Alcohol 47 (2013) 131e139

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Alcohol
journal homepage: http://www.alcoholjournal.org/

Diosmin protects against ethanol-induced hepatic injury via alleviation of inammation 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 OHamiza, Sarwat Sultana*
Molecular Carcinogenesis and Chemoprevention Division, Department of Medical Elementology and Toxicology, Faculty of Science, Jamia Hamdard (Hamdard University), Hamdard Nagar, New Delhi 110062, India

a r t i c l e i n f o
Article history: Received 11 April 2012 Received in revised form 17 December 2012 Accepted 20 December 2012

a b s t r a c t
The present investigation was designed to evaluate the efcacy of diosmin against ethanol-induced hepatotoxicity in rats by modulating various mechanisms including ethanol metabolizing enzymes, generation of free radicals, imbalance in oxidanteantioxidant status, oxidative damage to membrane lipids, activation of transcription factors and elevation in inammatory markers involved in ethanolinduced hepatic damage. Diosmin is a avone glycoside, having anti-inammatory and anti-cancer properties. Thirty female Wistar rats segregated in ve groups, each with six animals. Group I as control followed by Group II, III and IV were treated with ethanol for 28 days. While groups III and IV were administered with diosmin at 10 mg/kg b wt (D1) and 20 mg/kg b wt (D2) respectively prior to ethanol 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) signicantly increased by 77.82% and 32.32% in liver tissues respectively as compared with control group and this enhancement is signicantly normalized with diosmin administration. Diosmin administration (D1 & D2) signicantly (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 and LDH signicantly increased by 102.03, 116.91 and 45.20% in ethanol-treated group as compared with control group. Group III and IV animals showed signicant reduction in the serum toxicity markers. Diosmin further alleviated ethanol-induced NF-kB activation, enhanced expression of TNF-a, COX-2 and iNOS. Findings from the present study permit us to conclude that diosmin alleviates alcoholic liver injury via modulating ethanol metabolizing pathway, inhibition of oxidative stress markers and suppression of inammatory markers. This may represent a novel protective strategy against ethanol-induced liver diseases. 2013 Elsevier Inc. All rights reserved.

Keywords: Ethanol Cytochrome P450 2E1 Alcohol dehydrogenase Tumor necrosis factor-alpha Cyclooxygenase-2

Introduction Alcoholic liver diseases are the most challenging current health problems worldwide. Over the period of time, alcohol (ethanol) has evolved as one of the socially accepted addictive drugs worldwide (Guo & Ren, 2010). Ethanol consumption can lead to various alcoholic liver diseases ranging from steatohepatitis, cirrhosis (Lucey & Weinrieb, 2009), heart disease (George & Figueredo, 2010), Alzheimers disease (Marinho, Laks, Engelhardt, & Conn, 2006), stroke (Ohkubo, Metoki, & Imai, 2009), liver disease (Cederbaum, Lu, & Wu, 2009), respiratory disease (Morris, 1990), diabetes mellitus
* Corresponding author. Department of Toxicology, Jamia Hamdard, Hamdard Nagar, New Delhi 110062, India. Tel.: 91 11 26054685x5565/5566/26089688; fax: 91 11 26059663. E-mail address: sarwat786@rediffmail.com (S. Sultana). 0741-8329/$ e see front matter 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.alcohol.2012.12.010

(Baliunas et al., 2009), bone disease (Chen, Cui, Liao, & Huang, 2009) to cancer (Seitz & Becker, 2007), which may develop following chronic alcohol ingestion and contribute to the alcoholism-related high morbidity and mortality. Ethanol as such is a mild toxicant and its toxicity primarily depends upon its metabolism. Liver is the primary target for ethanol 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 lipids besides altering the enzyme activities (Niemel, 1999). Three main enzyme systems are involved in ethanol metabolism (Salaspuro, 1999): alcohol dehydrogenase (ADH), Cytochrome P450 2E1 (CYP

