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Alcohol 47 (2013) 131e139

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Alcohol

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Diosmin protects against ethanol-induced hepatic injury via alleviationof inflammation and regulation of TNF-a and NF-kB activation

Mir Tahir, Muneeb U. Rehman, Abdul Lateef, Rehan Khan, Abdul Quaiyoom Khan, Wajhul Qamar,Farrah Ali, Oday O’Hamiza, Sarwat Sultana*

Molecular Carcinogenesis and Chemoprevention Division, Department of Medical Elementology and Toxicology, Faculty of Science, Jamia Hamdard (Hamdard University), HamdardNagar, New Delhi 110062, India

a r t i c l e i n f o

Article history:Received 11 April 2012Received in revised form17 December 2012Accepted 20 December 2012

Keywords:EthanolCytochrome P450 2E1Alcohol dehydrogenaseTumor necrosis factor-alphaCyclooxygenase-2

* Corresponding author. Department of ToxicologyNagar, New Delhi 110062, India. Tel.: þ91 11 2605fax: þ91 11 26059663.

E-mail address: sarwat786@rediffmail.com (S. Sult

0741-8329/$ e see front matter � 2013 Elsevier Inc. Ahttp://dx.doi.org/10.1016/j.alcohol.2012.12.010

a b s t r a c t

The present investigation was designed to evaluate the efficacy of diosmin against ethanol-inducedhepatotoxicity in rats by modulating various mechanisms including ethanol metabolizing enzymes,generation of free radicals, imbalance in oxidanteantioxidant status, oxidative damage to membranelipids, activation of transcription factors and elevation in inflammatory markers involved in ethanol-induced hepatic damage. Diosmin is a flavone glycoside, having anti-inflammatory and anti-cancerproperties. Thirty female Wistar rats segregated in five groups, each with six animals. Group I ascontrol followed by Group II, III and IV were treated with ethanol for 28 days. While groups III and IVwere administered with diosmin at 10 mg/kg b wt (D1) and 20 mg/kg b wt (D2) respectively prior toethanol administration. Group V was given only higher dose of diosmin. In ethanol-treated group,ethanol metabolizing enzymes viz., CYP 450 2E1 and alcohol dehydrogenase (ADH) significantlyincreased by 77.82% and 32.32% in liver tissues respectively as compared with control group and thisenhancement is significantly normalized with diosmin administration. Diosmin administration (D1 &D2) significantly (p < 0.001) attenuates oxidative stress markers i.e., LPO, GSH, GPx, GR and XO by 90.77& 137.55%, 17.18 & 25%, 37.3 & 49.86%, 21.63 & 44.9% and 56.14 &77.19% respectively. Serum ALT, AST andLDH significantly increased by 102.03, 116.91 and 45.20% in ethanol-treated group as compared withcontrol group. Group III and IV animals showed significant reduction in the serum toxicity markers.Diosmin further alleviated ethanol-induced NF-kB activation, enhanced expression of TNF-a, COX-2 andiNOS. Findings from the present study permit us to conclude that diosmin alleviates alcoholic liver injuryvia modulating ethanol metabolizing pathway, inhibition of oxidative stress markers and suppression ofinflammatory markers. This may represent a novel protective strategy against ethanol-induced liverdiseases.

� 2013 Elsevier Inc. All rights reserved.

Introduction

Alcoholic liver diseases are the most challenging current healthproblems worldwide. Over the period of time, alcohol (ethanol) hasevolved as one of the socially accepted addictive drugs worldwide(Guo & Ren, 2010). Ethanol consumption can lead to various alco-holic liver diseases ranging from steatohepatitis, cirrhosis (Lucey &Weinrieb, 2009), heart disease (George & Figueredo, 2010), Alz-heimer’s disease (Marinho, Laks, Engelhardt, & Conn, 2006), stroke(Ohkubo, Metoki, & Imai, 2009), liver disease (Cederbaum, Lu, &Wu, 2009), respiratory disease (Morris, 1990), diabetes mellitus

, Jamia Hamdard, Hamdard4685x5565/5566/26089688;

ana).

ll rights reserved.

(Baliunas et al., 2009), bone disease (Chen, Cui, Liao, & Huang, 2009)to cancer (Seitz & Becker, 2007), which may develop followingchronic alcohol ingestion and contribute to the alcoholism-relatedhigh morbidity and mortality.

Ethanol as such is a mild toxicant and its toxicity primarilydepends upon its metabolism. Liver is the primary target forethanol toxicity as it is the main organ for ethanol metabolism(Lieber, 1988). Besides liver, other organs (Kidney, brain, and lungs)may also contribute to ethanol metabolism (Guidot & Roman,2002).

