnitrocarbamoyl)-d-glucosamine], anti-iNOS, anti-cytochrome c, anti-caspase-3, anti-cleaved caspase-3, anti-Apaf-1,anti-extracellular signal-regulated protein kinase (ERK) 1/2, and anti-phosphorylated ERK1/2 antibodies were purchased from Sigma–Aldrich Chemical Co. (St. Louis, MO, USA). Anti-NF-κB (p65 subunit),anti-phosphorylated NF-κB (p65 subunit), anti-Bcl-2, anti-Bad, anti-p38, and anti-phosphorylated p38 antibodies were purchased fromCell Signaling Technology (Beverly, MA, USA). Anti-PARP, anti-IκBα,and anti-phosphorylated IκBα were purchased from Santa Cruz Bio-technology (Santa Cruz, CA, USA).

Animals

Swiss albino male rats, weighing approximately 120–130 g, wereacclimatized under laboratory conditions for 2 weeks before theexperiments. They were maintained under standard conditions oftemperature (23±2 °C) and humidity (50±10%) with an alternating12-h light/dark cycle. All the experiments with animals were carriedout according to the guidelines of the institutional animal ethicalcommittee.

Extraction of AA

AAwas extracted from the bark of T. arjuna following themethod ofGhosh et al. [18]. Briefly, the bark of T. arjunawas cut into small pieces,dried, ground into powder, and extracted with petroleum followed bydiethyl ether. The crudematerialwasfiltered off and themother liquorwas concentrated under reduced pressure to yield a white amorphoussolid, which showed two spots on thin-layer chromatography. Thematerial was then subjected to separation on a silica gel column.Elution with chloroform:methanol yielded a compound later identi-fied as arjunolic acid (mp 325–328 °C). Approximately 250 mg of AAwas obtained from 1 kg of the bark. Purity of the compound wasconfirmed by using standard tools [such as NMR (1H, 13C), IR, massspectroscopy, HPLC, and optical rotation studies]. The detailedpurification data were included in our earlier communication [18].

Radical scavenging activity of AA in a cell-free system

Free radical-induced oxidative stress has been implicated in theetiology of diabetes and its associated complications. Excessive ROSgeneration destroys proteins, lipids, and DNA by oxidation. Moleculeshaving free radical scavenging properties could be a better choice forpreventing these diseases. In our study we therefore investigated thefree radical scavenging activity of AA in cell-free systems as describedbelow.

Hydroxyl radical scavenging activityThis assay was performed following the method of Elizabeth and

Rao [20] with a slightmodification. Hydroxyl radical was generated bythe Fe3+–ascorbate–EDTA–H2O2 system (Fenton reaction). The assayis based on the quantification of the 2-deoxyribose degradationproduct, by its condensation with Thiobarbituric acid.

Superoxide radical scavenging activityThe superoxide radical scavenging activity was measured following

the method of Siddhuraju and Becker [21]. The reaction mixturecontained 0.1 Mphosphate buffer, pH7.4, 150 μMnitroblue tetrazolium(NBT), 60 μM phenazine methosulfate, 468 μM NADH, and variousconcentrations of arjunolic acid. The mixture was incubated in the darkfor 10 min at25 °C and theabsorbancewas readat560 nm.Resultswereexpressed as percentage inhibition of the superoxide radical. Alldeterminations were performed in triplicate.

Peroxynitrite radical scavenging assayPeroxynitrite (ONOO−) was synthesized following the method of

Beckman et al. [22]. The Evans blue bleaching assay [23] was used tomeasure the peroxynitrite scavenging activity, with slight modifica-tion. The reaction mixture contained 50 mM phosphate buffer(pH 7.4), 0.1 mM Diethylenetriaminepentaacetic acid, 90 mM NaCl,5 mMKCl, 12.5 mM Evans blue, various doses of AA (0–1 mg/ml), and1 mM peroxynitrite in a final volume of 1 ml. After incubation at 25 °Cfor 30 min the absorbance was measured at 611 nm. The percentageof scavenging of ONOO− was calculated by comparing the results ofthe test and blank sample. All tests were performed six times.

Singlet oxygen (1O2) scavenging assaySinglet oxygen was determined by monitoring the bleaching of

N,N-dimethyl-4-nitrosoaniline following the method of Pedraza-Chaverrí et al. [24]. Briefly, the reaction mixture contained 45 mMsodium phosphate buffer (pH 7.1), 10 mM histidine, 10 mM NaOCl,10 mM H2O2, and 50 μM N,N-dimethyl-p-nitrosoaniline. The totalvolume of the assay mixture was 2.0 ml. The bleaching of N,N-dimethyl-4-nitrosoaniline was monitored by incubating the mixtureat 30 °C for 40 min. The extent of 1O2 production was determinedby measuring the decrease in the absorbance of N,N-dimethyl-p-nitrosoaniline at 440 nm. The relative scavenging efficiency (% inhi-bition of production of 1O2) was estimated from the difference inabsorbance of N,N-dimethyl-p-nitrosoaniline with and without theaddition of increasing amounts of AA.

Nitric oxide radical scavenging assayThe nitric oxide radical scavenging activity of arjunolic acid was

determined following the method of Green et al. [25]. Briefly, thereaction mixture contained 10 mM sodium nitroprusside, phosphate-buffered saline (pH 7.4), and various concentrations (0–1 mg/ml) ofAA in a final volume of 3 ml. After incubation for 150 min at 25 °C,1 ml sulfanilamide (0.33% w/v) was added to 0.5 ml of the incubatedsolution and allowed to stand for 5 min. Then 1 ml of naphthylethy-lenediamine dihydrochloride (0.1% w/v) was added and the mixturewas incubated for 30 min at 25 °C. The pink chromophore generatedduring diazotization of nitrite ions with sulfanilamide and subse-quent coupling with naphthylethylenediamine was measured spec-trophotometrically at 540 nm against a blank sample. Results wereexpressed as the percentage inhibition of nitric oxide. All determina-tions were performed in triplicate.

Hydrogen peroxide scavenging assayThe hydrogen peroxide scavenging activity was determined

following the method of Long et al. [26]. Aliquots of 50 mM H2O2

and various concentrations (0–1 mg/ml) of AA were mixed (1:1, v/v)and incubated for 30 min at room temperature. After incubation,90 ml of the H2O2 sample solution was mixed with 10 ml methanoland 0.9 ml FOX reagent (previously prepared by mixing 9 volof 4.4 mM butylated hydroxyltoluene in HPLC-grade methanol with1 vol of 1 mM xylenol orange and 2.56 mM ammonium ferrous sulfatein 0.25 M H2SO4). Then the reaction mixture was vortexed andincubated at room temperature for 30 min. The absorbance of ferric–xylenol orange complex was measured at 560 nm. All tests werecarried out six times.


Evaluation of antiglycation property

Oxidative stress and nonenzymatic glycation of proteins areclosely associated with the development of diabetes mellitus. Anti-oxidants protect against glycation-derived free radicals and may havetherapeutic potential. The antiglycation property of AA was investi-gated following the method of Tang et al. [27]. Bovine serum albumin(BSA) (10 mg/ml) in phosphate buffer (50 mM, pH 7.4) containing0.02% (w/v) sodium azide was preincubated with AA and vitamin C atvarious concentrations for 30 min at room temperature (25 °C).Glucose and fructose solutions (25 mM each) were added to thereaction mixture and incubated at 37 °C for 2 weeks. Fluorescencewas determined using a fluorescence spectrophotometer (Perkin–Elmer) with an excitation wavelength of 350 nm and an emissionwavelength of 450 nm. Antiglycation was also evaluated by deter-mining the fructosamine (the early glycation product) content by theNBT reduction method [28].