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2E1) and catalase. Alcohol dehydrogenase leads to the formation of acetaldehyde from ethanol with the simultaneous reduction of NAD to NADH which in turn elevates xanthine oxidase activity and leads to the enhanced production of superoxide (Lieber, 2005). Microsomal ethanol oxidizing system involves the participation of CYP 450 2E1 (Lieber, 2004). This system connects ethanol and nicotinamide adenine dinucleotide phosphate oxidation to the reduction of molecular oxygen to H2O2. In the third system, the oxidation of ethanol molecule into acetaldehyde is concurrent with the instantaneous decomposition of hydrogen peroxide in a catalase catalyzed reaction (Bradford, Seed, Handler, Forman, & Thurman, 1993). Ethanol metabolism is associated with free radical generation leading to oxidative stress (Cederbaum, 2001; Lu & Cederbaum, 2008). It has been previously demonstrated that oxidative stress plays a crucial role in the pathogenesis of ethanol-induced liver damage. Ethanol-induced oxidative stress is associated with the generation of reactive oxygen species (ROS) and reactive nitrogen intermediates (RNI) that leads to an imbalance in the pro-oxidant and antioxidant levels in the tissues (Arteel, 2003). Acetaldehyde, produced from the oxidative metabolite of ethanol leads to the peroxidative damage to membrane lipids leading to cellular damage. Moreover, acetaldehyde has the ability to interact with the cellular proteins such as enzymes, microsomal proteins and microtubules leading to the impairment of normal protein functions (Rintala et al., 2000), that has been proposed to play a key role in alcoholic liver diseases. Furthermore acetaldehyde can interact with DNA to form carcinogenic DNA adducts such as N2ethyl-20 -deoxyguanosine (Brooks & Theruvathu, 2005). Pathogenesis of ethanol-induced liver diseases has not only contributed to oxidative stress, abnormal methionine metabolism, malnutrition and activation of Kupffer cells due to ethanol-induced endotoxin production, but it has been recently found that lipopolysaccharide (LPS) signaling, innate immunity, transcription factors, alteration in epigenetic features, microRNAs (miRNAs) and stem cells could also contribute to the ethanol-induced liver diseases (Gao & Bataller, 2011). Ethanol toxicity is associated with the induction of signaling cascade leading to the activation of transcription of NF-kB that results in the expression of inammatory mediators including cytokines (TNF-a, IL-6, and IL-12) (Iimuro, Gallucci, Luster, Kono, & Thurman, 1997) chemokines, lipid mediators, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). TNF-a further initiates the production of reactive oxygen species (ROS) and reactive nitrogen intermediates (RNIs) causing liver damage due to oxidative stress (Barnes & Karin, 1997; Nanji et al., 1999). Diosmin (Diosmetin-7-O-rutinoside), a naturally occurring avones glycoside readily obtained by dehydrogenation of hesperidin, found abundantly in the pericarp of various citrus (Campanero, Escolar, Garcia-Quetglas, Sadaba, & Azanza, 2010). Diosmin has various biological activities including antioxidant activity (Cotelle et al., 1996), anti-inammatory effect (Crespo, Glvez, Cruz, Ocete, & Zarzuelo, 1999), anti-diabetic effect (Manuel, Keenoy, Vertommen, & De Leeuw, 1999) and antiproliferative and anti-cancer activities (Tanaka et al., 1997). Moreover, diosmin has been found to increase the venous tone, improves lymphatic drainage and reduces the capillary hyperpermeability, thereby, leading to reduction in the release of inammatory mediators (Lyseng-Williamson & Perry, 2003). Keeping in view the above biological activities of diosmin, the present study was designed to make an attempt to evaluate the preventive efcacy of diosmin against ethanol-induced hepatic damage in Wistar rats by modulating ethanol metabolizing enzymes (CYP 2E1, ADH and Catalase), inammatory cytokines (TNF-a, NF-kB, COX-2 and iNOS).