In liver, ethanol is metabolized to highly toxic metabolite e

acetaldehyde that interacts with the cellular macromolecules(lipids and proteins) and leads to the damage of membrane lipidsbesides altering the enzyme activities (Niemelä, 1999). Three mainenzyme systems are involved in ethanol metabolism (Salaspuro,1999): alcohol dehydrogenase (ADH), Cytochrome P450 2E1 (CYP

M. Tahir et al. / Alcohol 47 (2013) 131e139132

2E1) and catalase. Alcohol dehydrogenase leads to the formation ofacetaldehyde from ethanol with the simultaneous reduction ofNADþ to NADHwhich in turn elevates xanthine oxidase activity andleads to the enhanced production of superoxide (Lieber, 2005).Microsomal ethanol oxidizing system involves the participation ofCYP 450 2E1 (Lieber, 2004). This system connects ethanol andnicotinamide adenine dinucleotide phosphate oxidation to thereduction of molecular oxygen to H2O2. In the third system, theoxidation of ethanol molecule into acetaldehyde is concurrent withthe instantaneous decomposition of hydrogen peroxide in a cata-lase catalyzed reaction (Bradford, Seed, Handler, Forman, &Thurman, 1993).

Ethanol metabolism is associated with free radical generationleading to oxidative stress (Cederbaum, 2001; Lu & Cederbaum,2008). It has been previously demonstrated that oxidative stressplays a crucial role in the pathogenesis of ethanol-induced liverdamage. Ethanol-induced oxidative stress is associated with thegeneration of reactive oxygen species (ROS) and reactive nitrogenintermediates (RNI) that leads to an imbalance in the pro-oxidantand antioxidant levels in the tissues (Arteel, 2003). Acetaldehyde,produced from the oxidative metabolite of ethanol leads to theperoxidative damage to membrane lipids leading to cellulardamage. Moreover, acetaldehyde has the ability to interact withthe cellular proteins such as enzymes, microsomal proteins andmicrotubules leading to the impairment of normal protein func-tions (Rintala et al., 2000), that has been proposed to play a keyrole in alcoholic liver diseases. Furthermore acetaldehyde caninteract with DNA to form carcinogenic DNA adducts such as N2-ethyl-20-deoxyguanosine (Brooks & Theruvathu, 2005). Patho-genesis of ethanol-induced liver diseases has not only contributedto oxidative stress, abnormal methionine metabolism, malnutri-tion and activation of Kupffer cells due to ethanol-induced endo-toxin production, but it has been recently found thatlipopolysaccharide (LPS) signaling, innate immunity, transcriptionfactors, alteration in epigenetic features, microRNA’s (miRNAs)and stem cells could also contribute to the ethanol-induced liverdiseases (Gao & Bataller, 2011). Ethanol toxicity is associated withthe induction of signaling cascade leading to the activation oftranscription of NF-kB that results in the expression of inflam-matory mediators including cytokines (TNF-a, IL-6, and IL-12)(Iimuro, Gallucci, Luster, Kono, & Thurman, 1997) chemokines,lipid mediators, inducible nitric oxide synthase (iNOS) andcyclooxygenase-2 (COX-2). TNF-a further initiates the productionof reactive oxygen species (ROS) and reactive nitrogen interme-diates (RNI’s) causing liver damage due to oxidative stress (Barnes& Karin, 1997; Nanji et al., 1999).

Diosmin (Diosmetin-7-O-rutinoside), a naturally occurringflavones glycoside readily obtained by dehydrogenation ofhesperidin, found abundantly in the pericarp of various citrus(Campanero, Escolar, Garcia-Quetglas, Sadaba, & Azanza, 2010).Diosmin has various biological activities including antioxidantactivity (Cotelle et al., 1996), anti-inflammatory effect (Crespo,Gálvez, Cruz, Ocete, & Zarzuelo, 1999), anti-diabetic effect(Manuel, Keenoy, Vertommen, & De Leeuw, 1999) and anti-proliferative and anti-cancer activities (Tanaka et al., 1997). More-over, diosmin has been found to increase the venous tone,improves lymphatic drainage and reduces the capillary hyper-permeability, thereby, leading to reduction in the release ofinflammatory mediators (Lyseng-Williamson & Perry, 2003).Keeping in view the above biological activities of diosmin, thepresent study was designed to make an attempt to evaluate thepreventive efficacy of diosmin against ethanol-induced hepaticdamage in Wistar rats by modulating ethanol metabolizingenzymes (CYP 2E1, ADH and Catalase), inflammatory cytokines(TNF-a, NF-kB, COX-2 and iNOS).

Materials and methods

Chemicals

Glutathione reductase, oxidized (GSSG) and reduced gluta-thione (GSH), 1,2-dithio-bis-nitrobenzoic acid (DTNB), 1-chloro-2,4-dinitrobenzene (CDNB), bovine serum albumin (BSA), oxidizedand reduced nicotinamide adenine dinucleotide phosphate(NADP), (NADPH), diosmin, flavine adenine dinucleotide(FAD), 2,6-dichlorophenolindophenol, thiobarbituric acid (TBA),xanthine, ascorbic acid, ethylene diamine tetra acetate (EDTA)and glycine were obtained from SigmaeAldrich, USA. Ferricchloride, sodium chloride, triseHCl, ethanol, disodium hydrogenphosphate, sodium dihydrogen phosphate, hydrogen peroxide,sodium hydroxide, sulphosalicylic acid, sulfuric acid, sodiumazide were purchased from Merck India Ltd. Trichloroacetic acid(TCA) and perchloric acid (PCA) etc were purchased from CDH,India. p-Nitrophenol has been purchased from Thomas BakerChemicals Ltd. 4-nitrocatechol has been purchased from Lobachemicals.