Experimental induction of diabetes

The experimental animals were made diabetic with an intraper-itoneal injection of STZ at a dose of 65 mg/kg body wt dissolved incitrate buffer (0.1 M, pH 4.5). STZ-injected animals exhibited massiveglycosuria and hyperglycemia within a few days. Diabetes was con-firmed in the overnight-fasted rats by measuring blood glucose con-centration 96 h after injection with STZ. The rats with blood glucoseabove 240 mg/dl were considered to be diabetic and then they wereused for the experiments as necessary.

Animal treatments

The animals were divided into six groups, each consisting of sixrats, and they were treated as follows.

Group 1(normal control group)—animals received vehicle only;Group 2 (AA-treated group)—AAwas administered orally at a dose

of 20 mg/kg body wt for 14 days;Group 3(STZ-treated group)—diabetic control group;Group 4(AA-pretreated group)—AA was administered orally at a

dose of 20 mg/kg body wt for 14 days before the diabeticinduction;

Fig. 2. Schematic diagram of in

Group 5 (AA-posttreated group)—AA was administered orally at adose of 20 mg/kg body wt for 14 days after diabeticinduction;

Group 6(insulin-treated group)—a volume of 0.5 ml of a 2 mg/mlaqueous insulin solution was administered orally for14 days after diabetic induction.

Fig. 2 presents a diagram of the in vivo experimental protocol.After 31 days of treatment the animals were anesthetized first andthen sacrificed. From the sacrificed animals, blood was collected intubes containing a mixture of potassium oxalate and sodium fluoride(1:3) for the estimation of blood glucose. From all the animalsthe liver tissue was collected, stored at 4 °C, and used for the variousexperiments as needed.

The dose schedules of STZ and insulin were selected based on theearlier reports of Dias et al. [29] and Von Herrath et al. [30], respec-tively. In our previous studies [16] we found that at a dose of 20 mg/kg body wt, AA has an optimum antidiabetic role against STZ-inducedtype 1 diabetes. In the present study this particular dose of AA waschosen to investigate the protective role of the same against STZ-induced diabetic liver dysfunction as well as cell death.

Preparation of liver tissue homogenates

Liver tissues from experimental rats were homogenized using aglass homogenizer in 100 mM potassium phosphate buffer containing1 mM EDTA, pH 7.4, and centrifuged at 12,000×g for 30 min at 4 °C.The supernatant was collected and used for the experiments.

Determination of protein content

The protein contents of the liver tissue homogenates of the exper-imental animals were measured by the method of Bradford usingcrystalline BSA as standard [31].

Assessment of blood glucose, insulin, glycated hemoglobin (HbA1C), andserum-specific marker levels related to hepatic dysfunction

Blood samples collected from punctured rat hearts were keptovernight to clot and then centrifuged at 3000×g for 10 min. Bloodglucose and insulin levels were estimated by using standard kits (Span

vivo experimental protocol.


Diagnostic Ltd., India). Glycated hemoglobin concentration was deter-mined using Hemoglobin A1C micro columns (Bio-Rad, Hercules, CA,USA) and expressed as the percentage of total hemoglobin. Serum-specific markers ALT (alanine transaminase) and ALP (alkalinephosphatase), related to hepatic dysfunction, were also measuredusing kits (Span Diagnostic Ltd.).

Assay for the production of peroxynitrite

Intracellular peroxynitrite production was studied by the fluori-metric assay of 2,7-dichlorofluorescein (DCF) as described by Tannouset al. [32]. Peroxynitrite can oxidize the nonfluorescent molecule DCFdiacetate (DCFDA) to the fluorescent DCF. Briefly, the DCFDA-treatedcells were incubated with NO buffer (containing 100 μM l-arginine)for 15 min at 37 °C in the dark. The mixture was washed with PBS(pH 7.4) and then centrifuged for 2 min at 214×g. The supernatantfluorescence was measured in a fluorescence spectrometer (Hitachi,Model F4500) at an excitation wavelength of 475 nm and emissionwavelength of 520 nm.

Assay of advanced glycation end products in liver

Proteins are modified by glucose through the glycation reaction,resulting in the formation of advanced glycation end-products(AGEs). The contribution of AGEs to diabetes has received consider-able attention in recent years and free radicals have been shown toparticipate in AGE formation. The hepatic AGE level was determinedby the method of Nakayama et al. [33]. Minced liver tissue wasdelipidated with chloroform and methanol (2:1, v/v) overnight. Afterbeing washed, the tissue was homogenized in 0.1 N NaOH, followedby centrifugation at 8000×g for 15 min at 4 °C. The amount of AGEsin these alkali-soluble samples was determined by measuring thefluorescence at an emission wavelength of 440 nm and an excitationwavelength at 370 nm. BSA solution (1 mg/ml of 0.1 N NaOH) wasused as a standard, and its fluorescence intensity was defined as 1 unitof fluorescence. The fluorescence values of samples were measured ata protein concentration of 1 mg/ml and expressed in arbitrary unitscompared with a native BSA preparation.

Measurement of intracellular ROS production and serum NO (nitrate–nitrite) levels

Intracellular ROS production was estimated using DCFDA as aprobe in the method of LeBel and Bondy [34], followed by themodification introduced by Kim et al. [35]. The formation of DCFwas measured at the excitation wavelength of 488 nm and emissionwavelength of 610 nm for 10 min using a fluorescence spectrometer(Hitachi, Model F4500) equipped with a FITC filter.

For in vitro study, after treatment, the medium was aspirated andhepatocytes 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 mediumand maintained in 1 ml culture medium. Fluorescence was monitoredusing a fluorescence spectrometer (the intensity values werecalculated using Metamorph software) and they were also assessedusing a flow cytometer.

The levels of serum NO were measured by estimating theconcentrations of nitrate and nitrite with a modified nitrate reductaseand Griess reaction method [36]. Nitrate was reduced to nitrite by theaction of nitrate reductase in the presence of NADPH. Excess NADPHwas then consumed and the nitrite concentration measured by theaddition of equal volumes of 1% sulfanilamide in 5% concentratedphosphoric acid and 0.1% N-(1-naphthyl)ethylenediamine dihy-drochloride. The Griess color reaction forms a pink azo dye with an

absorbance wavelength of 540 nm. The assay was standardized usingvarious dilutions of sodium nitrate and nitrite (Sigma).

Assay of biochemical parameters including antioxidant enzymes, GSH/GSSG, lipid peroxidation, and protein carbonylation

Oxidative stress has been suggested to play an important role inthe pathogenesis of diabetic complications because oxidative stressincreases under diabetic conditions and induces cellular dysfunctionin a wide variety of cells. To protect against oxidative stress, cellsusually possess defensive mechanisms including their own intracel-lular antioxidants (such as antioxidant enzymes and cellular meta-bolites). In our study we, therefore, investigated the activities ofthe antioxidant enzymes SOD, CAT, GST, GR, and GPx; GSH/GSSGratio; and levels of lipid peroxidation and protein carbonylation in theexperimental animals following methods described elsewhere [16].