Materials and methods Chemicals Glutathione reductase, oxidized (GSSG) and reduced glutathione (GSH), 1,2-dithio-bis-nitrobenzoic acid (DTNB), 1-chloro-2, 4-dinitrobenzene (CDNB), bovine serum albumin (BSA), oxidized and reduced nicotinamide adenine dinucleotide phosphate (NADP), (NADPH), diosmin, avine adenine dinucleotide (FAD), 2,6-dichlorophenolindophenol, thiobarbituric acid (TBA), xanthine, ascorbic acid, ethylene diamine tetra acetate (EDTA) and glycine were obtained from SigmaeAldrich, USA. Ferric chloride, sodium chloride, triseHCl, ethanol, disodium hydrogen phosphate, sodium dihydrogen phosphate, hydrogen peroxide, sodium hydroxide, sulphosalicylic acid, sulfuric acid, sodium azide 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 Baker Chemicals Ltd. 4-nitrocatechol has been purchased from Loba chemicals. Animals Female Wistar rats (150e200 g), 6e8 weeks old, were obtained from the Central Animal House Facility of Hamdard University. Rats were housed in an animal care facility under room temperature (25 1  C) with 12 h light/dark cycles and were given free access to standard pellet diet and tap water. Before the treatment, rats were left for 7 days to acclimatize. Animals received humane care in accordance with the guide lines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India, and prior permission was sought from the Institutional Animal Ethics Committee (IAEC No: 173/CPCSEA, 28 January 2000). Experimental procedure In the present study, we have evaluated the preventive efcacy of diosmin against ethanol-induced hepatic toxicity. Thirty female Wistar rats divided into ve groups, each with six animals. Group I as control receive vehicle (distilled water) only, followed by Group II, III and IV were treated orally with sequential (per week) increased dose of ethanol 25%v/v (5, 8, 10 and 12 g/kg b wt per week in each group) for 28 days. While group III and IV were administered 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 given only diosmin (20 mg/kg b wt) (D2). After 28 days, rats were sacriced by cervical dislocation under mild anesthesia and blood has been taken by cardiac puncture for various serological parameters. Liver samples were taken at the same time for various biochemical parameters. Preparation of post mitochondrial supernatant (PMS), cytosolic and microsomal fractions Livers were removed and cleaned with ice-cold saline (0.85% sodium chloride). A 10% homogenate of liver tissues were obtained in a buffer solution containing 10 mM triseHCl, 250 mM sucrose pH 7.4 using a Potter Elvehjen homogenizer and were centrifuged at 3000 rpm for 10 min by Eltek Refrigerated Centrifuge (model RC 4100 D) to separate the nuclear debris. The aliquot so obtained was centrifuged at 12,000 rpm for 20 min to obtain post mitochondrial supernatant (PMS), which was used as a source of various enzymes. The supernatant obtained was further ultra-centrifuged at 34,000 rpm for 1 h to obtain cytosolic