Animals

Female Wistar rats (150e200 g), 6e8 weeks old, were obtainedfrom the Central Animal House Facility of Hamdard University. Ratswere housed in an animal care facility under room temperature(25�1 �C) with 12 h light/dark cycles and were given free access tostandard pellet diet and tap water. Before the treatment, rats wereleft for 7 days to acclimatize. Animals received humane care inaccordancewith the guide lines of the Committee for the Purpose ofControl and Supervision of Experiments on Animals (CPCSEA),Government of India, and prior permission was sought from theInstitutional Animal Ethics Committee (IAEC No: 173/CPCSEA, 28January 2000).

Experimental procedure

In the present study, we have evaluated the preventive efficacyof diosmin against ethanol-induced hepatic toxicity. Thirty femaleWistar rats divided into five groups, each with six animals. Group Ias control receive vehicle (distilled water) only, followed by GroupII, III and IV were treated orally with sequential (per week)increased dose of ethanol 25%v/v (5, 8,10 and 12 g/kg bwt per weekin each group) for 28 days. While group III and IV were adminis-tered with diosmin orally at 10 mg/kg b wt (D1) and 20 mg/kg b wt(D2) respectively 1 h prior to ethanol treatment. Group V was givenonly diosmin (20 mg/kg b wt) (D2).

After 28 days, rats were sacrificed by cervical dislocation undermild anesthesia and blood has been taken by cardiac puncture forvarious serological parameters. Liver samples were taken at thesame time for various biochemical parameters.

Preparation of post mitochondrial supernatant (PMS), cytosolic andmicrosomal fractions

Livers were removed and cleaned with ice-cold saline (0.85%sodium chloride). A 10% homogenate of liver tissues were ob-tained in a buffer solution containing 10 mM triseHCl, 250 mMsucrose pH 7.4 using a Potter Elvehjen homogenizer and werecentrifuged at 3000 rpm for 10 min by Eltek Refrigerated Centri-fuge (model RC 4100 D) to separate the nuclear debris. The aliquotso obtained was centrifuged at 12,000 rpm for 20 min to obtainpost mitochondrial supernatant (PMS), which was used asa source of various enzymes. The supernatant obtained wasfurther ultra-centrifuged at 34,000 rpm for 1 h to obtain cytosolic

M. Tahir et al. / Alcohol 47 (2013) 131e139 133

fraction for ADH activity. The precipitate obtained was washedwith homogenizing buffer to obtain the microsomal fraction forCYP 2E1 activity. All the experimental manipulations were carriedout at 4 �C.

Cytochrome P450 2E1 (CYP 2E1) activity

The catalytic activity of CYP 2E1 was analyzed by measuringp-nitrophenol hydroxylation (PNPH) as described by Reinke andMoyer (1985). The reaction mixtures contained a 100 mM potas-sium phosphate buffer (pH 6.8), 1.0 mM ascorbic acid, 1 mMNADPH, 1 mg hepatic microsomes, and 100 mM p-nitrophenol ina total volume of 1.0 ml. The 4-nitrocatechol that was formed wasdetermined spectro-photometrically at 511 nm. Data wereexpressed as nmol/mg/min.

Alcohol dehydrogenase (ADH) activity

ADH activity was determined by the method of Bonnichsen andBrink (1955). Briefly, ADH activity was measured in 50 mM glycine,pH 9.6, 0.8 mMNAD, 3 mM ethanol and 50 ml of cytosolic fraction ina final volume of 1 ml. Enzyme activity was measured at 340 nmand the activity was calculated as nmol NADH formed/min/mg protein using a molar extinction coefficient of 6.22 �106 M�1 cm�1.

Estimation of lipid peroxidation (LPO)

The assay of lipid peroxidation was done according to themethod of Wright, Colby, and Miles (1981). The reaction mixtureconsisted of 0.58 ml phosphate buffer (0.1 M, pH 7.4), 0.2 mlmicrosome, 0.2 ml ascorbic acid (100 mM) and 0.02 ml ferricchloride (100 mM) in a total of 1 ml. This reaction mixture wasthen incubated at 37 �C in a shaking water bath for 1 h. Thereaction was stopped by the addition of 1 ml of TCA (10%).Following addition of 1.0 ml TBA (0.67%), all the tubes were placedin a boiling water bath for a period of 20 min. The tubes wereshifted to ice bath and then centrifuged at 2500 � g for 10 min. Theamount of malondialdehyde (MDA) formed in each of the sampleswas assessed by measuring the optical density of the supernatantat 535 nm. The results were expressed as the nmol MDA formed/h/g tissue at 37 �C by using a molar extinction coefficient of1.56 � 105 M�1 cm�1.

Assay for xanthine oxidase activity

The activity of xanthine oxidase was assayed by the method ofAthar et al. (1996). The reaction mixture consisted of 0.2 ml PMSwhich was incubated for 5 min at 37 �C with 0.8 ml phosphatebuffer (0.1 M, pH 7.4). The reaction was started by adding 0.1 mlxanthine (9 mM) and kept at 37 �C for 20 min. The reactionwas terminated by the addition of 0.5 ml ice-cold perchloricacid (10% v/v). After 10 min, 2.4 ml of distilled water wasadded and centrifuged at 4000 rpm for 10 min and mg uric acidformed/min/mg protein was recorded at 290 nm.