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%nonfat dry milk to prevent nonspecific binding and then incubatedwith primary antibodies overnight at 4 °C. The primary antibodiesused in this study were anti-iNOS (1:500), anti-p38 (1:1000), anti-phosphorylated p38 (1:1000 dilution), anti-ERK1/2 (1:1000), anti-phosphorylated ERK1/2 (1:1000), anti-NF-κB (p65) (1:250 dilution),anti-phosphorylated NF-κB (p65) (1:500), anti-IκBα (1:1000), anti-phosphorylated IκBα (1:500), anti-cytochrome c (1:1000), anti-caspase-3 (1:1000), anti-cleaved caspase-3 (1:1000), anti-Apaf-1(1:1000), and anti-PARP (1:1000) antibodies. The membranes werewashed in 50 mmol/L Tris–HCl, pH 7.6, 150 mmol/L NaCl, 0.1% Tween20 for 30 min and incubated with appropriate horseradish peroxidase(HRP)-conjugated secondary antibody (1:2000) for 2 h at room tem-perature and developed by the HRP substrate 3,3′-diaminobenzidinetetrahydrochloride system (Bangalore Genei, Bangalore, India).

Preparation of nuclear extract from rat liver tissue

Nuclear proteins were extracted from the frozen liver samplesaccording to the method of Valen et al. [37]. Briefly, livers werehomogenized and lysis buffer was added. After incubation on ice,nuclei were collected bymeans of centrifugation for 1 min at 8000×g.The pellet was washed with 20 mM KCl buffer, centrifuged again, andresuspended in 20 mM KCl buffer. An equal part of 600 mM KCl wasadded to the pellet and kept on ice for 30 min. After centrifugation for15 min at 8000×g, the supernatant containing nuclear proteins wasobtained. This nuclear extract was subjected to immunoblotting asdescribed above to determine the activity of NF-κB in vivo.

Isolation of hepatocytes

Hepatocytes were isolated from liver tissue of experimental animalsby the perfusion technique as described by Ghosh and Sil [38]. Briefly,the animalswere anesthetizedwith ether and sacrificed and liverswerecollected. After collection, the organs were extensively perfused in situin phosphate-buffered saline to get rid of blood and irrigated in a buffercontaining Hepes (10 mM), KCl (3 mM), NaCl (130 mM), NaH2PO4 ·H2O (1 mM), and glucose (10 mM), pH7.4, and incubatedwith a secondbuffer containingCaCl2 (5 mM)and 0.05% collagenase type Imixedwiththe buffer previously described for about 45 min at 37 °C. The liversample was then passed through a wide-bore syringe, filtered, andcentrifuged and the pellet was suspended in DMEM containing 10% FBSand the suspension was adjusted to obtain ∼2×106 cells/ml. Only thepreparations with cell viability (measured by MTT assay) greater than90% were used for subsequent experiments.


Mitochondrial membrane potential

Reduction in the mitochondrial membrane potential (Δψ) andsubsequent activation of downstream signaling protein moleculesplay a key step in mitochondrial-dependent cell death. For thedetection of mitochondrial Δψ, the cells were incubated with rho-damine 123 (5 μg/ml final concentration) for 60 min in the dark at37 °C, harvested, and suspended in PBS. The mitochondrial Δψ wasmeasured by the fluorescence intensity (FL-1, 530 nm) of 10,000 cells[39] and the corresponding images were taken using a laser scanningconfocal microscope.

Assay of cytochrome c in the cytosolic and mitochondrial fraction

Depletion of the mitochondrial membrane potential causes therelease of cytochrome c and initiates the mitochondrial-dependentcell death pathway. The concentration of cytosolic cytochrome c wasmeasured with the cytochrome c enzyme immunometric assay kit(Quantikine, R&D Systems Inc., Minneapolis, MN, USA). The expres-sion of mitochondrial cytochrome c was investigated by immuno-blotting analysis using anti-cytochrome c antibody.

DNA fragmentation assay

The extent of DNA fragmentation (DNA ladder) was assayed byelectrophoresis of genomic DNA samples, isolated from normal aswell as experimental rat liver, on agarose/ethidium bromide gels bythe procedure described by Sellins and Cohen [40].

HPLC measurement of 8-hydroxy-2-deoxyguanosine (8-OHdG) and2-deoxyguanosine (2-dG)

DNA samples were isolated from the experimental liver tissueusing a standard kit. The isolated DNA samples were digestedfollowing the method of Kasai et al. [41]. The oxidative DNA adduct8-OHdGwasmeasuredwith HPLC equipped with electrochemical andUV detection following the methods described elsewhere [42]. Thewavelength for UV detection of 2-dG was set at 260 nm. The mobilephase was composed of 50 mM sodium acetate, 5% methanol, pH 5.2,and the flow rate was 1.0 ml/min.

Assay of intracellular NAD and ATP level

NAD and ATP play important roles in STZ-induced DNA fragmen-tation in the diabetic milieu. Levels of these parameters were there-fore determined in the experimental animals. NAD was assayed in thehomogenates by the colorimetric method as described by Matsumuraand Miyachi [43]. For the ATP assay tissue homogenate was mixedwith trichloroacetic acid (TCA) (6.5%, w/v) and left on ice for 10 min.The TCA extract was removed and used immediately for analysis ofATP as described by Kalbheim and Koch [44].

Immunocytochemistry

The cells plated on coverslips were fixed with 4% paraformalde-hyde for 30 min and permeabilized with 0.1% Triton X-100 in PBS for2.5 min. The cells were treated with blocking medium (3% BSA in PBS)for 1 h and incubated with anti-caspase-3 and anti-Apaf-1 antibodies(separately), diluted in blocking medium, for 2 h. The immuno-reactive primary antibody was detected by a 1:500 dilution of FITC-conjugated secondary antibody for 1 h. After being washed with PBS,the stained cells were mounted onto microscope slides in mountingmedium. The images were collected using the laser scanning confocalmicroscope.

Flow cytometry analysis

Theflow cytometric analysiswas done on a BD-LSRflow cytometer.Cell debris, characterized by a low FSC/SSC, was excluded fromanalysis. The data were analyzed using CellQuest software.

Assessment of apoptotic and necrotic cells

The apoptotic and necrotic cell distribution was analyzed byannexin V binding and propidium iodide (PI) uptake. Positioningof quadrants on annexin V/PI dot plots was performed, and livingcells (annexin V−/PI−), early apoptotic/primary apoptotic cells(annexin V+/PI−), late apoptotic/secondary apoptotic cells (annexinV+/PI+), and necrotic cells (annexin V−/PI+) were distinguished.Therefore, the total apoptotic proportion included the percentage ofcells with annexin V+/PI− and annexin V+/PI+ fluorescence. Thecells were suspended in 1 ml binding buffer (1×). An aliquot of 100 μlwas incubated with 5 μl annexin V–FITC and 10 μl PI for 15 min in thedark at room temperature, and 400 μl binding buffer (1×) was addedto each sample. The FITC and PI fluorescence was measured throughan FL-1 filter (530 nm) and FL-2 filter (585 nm), respectively, and10,000 events were acquired.

Histological analysis and immunohistochemistry

Livers 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 hematoxylin andeosin to evaluate the pathophysiological changes under a lightmicroscope. Immunohistochemistry was performed with antibodiesfor caspase-3 (Santa Cruz Biotechnology).