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fraction for ADH activity. The precipitate obtained was washed with homogenizing buffer to obtain the microsomal fraction for CYP 2E1 activity. All the experimental manipulations were carried out at 4  C. Cytochrome P450 2E1 (CYP 2E1) activity The catalytic activity of CYP 2E1 was analyzed by measuring p-nitrophenol hydroxylation (PNPH) as described by Reinke and Moyer (1985). The reaction mixtures contained a 100 mM potassium phosphate buffer (pH 6.8), 1.0 mM ascorbic acid, 1 mM NADPH, 1 mg hepatic microsomes, and 100 mM p-nitrophenol in a total volume of 1.0 ml. The 4-nitrocatechol that was formed was determined spectro-photometrically at 511 nm. Data were expressed as nmol/mg/min. Alcohol dehydrogenase (ADH) activity ADH activity was determined by the method of Bonnichsen and Brink (1955). Briey, ADH activity was measured in 50 mM glycine, pH 9.6, 0.8 mM NAD, 3 mM ethanol and 50 ml of cytosolic fraction in a nal volume of 1 ml. Enzyme activity was measured at 340 nm and the activity was calculated as nmol NADH formed/min/ mg protein using a molar extinction coefcient of 6.22 106 M1 cm1. Estimation of lipid peroxidation (LPO) The assay of lipid peroxidation was done according to the method of Wright, Colby, and Miles (1981). The reaction mixture consisted of 0.58 ml phosphate buffer (0.1 M, pH 7.4), 0.2 ml microsome, 0.2 ml ascorbic acid (100 mM) and 0.02 ml ferric chloride (100 mM) in a total of 1 ml. This reaction mixture was then incubated at 37  C in a shaking water bath for 1 h. The reaction was stopped by the addition of 1 ml of TCA (10%). Following addition of 1.0 ml TBA (0.67%), all the tubes were placed in a boiling water bath for a period of 20 min. The tubes were shifted to ice bath and then centrifuged at 2500 g for 10 min. The amount of malondialdehyde (MDA) formed in each of the samples was assessed by measuring the optical density of the supernatant at 535 nm. The results were expressed as the nmol MDA formed/h/ g tissue at 37  C by using a molar extinction coefcient of 1.56 105 M1 cm1. Assay for xanthine oxidase activity The activity of xanthine oxidase was assayed by the method of Athar et al. (1996). The reaction mixture consisted of 0.2 ml PMS which was incubated for 5 min at 37  C with 0.8 ml phosphate buffer (0.1 M, pH 7.4). The reaction was started by adding 0.1 ml xanthine (9 mM) and kept at 37  C for 20 min. The reaction was terminated by the addition of 0.5 ml ice-cold perchloric acid (10% v/v). After 10 min, 2.4 ml of distilled water was added and centrifuged at 4000 rpm for 10 min and mg uric acid formed/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 ml PMS, 1.0 ml hydrogen peroxide (0.019 M), 1.95 ml phosphate buffer (0.1 M, pH 7.4), in a total volume of 3 ml. Changes in absorbance were recorded at 240 nm and the change in absorbance was calculated as nmol H2O2 consumed per min per mg protein.

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 with 1.0 ml of (4%) sulphosalicylic acid, Then incubated at 4  C for a minimum time period of 1 h and then centrifuged at 4  C at 1200 g for 15 min. Briey reaction mixture has 0.4 ml supernatant, 2.2 ml phosphate buffer (0.1 M, pH 7.4) and 0.4 ml DTNB (4 mg/ ml) in a total volume of 3.0 ml. The yellow color developed was read immediately at 412 nm on spectrophotometer (Perkin Elmer, lambda EZ201). The reduced glutathione concentration was calculated as nmol GSH conjugates/g tissue. Assay for glutathione peroxidase activity The activity of glutathione peroxidase was calculated by the method of Mohandas, Marshall, Duggin, Horvath, and Tiller (1984). A total of 2 ml volume consisted of 0.1 ml EDTA (1 mM), 0.1 ml sodium 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 glutathione (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 nm was recorded at 25  C. Activity of the enzyme was calculated as nmol NADPH oxidized/min/mg protein with the molar extinction coefcient of 6.22 103 M1 cm1. Assay for glutathione reductase activity Glutathione reductase activity was measured by the method of Carlberg and Mannervik (1975). The reaction mixture consisted of 1.65 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) and 0.1 ml 10% PMS in a total volume of 2 ml. Enzyme activity was assessed at 25  C by measuring disappearance of NADPH at 340 nm and was calculated as nmol NADPH oxidized/min mg protein using molar extinction coefcient of 6.22 103 M1 cm1. Assay for serum aspartate aminotransferase and alanine aminotransferase (AST & ALT) activity AST and ALT activity were determined by the method of Reitman and Frankel (1957). Each substrate (0.5 ml) (2 mM a-ketoglutarate and either 200 mM L-alanine or L-aspartate) was incubated for 5 min at 37  C in a water bath. Serum (0.1 ml) was then added and the volume was adjusted to 1.0 ml with 0.1 M, pH 7.4 phosphate buffer. The reaction mixture was incubated for exactly 30 and 60 min at 37  C for ALT and AST, respectively. Then to the reaction mixture, 0.5 ml of 1 mM DNPH was added, after another 30 min at room temperature, the color was developed by addition of 5.0 ml of NaOH (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 of Korenberg (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 volume of 3 ml. Enzyme activity was recorded at 340 nm and activity was calculated as nmol NADH oxidized/min/mg protein. Assay for tumor necrosis factor-alpha (TNF-a) TNF-a protein level was measured by enzyme linked immunosorbent assay (ELISA) kit (eBioscience: Inc., San Diego., USA). Samples were prepared in phosphate buffer saline (1 PBS)