Assay for catalase activity

The Catalase activity was done by the method of Claiborne(1985). In short the reaction mixture comprised of 0.05 mlPMS, 1.0 ml hydrogen peroxide (0.019 M), 1.95 ml phosphatebuffer (0.1 M, pH 7.4), in a total volume of 3 ml. Changes inabsorbance were recorded at 240 nm and the change in absor-bance was calculated as nmol H2O2 consumed per min per mgprotein.

Estimation of reduced glutathione

Reduced GSH was assessed by the method of Jollow, Mitchell,Zampaglione, and Gillette (1974). 1.0 ml of 10% (PMS) mixed with1.0 ml of (4%) sulphosalicylic acid, Then incubated at 4 �C fora minimum time period of 1 h and then centrifuged at 4 �C at1200 � g for 15 min. Briefly reaction mixture has 0.4 ml superna-tant, 2.2ml phosphate buffer (0.1M, pH 7.4) and 0.4ml DTNB (4mg/ml) in a total volume of 3.0ml. The yellow color developed was readimmediately at 412 nm on spectrophotometer (Perkin Elmer,lambda EZ201). The reduced glutathione concentration was calcu-lated as nmol GSH conjugates/g tissue.

Assay for glutathione peroxidase activity

The activity of glutathione peroxidase was calculated by themethod of Mohandas, Marshall, Duggin, Horvath, and Tiller (1984).A total of 2 ml volume consisted of 0.1 ml EDTA (1 mM), 0.1 mlsodium azide (1 mM), 1.44 ml phosphate buffer (0.1 M, pH 7.4),0.05 ml glutathione reductase (1 IU/ml), 0.05 ml reduced gluta-thione (1 mM), 0.1 ml NADPH (0.2 mM) and 0.01 ml H2O2(0.25 mM) and 0.1 ml 10% PMS. The depletion of NADPH at 340 nmwas recorded at 25 �C. Activity of the enzyme was calculated asnmol NADPH oxidized/min/mg protein with the molar extinctioncoefficient of 6.22 � 103 M�1 cm�1.

Assay for glutathione reductase activity

Glutathione reductase activity was measured by the method ofCarlberg and Mannervik (1975). The reaction mixture consisted of1.65 ml phosphate buffer (0.1 M, pH 7.6), 0.1 ml NADPH (0.1 mM),0.05 ml oxidized glutathione (1 mM), 0.1 ml EDTA (0.5 mM) and0.1 ml 10% PMS in a total volume of 2 ml. Enzyme activity wasassessed at 25 �C by measuring disappearance of NADPH at 340 nmand was calculated as nmol NADPH oxidized/min mg protein usingmolar extinction coefficient of 6.22 � 103 M�1 cm�1.

Assay for serum aspartate aminotransferase and alanineaminotransferase (AST & ALT) activity

AST and ALT activity were determined by themethod of Reitmanand Frankel (1957). Each substrate (0.5 ml) (2 mM a-ketoglutarateand either 200 mM L-alanine or L-aspartate) was incubated for5 min at 37 �C in a water bath. Serum (0.1 ml) was then added andthe volume was adjusted to 1.0 ml with 0.1 M, pH 7.4 phosphatebuffer. The reaction mixture was incubated for exactly 30 and60 min at 37 �C for ALT and AST, respectively. Then to the reactionmixture, 0.5 ml of 1 mM DNPH was added, after another 30 min atroom temperature, the color was developed by addition of 5.0 ml ofNaOH (0.4 N) and the product read at 505 nm.

Assay for lactate dehydrogenase (LDH) activity

LDH activity has been estimated in serum by the method ofKorenberg (1955). The assay mixture consisted of serum (0.2 ml),(0.1 ml) 0.02 M NADH, (0.1 ml) 0.01 M sodium pyruvate, (1.1 ml)0.1 M, pH 7.4 phosphate buffer and distilled water in a total volumeof 3 ml. Enzyme activity was recorded at 340 nm and activity wascalculated as nmol NADH oxidized/min/mg protein.

Assay for tumor necrosis factor-alpha (TNF-a)

TNF-a protein level was measured by enzyme linked immuno-sorbent assay (ELISA) kit (eBioscience: Inc., San Diego., USA).Samples were prepared in phosphate buffer saline (1� PBS)

Fig. 1. Average increase in body weight (g) per week. Representative graph shows theaverage increase in the body weight per week of the rats fed on different treatmentregimen as explained in the Materials and methods section. Only the rats of group IIshowed the slight depletion in the body weight of the rats. Each value representsmean. n ¼ 6.

Fig. 3. CYP 2E1 activity in liver. Representative figure showed the ethanol metabolizingenzyme (CYP 2E1) activity. It has been found that ethanol administration significantlyelevates CYP 2E1 activity (Ethanol group). Diosmin administration at both dosessignificantly restores the ethanol-induced CYP 2E1 activity. Each value representsmean � S.E., n ¼ 6. #p < 0.001 compared with the corresponding value for controlgroup. **p < 0.01 and ***p < 0.001 compared with the corresponding value for ethanol-treated group.