Statistical analysis

All the values are expressed as means±SD (n=6). Significantdifferences between the groups were determined with SPSS 10.0software (SPSS Inc., Chicago, IL, USA) for Windows using one-wayanalysis of variance and the groupmeans were compared by Duncan'smultiple range test. A difference was considered significant at thePb0.05 level.

Results

The chemical structure of AA is presented in Fig. 1A. It possessesthree hydroxyl (one primary and two secondary) and one carboxylicacid groups. In addition, it has one double bond.

Free radical scavenging and antiglycation properties of AA in a cell-freesystem

The inhibition of the production of free radicals (peroxynitrite, nitricoxide, superoxide, hydroxyl), hydrogen peroxide, singlet oxygen, andAGEs was carried out in a cell-free system using various concentra-tions of AA (Figs. 1B–E). The results suggest that at a concentrationof 0.4 mg/ml AA shows its maximum radical scavenging as well asglycation inhibitory property.

Effects of AA against STZ-induced hepatotoxicity under hyperglycemia

Reduction in body weight and an increase in blood glucose level,increase in in vivo AGE formation, and decrease in insulin level are themarkers for the development of diabetes. This pathophysiology wasobserved in STZ-treated animals (Table 1), suggesting their diabeticnature. AA supplements, however, significantly lowered the bloodglucose level and formation of HbA1C as well as AGEs and prevented

Table 1Effects of streptozotocin (STZ) and arjunolic acid (AA) on body weight; levels blood glucose, insulin, HbA1C, and AGEs; and activities of serum marker enzymes

Parameter Normal control AA treated STZ treated AA+STZ STZ+AA STZ+insulin

Body weight (g) 135.42±6.81 133.51±6.72 110.14±5.55a 126.72±6.35b 122.47±6.15c 130.51±6.57d

Blood glucose (mg/dl) 86.33±4.35 89.14±4.55 275.15±14.04a 109.24±5.45b 122.17±6.16c 95.35±4.81d

Plasma insulin (μg/dl) 2.85±0.15 2.83±0.14 1.12±0.06a 2.27±0.12b 2.15±0.11c 2.55±0.12d

HbA1C (%) 12.34±0.62 12.59±0.65 26.37±1.35a 15.14±0.81b 18.97±0.95c 14.41±0.75d

Hepatic AGEs (AU) 3.47±0.19 3.45±0.17 5.45±0.28a 3.75±0.21b 3.94±0.22c 3.61±0.18d

ALT (IU/L) 25.38±1.27 23.79±1.19 57.78±2.91a 30.52±1.55b 38.45±1.95c 29.35±1.47d

ALP (KA units) 30.15±1.55 29.88±1.51 65.35±3.28a 39.75±2.01b 44.12±2.22c 33.34±1.69d

Values are expressed as means±SD for six animals in each group.a Pb0.05, significant difference between the normal control and the STZ-treated group.b Pb0.05, significant difference between the STZ-treated and the AA-pretreated group (AA+STZ).c Pb0.05, significant difference between the STZ-treated and the AA-posttreated group.d Pb0.05, significant difference between the STZ-treated and the insulin-treated group (STZ+insulin).


the reduction in insulin level as well as the loss in body weightcompared to the diabetic control group.

The status of the serum-specificmarker enzymes related to hepaticdysfunction is shown in Table 1. STZ-induced hyperglycemia caused asignificant increase in the activities of ALT and ALP (Table 1). Treat-ment with AA, however, could keep these levels close to normal.

Effects on intracellular ROS and peroxynitrite as well as the levels ofserum nitrate and nitrite, lipid peroxidation, and protein carbonylcontent

Several investigations support the fact that increased production ofROS as well as RNS induces organ pathophysiology under STZ-inducedhyperglycemic conditions. STZ exposure caused alterations in the levelsof intracellularROSproduction aswell as serumnitrate andnitrite levels.Under hyperglycemic conditions, the production of ROS and peroxyni-trite, as well as serum nitrate and nitrite levels (Table 2), increased bya large amount. AA administration at a dose of 20 mg/kg body wtfor 14 days, however, could prevent the hyperglycemia-induced ROSproduction as well as the increase in serum nitrate and nitrite levels.

Table 2ROS and NO production (expressed by nitrate–nitrite level), peroxynitrite level, lipid peroxitreated liver tissue

Parameter Normal control AA treate

Rate of ROS production (nmol DCF/min/mg protein) 35.47±1.76 33.54±1Serum NO level (μmol/L) 16.37±0.85 16.45±0Peroxynitrite level (nmol/mg protein) 0.74±0.041 0.82±0Lipid peroxidation (nmol/mg protein) 5.35±0.26 5.27±0Protein carbonylation (nmol/mg protein) 1.75±0.08 1.63±0

Values are expressed as means±SD for six animals in each group.a Pb0.05, significant difference between the normal control and the STZ-treated group.b Pb0.05, significant difference between the STZ-treated and the AA-pretreated group (Ac Pb0.05, significant difference between the STZ-treated and the AA-posttreated group.d Pb0.05, significant difference between the STZ-treated and the insulin-treated group (

Table 3Activities of antioxidant enzymes in the diabetic and AA-treated liver tissue

Parameter Normal control AA treated ST

SOD (unit/mg protein) 32.33±1.63 31.47±1.58 15CAT (μmol/min/mg protein) 75.23±3.77 74.39±3.73 39GST (μmol/min/mg protein) 5.75±0.29 5.69±0.28 3GR (nmol/min/mg protein) 142.31±7.15 137.26±6.88 76GPx (nmol/min/mg protein) 155.29±7.78 151.37±7.57 85

Values are expressed as means±SD for six animals in each group.a Pb0.05, significant difference between the normal control and the STZ-treated group.b Pb0.05, significant difference between the STZ-treated and the AA-pretreated group (Ac Pb0.05, significant difference between the STZ-treated and the AA-posttreated group.d Pb0.05, significant difference between the STZ-treated and the insulin-treated group (

Lipid peroxidation and protein carbonyl content are widely usedas markers of cell membrane damage and oxidative modification ofproteins, respectively. In this study the lipid peroxidation was mea-sured by estimating the concentration of MDA (lipid peroxidation endproduct). STZ intoxication increased the levels of MDA and proteincarbonylation in the liver tissue of the experimental animals (Table 2).Treatment with AA was effective in preventing the STZ-inducedhepatotoxicity under hyperglycemic conditions.

Effects on GSH/GSSG and 8-OHdG/2-dG ratios

GSH plays a critical role in scavenging intracellular reactive inter-mediates. A massive amount of GSH is consumed to accomplish thistask, thus shifting the redox status of the cell. Whenever the GSH leveldecreases below the threshold level, the concentration of reactiveradicals becomes elevated and causes oxidative stress as well as DNAdamage. In our study we found that hyperglycemia decreased theGSH/GSSG ratio, with a simultaneous increase in DNA oxidation(as measured by 8-OHdG/2-dG ratio) (Table 3). AA treatment could,however, prevent those alterations.

dation (measured by MDA content), and protein carbonylation in the diabetic and AA-

d STZ treated AA+STZ STZ+AA STZ+insulin

.69 82.54±3.98a 40.37±2.03b 48.63±2.47c 39.43±1.98d

.92 30.17±1.62a 20.31±1.05b 22.87±1.16c 18.21±1.01d

.044 2.54±0.133a 1.35±0.071b 1.67±0.085c 1.19±0.062d

.25 12.63±0.65a 7.73±0.39b 8.31±0.42c 6.25±0.37d

.09 4.39±0.25a 2.12 ± 0.12b 2.75±0.15c 1.95±0.11d

A+STZ).