M. Tahir et al. / Alcohol 47 (2013) 131e139134

containing protease inhibitor cocktail. Analysis was performedaccording to the manufacturer’s instruction. The results wereexpressed as pg/mg tissue protein.

Immunohistochemical staining of NF-kB, iNOS and COX-2

The liver tissues were fixed in formalin and embedded inparaffin. Sections of 5 mm thickness were cut onto poly-lysinecoated glass slides. Sections were deparaffinize three times(5 min) in xylene followed by dehydration in graded ethanol andfinally rehydrated in running tap water. For antigen retrieval,sections were boiled in 10 mM citrate buffer (pH 6.0) for 5e7 min.Sections were incubated with hydrogen peroxide for 15 min tominimize non-specific staining and then rinsed three times (5 mineach) with 1� PBST (0.05% Tween-20). Blocking solution wasapplied for 10 min then sections were incubated with diluted(1:100 for NF-kB and COX-2; 1:500 for iNOS) primary antibodies,purified rabbit polyclonal anti-NF-kB antibody (BioLegend), rabbitpolyclonal anti-iNOS antibody (Enzo Life Sciences) and rabbitpolyclonal anti-COX-2 antibody (Bio Vision), overnight at 4 �Cin humid chamber. Further processing was done according tothe instructions of Ultra Vision plus Detection System Anti-Polyvalent, HRP/DAB (Ready-To-Use) staining kit (Thermo scien-tific system). The peroxidase complex was visualized with 3,30-diaminobenzidine (DAB). Lastly the slides were counterstainedwith hematoxylin, cleaned in xylene, dehydrated with ethanol and

Fig. 2. Percent liver body weight ratio. Representative figure shows the relative liverweight of the rats. A slight depletion in the relative liver weight has been observed inthe rats fed on ethanol (group II). No alteration in relative liver weights has beenobserved in diosmin administration to rats (Group III and IV) showed n ¼ 6.

after DPX mounting microscopic (BX 51 Olympus) analysis wasdone at 400� magnification.

Histological investigation

For histopathology study, the liver was removed and immedi-ately fixed in freshly prepared 10% neutral buffered formalin at 4 �C.Then, the skin was embedded in paraffin wax. A section of liver(5 mm thick) was cut and stained with hematoxylin and eosin (H &E). Inflammatory response around the central vein in terms ofinfiltration of inflammatory cells, vacuolar degeneration andpronounced necrosis around the central vein were observed as anindicator of histological changes with microscope (fluorescentmicroscope, Olympus) at least in six different regions.

Estimation of protein

The protein concentration in all samples was determined by themethod of Lowry, Rosebrough, Farr, and Randall (1951), usingbovine serum albumin (BSA) as standard.

Statistical analysis

Differences between groups were analyzed using analysis ofvariance (ANOVA) followed by Dunnet’s multiple comparisons test.

Fig. 4. ADH activity in liver tissue. Alcohol dehydrogenase (ethanol metabolizingenzyme) activity. Ethanol treatment showed enhanced ADH activity and diosminadministration at a dose of 20 mg/kg b wt (D2) restores this elevation in ADH activity.Each value represents mean � S.E., n ¼ 6. þþp < 0.01 compared with the corre-sponding value for control group. NS ¼ non-significant, *p < 0.05 compared with thecorresponding value for ethanol-treated group.

Fig. 5. Inhibition of LPO by diosmin in liver. Representative figure showed ameliorationof ethanol-induced hepatic lipid peroxidation by diosmin. Group II (Ethanol group)showed increased lipid peroxidation and groups administered with diosmin in addi-tion to ethanol showed depletion in lipid peroxidation. Each value representsmean � S.E., n ¼ 6. #p < 0.001 compared with the corresponding value for controlgroup. **p < 0.01 and ***p < 0.001 compared with the corresponding value for ethanol-treated group.

M. Tahir et al. / Alcohol 47 (2013) 131e139 135

All data points are presented as the treatment groupsmean � standard error of the mean (S.E.).

Results

Effect of diosmin on body weight and relative liver weight of rats

Figs. 1 and 2 shows respective body weight and relative organweight of rats treated with diosmin (10 and 20 mg/kg b wt) for 4weeks. Our results demonstrate that there was a slight decrease inthe body weight (per week) and liver body weight ratio of rats thatingested alcohol only compared to normal group. Diosmin admin-istration prevented ethanol-induced weight loss.

Effect of diosmin on ethanol metabolizing enzymes

CYP2E1 induction in hepatic tissues from ethanol-treated groupwas increased 77.82% compared to control animals (23.42 nmol/mg � 1.15 vs 13.17 nmol/mg � 0.68, p < 0.001). Treatment withdiosminatdoseD1andD2significantlybroughtback the level of CYP2E1 by 44.79% (p< 0.01) and 68.33% (p< 0.001) respectively (Fig. 3).

In ethanol-treated groups (Fig. 4), liver tissue showed significant(p< 0.01) enhancement in ADH activities (9.58� 1.02) as comparedwith control group (7.24 � 1.24). Only high dose of diosmin (D2)significantly suppressed the ethanol-induced ADH activity by31.35%.