STZ+insulin).

Z treated AA+STZ STZ+AA STZ+insulin

.12±0.79a 25.63±1.29b 24.47±1.25c 30.87±1.55d

.81±1.98a 63.24±3.18b 55.12±2.78c 70.39±3.52d

.47±0.18a 5.12±0.26b 4.89±0.25c 5.49±0.27d

.97±3.85a 115.43±5.79b 108.36±5.42c 121.47±6.11d

.76±4.31a 129.62±6.51b 112.45±5.65 143.39±7.15d

A+STZ).

STZ + insulin).

Table 4Status of the thiol-based antioxidant in the diabetic as well as AA-treated liver tissue

Parameter Normal control AA treated STZ treated AA+STZ STZ+AA STZ+insulin

GSH (nmol/mg protein) 8.57±0.45 8.45±0.44 5.67±0.31a 8.11±0.41b 7.85±0.39c 8.25±0.43d

GSSG (nmol/mg protein) 0.18±0.011 0.21±0.012 0.74±0.042a 0.26±0.015b 0.31±0.018c 0.230.014d

Redox ratio (GSH/GSSG) 47.61±1.85 40.24±1.68 7.66±0.25a 31.19±1.57b 25.32±1.27c 35.87±1.81d

(8-OHdG/2-dG) × 105 7.37±0.38 8.14±0.42 24.21±1.25a 10.81±0.57b 15.32±0.75c 10.05±0.52d

Values are expressed as means±SD for six animals in each group.a Pb0.05, significant difference between the normal control and the STZ-treated group.b Pb0.05, significant difference between the STZ-treated and the AA-pretreated group (AA+STZ).c Pb0.05, significant difference between the STZ-treated and the AA-posttreated group.d Pb0.05, significant difference between the STZ-treated and the insulin-treated group (STZ+insulin).


Activities of hepatic antioxidant enzymes

Oxidative stress is the result of a redox imbalance between thegeneration of ROS and the compensatory response from the endog-enous antioxidant network. To investigate whether the radical scav-enging activity of AA was mediated by the activities of antioxidantenzymes, we measured the activities of the intracellular antioxidantenzymes SOD, CAT, GST, GR, and GPx. Table 4 presents the activities ofthe antioxidant enzymes in liver tissue of the experimental animals.Our study revealed that STZ intoxication significantly attenuatedthe activities of the antioxidant enzymes. The results suggest that AAtreatment could be able to prevent as well as protect against thereduction in antioxidant activities in hyperglycemia.

In this study the antidiabetic action of AA was compared with agroup treated with insulin (a potent antidiabetic drug commerciallyused for type 1 diabetes). It was observed that the antidiabetic aswell as antioxidant properties of AA were comparable to those ofinsulin.

Effect of AA against STZ-induced activation of NO signaling

Increased production of NO, ROS, and peroxynitrite and theoverexpression of nitric oxide synthase play important roles in theSTZ-induced diabetic milieu and its associated complications. In ourstudy we found that STZ exposure not only increased the productionof ROS, NO, and peroxynitrite, it also caused an overexpression of iNOS(Fig. 3). Treatment with AA both before and after diabetes, on theother hand, prevented the NO signaling pathways and thereby helpedthe organ back to its physiological state.

Fig. 3. Western blot analysis of iNOS in the liver tissue of diabetic and AA-treated animals. Tcontrol band was given an arbitrary value of 1. Data represent the averages±SD of six sepcontrol and the diabetic group; bPb0.05, significant difference between the diabetic anddiabetic and the AA-posttreated group (STZ+AA).

Effect on the activity of NF-κB and IκBα

NF-κB, a ubiquitous transcription factor, can be activated by avariety of stimulating signals relevant to pathophysiology. Transcrip-tional activity of NF-κB can be regulated by the phosphorylation of IκBas well as the NF-κB p65 subunit. In our study, we used Westernimmunoblotting to determine whether activation of NF-κB (in thenuclear extract) or IκBα (cytosolic part) is involved in the hypergly-cemic hepatic damage and its prevention by AA. We observed thathyperglycemia elevated the expression of phosphorylated IκBα andphosphorylated NF-κB p65 (Fig. 4). AA treatment, however, waseffective in preventing the phosphorylation of IκBα and NF-κB p65.

Effect on the activity of MAPKs

MAPKs are the critical upstream signaling proteins. There are threedistinct subfamilies of MAPKs: ERK, p38-MAPK, and JNK. To assess theeffects of STZ and AA exposure on the activation of the MAPKsubfamilies, the liver tissue homogenates were analyzed for both totaland phosphorylated forms of p38 and ERK1/2 by immunoblotting. Fig. 5shows that in the diabetic animals, phosphorylated p38 and ERK wereactivated, leaving their total levels unchanged. Treatmentwith AA couldprevent the hyperglycemia-induced phosphorylation of ERK and p38.

Effect of AA on the Bcl-2 family of proteins

Bcl-2 family proteins are upstream regulators of the mitochondrialmembrane potential. Fig. 6 presents the expression of Bcl-2 familyproteins developed under STZ-induced hyperglycemia. From theimmunoblotting study, it was observed that STZ treatment upregulated

he relative intensities of the bands were determined using NIH Image software and thearate experiments in each group. aPb0.05, significant difference between the normal

the AA-pretreated group (AA+STZ); and cPb0.05, significant difference between the

Fig. 4.Western blot analysis of (A) NF-κB and (B) IκBα in the liver tissue of diabetic and AA-treated animals. The relative intensities of the bands were determined using NIH Imagesoftware and the control band was given an arbitrary value of 1. Data represent the averages±SD of six separate experiments in each group. aPb0.05, significant difference betweenthe normal control and the diabetic group; bPb0.05, significant difference between the diabetic and the AA-pretreated group (AA+STZ); and cPb0.05, significant difference betweenthe diabetic and the AA-posttreated group (STZ+AA).


proapoptotic (Bad) and downregulated antiapoptotic (Bcl-xL) Bcl-2proteins in liver tissue. Treatmentwith AA, however, could significantlyrepress hyperglycemia-induced alterations in these parameters.

Effect on the ROS production

ROS play a major role in diabetes and its associated complications.To assess changes in the hepatocyte morphology under oxidativestress, hepatocytes were isolated from the experimental animals andthe ROS production was assayed using DCFDA under fluorescencemicroscopy and by FACS analysis. Fig. 7 shows the effects ofhyperglycemia-induced ROS production in isolated hepatocytes andits reversal by AA treatment. Here, we observed (Fig. 7) that theintracellular glucose burden increased the level of ROS production(as revealed by the increased number of green fluorescent cells),with simultaneous alterations in their morphology. FACS analysis alsosupported this result. Treatment with AA prevented these alterations.

Effect of AA against STZ-induced mitochondrion-dependent cell deathpathway

Oxidative stress-induced apoptosis is directly related to mitochon-drial dysfunction. Disruption of the mitochondrial membrane potentialand subsequent release of cytochrome c are the novel biomarkers of

oxidative stress-induced cell death via the mitochondria-dependentpathway. Release of cytochrome c causes the activation of caspases viathe formation of apoptosomes. Therefore, we assessed the mitochon-drial membrane potential and leakage of cytochrome c into the cytosol,aswell as its level in themitochondrial fraction andthe status of caspases(initiator caspase-9 and effector caspase-3) and Apaf-1. Immunoblotanalyses (Fig. 8) showed that hyperglycemia reduced themitochondrialmembrane potential (Fig. 8A), decreased the activity of cytochrome c inthe mitochondrial fraction (Fig. 8B), elevated the concentration ofcytosolic cytochrome c (Fig. 8D), anddecreased theApaf-1 level (Fig. 8C)in association with upregulating caspase-9 (Fig. 8F), caspase-3, andcleaved caspase-3 (Fig. 8E), indicating the involvement of the mito-chondrial apoptotic pathway in this pathophysiology. Immunofluores-cence study (Fig. 8G) also showed that hyperglycemia increased theexpression of caspase-3 and Apaf-1. Treatment with AA, however, couldsignificantly inhibit hyperglycemia-induced mitochondria-dependentcell death via the apoptotic mode.

Effect of AA against STZ-induced DNA fragmentation via the PARP-mediated pathway

The PARP-mediated reduction in NAD as well as ATP levels playsan important role in STZ-induced DNA fragmentation in the diabeticmilieu. We also investigated PARP expression and its downstream

Fig. 5.Western blot analysis of MAPKs [(A) p38; (B) ERK1/2] in the liver tissue of diabetic and AA-treated animals. The relative intensities of the bands were determined using NIHImage software and the control band was given an arbitrary value of 1. Data represent the average±SD of 6 separate experiments in each group. aPb0.05, significant differencebetween the normal control and the diabetic group; bPb0.05, significant difference between the diabetic and the AA-pretreated group (AA+STZ); and cPb0.05, significant differencebetween the diabetic and the AA-posttreated group (STZ+AA).


effects (Fig. 9) in both diabetic and AA-treated animals. We observedthat bioactivation of STZ increased the expression of PARP anddecreased the levels of NAD as well as ATP in the liver tissue of the

Fig. 6.Western blot analysis of Bad and Bcl-2 in the liver tissue of diabetic and AA-treated animsoftware and the control band was given an arbitrary value of 1. Data represent the averagesthe normal control and the diabetic group; bPb0.05, significant difference between the diabetthe diabetic and the AA-posttreated group (STZ+AA).

diabetic animals. Treatmentwith AA both before and after the diabeticinduction attenuated STZ-induced DNA fragmentation by preventingthe activation of PARP and its downstream effects.

als. The ratios of the relative intensities of the bands were determined using NIH Image±SD of six separate experiments in each group. aPb0.05, significant difference betweenic and the AA-pretreated group (AA+STZ); and cPb0.05, significant difference between

Fig. 7. The intracellular ROS production was detected by the DCFDA method. (A) DCF fluorescence was measured using a flow cytometer with an FL-1 filter. (B) Representativeimages under the fluorescencemicroscope (original magnification×400). Cont, cells isolated from the liver tissue of normal animals; AA, cells isolated from the liver tissue of animalstreated with AA; STZ, cells isolated from the liver tissue of the diabetic animals; AA+STZ, cells isolated from the liver tissue of animals treated with AA before diabetic induction; andSTZ+AA, cells isolated from the liver tissue of animals treated with AA after diabetic induction. The measurements were taken six times.


Effects of STZ and AA on cell death pathway

To show the apoptotic changes, next we examined the DNA frag-mentation pattern. STZ caused a DNA ladder fragmentation, a hall-mark of apoptosis (Fig. 10A). AA treatment, however, could effectivelyprevent the DNA laddering.

Next we focused on the morphological changes in hepatocytesundergoing apoptosis using DAPI and PI double staining. As shownin Fig. 10B, hepatocytes from control animals exhibited normalmorphology, whereas cells from STZ-exposed animals were observedto have condensed chromatin and fragmented nuclei. AA treatment,

however, could restore the normal morphology of these cells,suggesting that AA might act as a cytoprotective agent againsthyperglycemia.

The annexin V binding assay, which measured the fluorescencegenerated by annexin binding to externalized phosphatidylserine ofapoptotic cells, corroborated the apoptotic DNA data. The annexinV binding assay, which also incorporates PI, reflected necrotic cells aswell. To understand the nature of cell death in hyperglycemia, thedouble-labeling technique using annexin V/PI to distinguish betweenapoptotic andnecrotic cellswasutilized. Flow cytometric data (Fig. 10C)revealed that, in comparison with control untreated hepatocytes,


hyperglycemia increased the number of annexinV-staininghepatocytesbut there was very little PI binding, indicating that the majority of celldeath occurred via the apoptotic pathway. Treatment with AA showedfewer poptotic cells, indicating that AA treatment protected againstapoptotic cell death under hyperglycemic conditions.

Histological assessment

Histological assessments of various liver segments of the normaland experimental animals are presented in Fig. 11. Hepatic lobulararchitecture was clear and intact without any abnormalities in the

Fig. 8. (A)Study of the mitochondrial membrane potential by flow cytometry analysis and confocal microscopic analysis. (Top) Confocal images and (bottom) histogram plot. Cont,mitochondria isolated from the liver tissue of normal animals; AA, mitochondria isolated from the liver tissue of animals treated with AA; STZ, mitochondria isolated from the livertissue of diabetic animals; AA+STZ, mitochondria isolated from the liver tissue of animals treated with AA before diabetic induction; and STZ+AA, mitochondria isolated from theliver tissue of animals treated with AA after diabetic induction. The measurements were made six times. (B) Western blot analysis of cytochrome c in the mitochondrial fraction ofthe liver tissue of diabetic and AA-treated animals. The relative intensities of the bands were determined using NIH Image software and the control bandwas given an arbitrary valueof 1. Data represent the averages±SD of six separate experiments in each group. (C) Western blot analysis of Apaf-1 in the liver tissue of diabetic and AA-treated animals. Therelative intensities of the bands were determined using NIH Image software and the control band was given an arbitrary value of 1. Data represent the averages ± SD of six separateexperiments in each group. (D) Study on the concentration of cytosolic cytochrome c. The measurements were made six times. Data represent the averages ± SD of six separateexperiments in each group. (E, F) Western blot analysis of caspase-3 and cleaved caspase-3(E) and caspase-9 (F) in the liver tissue of diabetic and AA-treated animals. The relativeintensities of the bands were determined using NIH Image software and the control band was given an arbitrary value of 1. Data represent the averages ± SD of sixseparate experiments in each group. aPb0.05, significant difference between the normal control and the diabetic groups, bPb0.05, significant difference between the diabetic and theAA-pretreated groups (AA+STZ); and cPb0.05, significant difference between the diabetic and the AA-posttreated groups (STZ+AA). (G) Representative confocal microscopyimages. The activation of the apoptotic program was assessed by measuring fluorescence changes in caspase-3 (green fluorescence) and Apaf-1 (red fluorescence) activity.


Fig. 8 (continued).