Effect of diosmin on xanthine oxidase activity

Hepatic xanthine oxidase (XO) activity reflected a significantincrease of 82.45% in ethanol-treated group (p < 0.001) as

Table 1Effect of diosmin administration on ethanol mediated depletion of hepatic reduced gluxanthine oxidase activity in rats.

Groups Reduced glutathione(mmol GSH conjugate/gtissue)

Glutathione peroxidase(nmol NADPH oxidized/min/mg protein)

Control 0.64 � 0.01 447.6 � 32.37Ethanol 0.28 � 0.02# (56.2%) 236.7 � 24.5# (47.11%)Diosmin D1 þ ethanol 0.39 � 0.02NS (17.2%) 403.8 � 16.3** (37.3%)Diosmin D2 þ Ethanol 0.44 � 0.02** (25%) 459.9 � 36.87*** (49.86%)Only diosmin D2 0.59 � 0.02 448.6 � 40.92

Each value represents mean � S.E., n ¼ 6.#p < 0.001 compared with the corresponding value for control group.*p < 0.05, **p < 0.01 and ***p < 0.001 compared with the corresponding value for ethanNS ¼ Non significant compared with the corresponding value for ethanol treated group.

compared with control group. Diosmin significantly restores thelevel of xanthine oxidase (XO) activity by 56.14 and 77.19% at doseD1 and D2 respectively. Only D2 group showed no significantchange as compared to control group.

Effect of diosmin on hepatic membrane damage (lipid peroxidation)

MDA formation was measured to demonstrate the oxidativedamage in ethanol-induced liver injury of Wistar rats. A significantamplification by 177.1% of ethanol-induced MDA formation wasfound as compared with control group (25.83 nmol/g � 1.15 vs9.32 nmol/g � 1.23, p < 0.001). We have observed that treatmentwith diosmin leads to the significant restoration of membraneintegrity by 90.77 and 137.55% at dose D1 (p < 0.01) and D2(p < 0.001) respectively (Fig. 5). Diosmin alone did not show anysignificant difference as compared to control.

Diosmin treatment restores the activities of hepatic antioxidants

Ethanol treatment was found to diminish hepatic antioxidantsGSH, GPx, GR and Catalase by 56.25%, 47.11%, 48.83% and 36.72% ascompared to corresponding control group (p< 0.001). Treatment ofdiosmin significantly increases the level of GSH, GPX, GR andCatalase in liver at dose D1 and D2 by 17.18 & 25%, 37.3 & 49.86%,21.63 & 44.90% and 32.65 & 40.51% respectively (Table 1), whichindicates antioxidant property of diosmin against ethanol-inducedoxidative stress.

Diosmin attenuates ethanol-induced hepatotoxicity

Ethanol-treated groups showed significant increase in theserum AST, ALT (Fig. 6) and LDH (Fig. 7) levels by 116.91% (p< 0.01),102.03% (p< 0.001) and 45.2% (p< 0.001) respectively as comparedwith the control group. Administration with diosmin was foundeffective in the normalization in these serum toxicity markers by44.18, 33.35 and 15.58% at dose D1 and 97.7, 81.1 and 38.32% at doseD2 respectively.

Restoration of TNF-a level by diosmin

Ethanol-treated groups showed significant (p < 0.001) elevationof 28.05% in hepatic TNF-a levels as compared with the controlgroup (307.22 � 12.89 vs 39.9 � 8.6), while diosmin administrationat dose D1 and D2 restores the elevated levels of TNF-a by 21.30%(256.1 � 10.89, p < 0.01) and 27.63% (240.9 � 9.15, p < 0.001)respectively (Fig. 8).

Expression of NF-kB, COX-2 and iNOS

Hepatic expressions of NF-kB, COX-2 and iNOS have beenshown in Figs. 9e11 respectively. In the ethanol-treated group,

tathione, glutathione peroxidase, glutathione reductase, catalase and elevation in

Glutathione reductase(nmol NADPH oxidized/min/mg protein)

Catalase (nmol H2O2

consumed/min/mgprotein)

Xanthine oxidase (mg ofuric acid formed/min/mgprotein)

465.9 � 18.8 73.8 � 4.3 0.57 � 0.02238.4 � 12.6# (48.83%) 46.7 � 2.4# (36.7%) 1.04 � 0.07# (82.45%)339.2 � 12.6** (21.63%) 70.8 � 4.2** (32.65%) 0.72 � 0.07** (56.14%)447.6 � 16.85***(44.9%) 76.6 � 4.2*** (40.5%) 0.60 � 0.38*** (77.19%)458.7 � 15.64 76.5 � 4.5 0.67 � 0.10

ol-treated group.

Fig. 6. Serum markers of liver toxicity AST and ALT. Representative figure showedelevation in ethanol-induced AST and ALT levels. Enhanced levels of AST and ALT arethe key serum markers of liver toxicity. Diosmin administration significantlyrestores the levels of ethanol-induced AST and ALT. Each value representsmean � S.E., n ¼ 6. #p < 0.001 compared with the corresponding value for controlgroup. **p < 0.01 and ***p < 0.001 compared with the corresponding value forethanol-treated group.

Fig. 8. Inhibition of TNF-a by diosmin in liver. Representative figure showed the effectof ethanol and diosmin treatment on TNF-a. Each value represents mean � S.E., n ¼ 6.#p < 0.001 compared with the corresponding value for control group. **p < 0.01 and***p < 0.001 compared with the corresponding value for ethanol-treated group.

M. Tahir et al. / Alcohol 47 (2013) 131e139136

there was higher number of cells showing expression of theseproteins as indicated by the brown stains. Expression of theseproteins (NF-kB, COX-2 and iNOS) is markedly suppressed in thediosmin-treated groups. For immunohistochemical analysis,brown color indicates specific immune staining of these proteinsand light blue color indicates hematoxylin staining (originalmagnification: 400�).

Histopathology of liver tissue

Analysis of tissue sections of animals from different treatmentgroups under microscope (400� magnification) revealed markedchangeswhen comparedwith control group animals (Fig.12). In theethanol-treated animals, there was an evident of vacuolar degen-eration and pronounced necrosis around the central vein. Moreoverethanol causes apparent inflammatory response around the centralvein in terms of infiltration of inflammatory cells (Fig. 12B) in livertissue. In contrast, diosmin administration at both the doses (10 and20 mg/kg b wt) protected the liver histology against ethanol-induced alterations (Fig. 12C and D). Only diosmin administration(20 mg/kg b wt) does not show any alterations from normal liverhistology (Fig. 12E).

Fig. 7. Effect of administration of diosmin on cytotoxicity marker LDH. Representativefigure showed the lactate dehydrogenase (LDH) activity in serum. LDH is an impor-tant cytotoxicity marker. Ethanol administration leads to the cellular toxicity andresulted in the release of LDH into the circulation. Hence estimation of LDH activity isindicator of cytotoxicity. Diosmin at both doses (D1 and D2) showed depleted LDHactivity as compared with the only ethanol-treated group. Each value representsmean � S.E., n ¼ 6. #p < 0.001 compared with the corresponding value for controlgroup.*p < 0.05, **p < 0.01 compared with the corresponding value for ethanol-treated group.

Discussion

Alcohol is one of the main causes of end-stage liver diseases.From our previous work (Tahir & Sultana, 2011), in fact it is clearthat sequential increase in ethanol dose (5e12 g/kg b wt) inducesmaximum tissue damage and also over comes the toleranceproduced by the ethanol consumed at the same dose. In the presentstudy, it has been observed, that the per week sequential increase inthe dose of ethanol administration, there is a reduction in the rateof body weight gain and relative liver weights in the animals fedwith ethanol only. This reduction in the rate of body weight gainand relative liver weight has also been reported by Aruna,Rukkumani, Varma, and Menon (2005). This decrease in bodyweight gain has been attributed to the reduction in the adiposetissue content due to ethanol consumption (Aruna et al., 2005). Incontrast, diosmin administration prevented the ethanol-inducedweight loss and relative liver weights significantly. The generationof oxygen metabolites such as superoxide (�O2

�), hydrogen peroxide(H2O2) and hydroxyl radical (�OH�) during ethanol metabolism isbelieved to be the main cause in the pathogenesis of alcoholic liverinjury (Zima et al., 2001). Increased generation of free radicalsresults in the loss of membrane integrity and function via lipidperoxidation. Oxidation of ethanol via ADH and CYP 2E1 leads tothe generation of NADH (Lieber, 2004); that in turn elevates thexanthine oxidase activity (Zima et al., 2001). Generation of NADH inthis pathway of ethanol oxidation is concomitant with the utiliza-tion of NADPH that suppresses the reduction of oxidized gluta-thione by glutathione reductase and subsequently depletes thereduced glutathione content, thereby depleting other glutathionedependent enzymes. Depletion of reduced glutathione cannot onlyimpair the cellular defense but also result in the enhanced ethanol-induced oxidative stress and oxidative injury (Hayes & Pulford,1995). In the present study, our findings have been in completeconformity with the above facts related to ethanol-induced hepatictoxicity. Our results also demonstrated that ethanol consumption isassociated with the depletion in glutathione and dependentenzymes leading to the imbalance in the oxidant: antioxidant statusin the cells and diosmin administration suppresses the activities ofethanol metabolizing enzymes (ADH, CYP 2E1), thereby reducingthe ethanol-induced hepatic toxicity.

Ethanol consumption is notably associated with hepatic damageand the prominent sign of hepatic injury is the leakage of cellularenzymes (AST, ALT and LDH) in to the serum (Sehrawat & Sultana,2006). Ethanol-treated rats show higher levels of these toxicitymarker enzymes, indicating increased membrane permeability,cellular damage and/or necrosis of hepatocytes (Baldi, Burra,

Fig. 9. Hepatic sections showing NF-kB expression by immunohistochemistry. Effect of diosmin on ethanol-induced NF-kB expression in rat liver. (A): liver sections of control rats.(B): showing hepatic sections of ethanol fed rats showing higher expression of NF-kB (arrows). (C & D): liver sections of diosmin-treated groups (D1 and D2 respectively). (E): onlydiosmin D2-treated group.

M. Tahir et al. / Alcohol 47 (2013) 131e139 137

Plebani, & Salvagnini, 1993; Tahir & Sultana, 2011). Diosminadministration to rats showed depression in these liver injurymarker enzymes, thus suggesting its cell membrane stabilizingproperty and hepatoprotective efficacy against ethanol-inducedliver damage.