Fig. 9. (A) Western blot analysis of PARP in the liver tissue of diabetic and AA-treated animals. The relative intensities of the bands were determined using NIH Image software andthe control band was given an arbitrary value of 1. Data represent the averages±SD of six separate experiments in each group. (B, C) Intracellular NAD (B) and ATP (C) levels weremeasured in the liver tissue of diabetic and AA-treated animals. Values are expressed as means ± SD for six animals in each group. Cont, levels in normal animals; AA, levels in theanimals treated with AA; STZ, levels in diabetic animals; AA+STZ, levels in the animals treated with AA before diabetes induction; and STZ+AA, levels in the animals treatedwith AA after diabetes induction. aPb0.05, significant difference between the normal control and the STZ-treated group; bPb0.05, significant difference between the STZ-treated andAA-pretreated group (AA+STZ); cPb0.05, significant difference between the STZ-treated and the AA-posttreated group.


liver section of control group. The most easily recognizable featureof the apoptotic cells is the condensed nuclear chromatin thatappears either as a darkly staining single nucleus or as a fragmentednucleus (several small round balls of dark blue/black-stainingcondensed chromatin). Hyperglycemia caused obvious swelling ofliver cells, chromatin condensation, and formation of apoptoticbodies. The apoptotic cells may also be seen within vacuoles (withan apparently empty space surrounding the apoptotic bodies withinthe vacuole) inside neighboring cells (macrophages in the germinalcenters). AA treatment prevented the nuclear chromatin condensa-tion, DNA fragmentation, and formation of apoptotic bodies andthus showed improvement in liver morphology. Immunohisto-chemical study with anti-caspase-3 antibody revealed the signifi-cant overexpression of caspase-3 in the liver tissue of the diabeticanimals and this observation clearly suggests that the STZ-inducedapoptotic cell death occurs via caspase-dependent pathways. Over-expression was less pronounced in the liver section of the othergroups of animals.

Discussion

This study demonstrates the prophylactic role of AA againsthyperglycemic hepatic complications. Destruction of pancreatic

β cells (by STZ exposure) causes a reduction in insulin secretion.Glycogenesis is thus inhibited and the blood glucose level goes up. Ingeneral, increased blood glucose levels, glycated hemoglobin (HbA1C),AGE formation, decreased insulin levels, and severe loss in bodyweight characterize STZ-induced diabetes. The results of this study areconsistent with these observations in diabetic animals. Treatmentwith AA, before as well as after the induction of diabetes, enhancedglycogenesis and prevented glucose accumulation in blood as well asglycation of hemoglobin (HbA1C). The weight loss detected in thediabetic animals is most likely due to the induction of insulitis and/orthe lack of insulin. Oxidative stress is suggested to play a central rolein the pathogenesis and progression of diabetes and its associatedcomplications [1]. STZ itself is an NO donor. During intracellularmetabolization, STZ liberates NO, which, in combination with ROS,produces many reactive intermediates, causing increased expressionof proinflammatory cytokines [45]. In our study, we also observed thatintracellular glucose overload increased the production of NO, ROS,and peroxynitrite and overexpression of iNOS. Being an antioxidant,AA could scavenge the ROS aswell as RNS and thereby prevent hepaticoxidative stress under hyperglycemia. Both oxidative and nitrosativestress, developed in hyperglycemia, altered enzymatic and non-enzymatic antioxidant defenses. Among the various antioxidantmolecules, SOD and CAT mutually function as important enzymes in

Fig. 10. (A) DNA fragmentation on agarose/ethidium bromide gel. DNA isolated from experimental liver tissues was loaded onto 1% (w/v) agarose gels. Lanes 1, marker (1 kb DNAladder); 2, DNA isolated from normal liver; 3, DNA isolated from the liver tissue of animals treated with AA; 4, DNA isolated from the liver tissue of the diabetic animals (arrowindicates the DNA laddering); 5, DNA isolated from the liver tissue of the animals treated with AA before diabetic induction; and 6, DNA isolated from the liver tissue of the animalstreated with AA after diabetic induction. (B) Confocal imaging of DAPI and PI double staining. Apoptotic and necrotic cells were imaged by using DAPI (blue) and PI (red) asfluorescent probes. Cont, cells isolated from the liver tissue of normal animals; AA, cells isolated from the liver tissue of animals treated with AA; STZ, cells isolated from the livertissue of diabetic animals; AA+STZ, cells isolated from the liver tissue of animals treated with AA before diabetic induction; and STZ+AA, cells isolated from the liver tissue of theanimals treated with AA after diabetic induction. (C) Effects of STZ and AA on percentage distribution of apoptotic and necrotic cells. Cell distribution was analyzed using annexinV binding and PI uptake. The FITC and PI fluorescence was measured using a flow cytometer with FL-1 and FL-2 filters, respectively. Results are expressed as a dot plot representingone of six independent experiments.


Fig. 11. (A) Histopathological changes in liver tissue (stained with hematoxylin and eosin dye). Cont, liver section of normal rat (original magnification ×200); AA, liver section fromanimal treated with AA; STZ, liver section from the diabetic group, arrows indicate the apoptotic changes (original magnification ×200); AA+STZ, liver from an animal treated withAA before diabetic induction (original magnification ×200) showing almost normalmorphology; and STZ+AA, liver from an animal treatedwith AA after diabetic induction (originalmagnification×200) showing normalmorphology. (B) Immunohistochemistrywith antibodies for caspase-3. Cont, liver section of normal rat (originalmagnification ×100); AA, liversection from an animal treated with AA; STZ, liver section from the diabetic group, showing increased expression of caspase-3 marked with brown or black spots (originalmagnification ×100); AA+STZ, liver from an animal treatedwith AA before diabetic induction (originalmagnification ×100) showingminimumcaspase-3 expression; and STZ+AA,liver from an animal treated with AA after diabetic induction (original magnification ×100) showing less caspase-3 activation.


the elimination of ROS. On the other hand, GSH maintains the intra-cellular redox status against pro-oxidative stress by detoxifyingvarious xenobiotics as well as by scavenging free radicals. GST andGPx also utilize GSH during their reactions. Decreases in GSH contentdue to organ pathophysiology simultaneously decrease the activitiesof GST and GPx, with a concomitant decrease in the activity of theGSH-regenerating enzyme, GR. In this context, we assayed the activ-ities of the antioxidant enzymes SOD, CAT, GST, GR, GPx, and G6PDand the levels of the nonenzymatic antioxidant molecule GSH and itsmetabolite GSSG. Production of increased free radicals caused theoxidation of DNA molecules. We observed that intracellular glucoseburden decreased the activities of the antioxidant enzymes andthe GSH/GSSG as well as 8-OHdG/2-dG ratio, along with increasedlipid peroxidation and protein carbonylation. Treatment with AAaugmented the intracellular antioxidant defense.

In addition to hyperglycemia, type 1 diabetic patients alsofrequently experience hyperketonemia (ketosis) because duringinsulin deficiency the fat supplies the body with fuel [46,47]. Earlierinvestigations [48–50] reported that ketosis increases ROS produc-tion and causes oxidative stress as well as cellular dysfunction.However, the mechanism by which the ketone bodies (acetoacetate,β-hydroxybutyrate, and acetone) generate ROS is not clear. Thus, intype 1 diabetes, both hyperglycemia and hyperketonemia causeoxidative stress. Recent studies reported that diabetic hyperketo-nemia also coexists with hyperglycemia among older type 2 diabeticpatients [51]. In our study, the earlier experimentally establishedradical scavenging (both in vivo and in vitro) and antioxidantproperty of AA might be helpful in preventing the hyperketonemia-induced oxidative stress in type 1 diabetes [16–18].