Numerous reports have demonstrated that TNF-a plays a pivotalrole in the ethanol-induced liver pathology (Honchel et al., 1992; Ji,Deng, & Kaplowitz, 2004). Activation of transcription factor NF-kBby TNF-a is one of the myriad actions of TNF-a that causes genes togenerate potentially cell damaging oxidative enzymes such asNADP oxidase, cyclooxygenase (COX-2) and iNOS as well as further

Fig. 10. Expression of COX-2 in liver tissue by immunohistochemistry. Immunohistochemistra brown-colored product (arrows). (A): vehicle-treated control rat, �400; (B): ethanol treatekg b wt) plus ethanol treated, �400; (E): only diosmin (20 mg/kg b wt) treated, �400. (For inweb version of this article.)

release of TNF-a and other pro-inflammatory cytokines (Nanji et al.,2003).

In the present strategy of inducing hepatic injury by chronicethanol administration, our results are in complete accordancewith the above reports that the levels of necrosis and inflam-matory markers (TNF-a, NF-kB, iNOS and COX-2) increased in therats treated with ethanol only. Furthermore, diosmin adminis-tration in the present study has been found to restore theseelevated levels of inflammatory cytokines and cell necrosismarkers. Our results directly show that diosmin alters theethanol metabolizing enzymes thereby inhibiting the production

y of COX-2 in the liver of diosmin-treated rats. Positively stained COX-2 staining yieldedd, �400; (C): diosmin 10 mg/kg b wt plus ethanol treated, �400; (D): diosmin (20 mg/terpretation of the references to color in this figure legend, the reader is referred to the

Fig. 11. Expression of iNOS in liver tissue by IHC. Immunohistochemistry of iNOS in the liver of diosmin-treated rats. Positively stained iNOS staining yielded a brown-coloredproduct (arrows). (A): vehicle-treated control rat, �400; (B): ethanol treated, �400; (C): diosmin 10 mg/kg b wt plus ethanol treated, �400; (D): diosmin (20 mg/kg b wt) plusethanol treated, �400; (E): only diosmin (20 mg/kg b wt) treated, �400. (For interpretation of the references to color in this figure legend, the reader is referred to the web version ofthis article.)

M. Tahir et al. / Alcohol 47 (2013) 131e139138

of ethanol mediated ROS, inflammatory cytokines, which therebyconfirms its significant efficacy against ethanol-induced oxidativestress.

The above mentioned findings corroborated with the histo-logical data which exhibited the protective effects of diosminagainst ethanol-induced toxicity. Clinically, ethanol-inducedhistopathological changes in liver tissue vary depending on theextent and state of injury and additionally due to concentrationand duration of the exposure. In humans, in addition to fataccumulation in liver tissue, other pathological changes may beobserved which include lobular inflammation, periportal fibrosis,and nuclear vacuolization etc. Most of these symptoms are

Fig. 12. Effects of diosmin on ethanol-induced histological changes in the livers of rats. (A):(400�). (B): ethanol-treated group showing inflammatory response around central vein (arr(at a dose of 10 mg/kg b wt) with ethanol group showing lesser vacuolar degeneration andiosmin (dose 20 mg/kg b wt) showing almost normal architecture (400�). (E): slide showi

reported to mainly arise in heavy drinkers. In the present study,we also found that ethanol causes apparent inflammatoryresponse around the central vein in terms of infiltration ofinflammatory cells and portal expansion in rat liver tissue. Inaddition to the histopathological changes, all animal modelsestablished so far, depict that ethanol leads to the induction ofethanol metabolizing enzymes, oxidative stress and suppressionin the natural cellular antioxidant defense system. Our model ofethanol-induced liver injury is also in complete conformity withthese alterations occurred during ethanol-induced liver toxicity.Although there is no single animal model that completely mimicsalcoholic liver disease as it occurs in humans (Nanji & French,

liver section of rat control group showing structural intactness and normal architectureow) with vacuolar degeneration and necrosis (400�). (C): co-administration of diosmind lesser inflammatory response around central vein (400�). (D): co-administration ofng liver histology of rats treated with only diosmin at a dose of 20 mg/kg b wt (400�).

M. Tahir et al. / Alcohol 47 (2013) 131e139 139

2003). In order to simulate the ethanol toxicity from preclinical toclinical aspects, researchers have used various animal models toseparately address specific questions about alcohol liver disease.Our present work suggested likely changes in humans whenexposed to alcohol.

In the present study, several mechanisms by which ethanol leadto hepatotoxicity were evaluated. Among them, generation of freeradicals, imbalance in redox state, damage to mitochondria, per-oxidation of membrane lipids and induction of TNF-a and activationof the NF-kB and its translocation to nucleus are the major mech-anism revealing ethanol inducing liver toxicity. All these mecha-nisms lead to cell death and require ethanol metabolism toacetaldehyde. Findings from the present studies permit us toconclude that diosmin alleviates alcoholic liver injury via modu-lating ethanol metabolizing pathway, inhibition of oxidative stressand repression of inflammation. This may represent a novelprotective strategy against ethanol-induced liver diseases.

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