Oxidative stress causes alterations in gene expression possiblythrough posttranslational modification of redox-sensitive transcrip-tion factors, NF-κB, and activator protein-1. NF-κB is a transcriptionfactor implicated in pathological responses that develop duringdiabetes mellitus. In hyperglycemia, early activation of NF-κB causesregulation of a number of genes leading to inflammatory, fibrogenic,and carcinogenic responses [52]. Cumulative evidence suggests thatNF-κB-dependent NO production plays a major role in STZ-induceddiabetes and its associated complications. The NF-κB transcriptionalfactors are composed of homodimers or heterodimers of Rel protein,of which p65/p50 is the predominant complex. In this study weobserved a significantly increased phosphorylation of the p65 subunitof NF-κB in the hyperglycemic liver tissue. This observation is sup-ported by the fact that phosphorylation at multiple serine sites of thep65 subunit increases the transcriptional activity of NF-κB in thenucleus [53]. In the cytoplasm, NF-κB is present in an inactive formand is linked with its inhibitor IκB through the p65 subunit. Tran-scriptional activity of NF-κB is thus regulated by the phosphorylationof IκB as well as its p65 subunit. Exposure to various extracel-lular signals leads to the phosphorylation and degradation of IκB. FreeNF-κB dimers are then rapidly translocated to the nucleus, whereinthey bind to the DNA and activate transcription of target genes. Inthis study, we observed that NF-κB activation (p65 subunit) (in thenuclear extract) under hyperglycemic conditions is linked with thesimultaneous increased phosphorylation of IκBα in the cytosol.Treatment with AA, however, could reduce hyperglycemia-inducedNF-κB-mediated hepatic oxidative disorders.

MAPKs are a family of serine/threonine kinases that are activatedby dual phosphorylation on threonine and tyrosine residues. The

Fig. 12. (A, B) Probable mechanisms for the free radical scavenging activity of AA and its protection activity by functional group modification. (C) Schematic diagram of the STZ-induced hyperglycemic liver damage and its prevention by AA.



MAPKs play a pivotal role in transducing extracellular stimuli intointracellular posttranslational and transcriptional responses. All threegroups of MAPKs are activated by osmotic perturbations derived fromglucose itself or by glucose-induced oxidative stress and by AGEs viathe receptors for AGEs [54]. MAPK signaling has been implicated inthe activation of NF-κB via the phosphorylation of its inhibitor, IκBα.In agreement with the earlier investigations we also observed thathyperglycemia caused the activation of phosphorylated ERK as well asphosphorylated p38, leaving the activation of total MAPKs unchanged.Treatment of the animals with AA prevented the activation of phos-phorylated MAPKs and thus prevented the cellular damage via theMAPK signaling pathways.

Apoptosis is known to be a delicately controlled programmed celldeath pathway. Several lines of earlier investigation suggest that thechange in mitochondrial transmembrane potential is able to switchthe committed cells to apoptotic death with oxidative stress as themediator [55]. Verzola et al. [56] reported that Bcl-2 family proteinsact on the mitochondria to regulate mitochondria-dependent celldeath. There are two classes of regulatory proteins in the Bcl-2 familythat confer opposite effects on apoptosis: the antiapoptotic members(e.g., Bcl-2, Bcl-xL) protect cells against some forms of apoptosis,whereas the proapoptotic members (e.g., Bax, Bad) promote prog-rammed cell death. The proapoptotic members of the Bcl-2 familyphysically interact to form oligomers that can move onto the mito-chondrial membrane and release cytochrome c from mitochondria tothe cytosol. In the cytosol, Apaf-1, procaspase-9, and released cyto-chrome c interact to form the apoptosome that triggers the activationof caspase-3. To confirm an antiapoptotic role for AA against hyper-glycemia, we determined the mitochondrial membrane potential andstudied the expression of Bcl-2 family proteins, cytochrome c, Apaf-1,caspase-9, and caspase-3. In our study, we observed that hypergly-cemia upregulated proapoptotic (Bad) and downregulated antiapop-totic (Bcl-2) proteins, causing a reduction in the mitochondrialmembrane potential and release of cytochrome c followed by a de-creased level of Apaf-1 in the cytosol. Hyperglycemia-induced loss inmitochondrial membrane potential and formation of apoptosomessubsequently activated cell death executor caspases (caspase-3 andcaspase-9). The immunofluorescence study using anti-caspase-3 andanti-Apaf-1 also supports the immunoblot analysis. Treatment withAA could attenuate apoptotic cell death by regulating the activationof Bcl-2 family proteins and their effects on the mitochondria-dependent cell death pathway.

DNA strand breaks and overactivation of PARP are the primemodes of STZ-induced cell death [57]. It is also known that antioxidantsupplements such as metallothionein and poly(ADP-ribose) poly-merase inhibitors have resulted in prevention of β-cell destruction invitro and in vivo [57,58]. Our investigation on PARP signaling path-ways and DNA fragmentation studies revealed that treatment withAA protected against STZ-induced DNA strand breaks, indicating thatAA-induced protection occurs upstream of the DNA damage. Hence, itis likely that AA treatment protected the liver tissue from the adverseeffects of diabetes via the prevention of STZ-induced overactivation ofPARP.

Diabetic liver injury is also supported by the histological assess-ments. Intracellular glucose burden increased the nuclear chromatincondensation (dark blue or black staining) around the edge of thenucleus. AA treatment prevented the formation of apoptotic bodiesand thereby protected the organ from diabetes and its associatedcomplications.

In this study, oxidative stress-responsive cell signaling in hyper-glycemia caused hepatic cell death mainly via the apoptotic pathway.In agreement with the cell-signaling studies, we also observed that indiabetic liver tissue, tiny DNA fragments appeared in the agarose gel.Studies under the confocal microscope (using DAPI and PI doublestaining) revealed that hyperglycemia increased chromatin conden-sation and the numbers of fragmented nuclei. Finally FACS analysis

with annexin V/PI double staining showed that hyperglycemiaincreased apoptotic cell death. AA treatment, however, could preventthe chromatin condensation, DNA fragmentation, and apoptotic celldeath. STZ-induced apoptotic cell death was also investigated byimmunohistochemical studies using anti-caspase-3 antibody. Weobserved that STZ-induced diabetic exposure caused overexpressionof caspase-3 and treatment with AA lessened the diabetic liverdamage by reducing its overactivation.

Our preliminary studies revealed that blocking the carboxylichydrogen of AA with an alkyl group reduced the free radical scav-enging property of AA in both cell-free and cellular systems (datanot shown). Again, blocking the primary as well as the secondaryhydroxyl group also reduced the free radical scavenging property ofAA (data not shown). These functional groups thus play importantroles in the radical scavenging property of AA and also are consideredto be responsible for the preventive action of AA against STZ-inducedhyperglycemic liver disease. However, when AA was administeredafter the diabetic induction it also protected the liver from diabeticdamage by reducing the overactivation of PARP, reducing DNA frag-mentation, and augmenting intracellular NAD and ATP levels (Fig. 12).

In conclusion, the results of our study suggest that hyperglycemiacaused liver dysfunction as well as hepatic cell death via oxidativestress-induced cell signaling pathways including signals from themitochondria. Treatment with AA, on the other hand, could preventthe development of diabetic liver dysfunction and cell death. Inaddition, the present research status of AA could imply that the anti-oxidant and antiglycation properties of this molecule make it acandidate for treatment of diabetes and its associated complications.

Acknowledgment

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

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