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Fig. 1. Average increase in body weight (g) per week. Representative graph shows the average increase in the body weight per week of the rats fed on different treatment regimen as explained in the Materials and methods section. Only the rats of group II showed the slight depletion in the body weight of the rats. Each value represents mean. n 6.

containing protease inhibitor cocktail. Analysis was performed according to the manufacturers instruction. The results were expressed as pg/mg tissue protein. Immunohistochemical staining of NF-kB, iNOS and COX-2 The liver tissues were xed in formalin and embedded in parafn. Sections of 5 mm thickness were cut onto poly-lysine coated glass slides. Sections were deparafnize three times (5 min) in xylene followed by dehydration in graded ethanol and nally 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 to minimize non-specic staining and then rinsed three times (5 min each) with 1 PBST (0.05% Tween-20). Blocking solution was applied for 10 min then sections were incubated with diluted (1:100 for NF-kB and COX-2; 1:500 for iNOS) primary antibodies, puried rabbit polyclonal anti-NF-kB antibody (BioLegend), rabbit polyclonal anti-iNOS antibody (Enzo Life Sciences) and rabbit polyclonal anti-COX-2 antibody (Bio Vision), overnight at 4  C in humid chamber. Further processing was done according to the instructions of Ultra Vision plus Detection System AntiPolyvalent, HRP/DAB (Ready-To-Use) staining kit (Thermo scientic system). The peroxidase complex was visualized with 3, 30 -diaminobenzidine (DAB). Lastly the slides were counterstained with hematoxylin, cleaned in xylene, dehydrated with ethanol and

Fig. 3. CYP 2E1 activity in liver. Representative gure showed the ethanol metabolizing enzyme (CYP 2E1) activity. It has been found that ethanol administration signicantly elevates CYP 2E1 activity (Ethanol group). Diosmin administration at both doses signicantly restores the ethanol-induced CYP 2E1 activity. Each value represents mean S.E., n 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 ethanoltreated group.

after DPX mounting microscopic (BX 51 Olympus) analysis was done at 400 magnication. Histological investigation For histopathology study, the liver was removed and immediately xed in freshly prepared 10% neutral buffered formalin at 4  C. Then, the skin was embedded in parafn wax. A section of liver (5 mm thick) was cut and stained with hematoxylin and eosin (H & E). Inammatory response around the central vein in terms of inltration of inammatory cells, vacuolar degeneration and pronounced necrosis around the central vein were observed as an indicator of histological changes with microscope (uorescent microscope, Olympus) at least in six different regions. Estimation of protein The protein concentration in all samples was determined by the method of Lowry, Rosebrough, Farr, and Randall (1951), using bovine serum albumin (BSA) as standard. Statistical analysis Differences between groups were analyzed using analysis of variance (ANOVA) followed by Dunnets multiple comparisons test.

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

Fig. 4. ADH activity in liver tissue. Alcohol dehydrogenase (ethanol metabolizing enzyme) activity. Ethanol treatment showed enhanced ADH activity and diosmin administration at a dose of 20 mg/kg b wt (D2) restores this elevation in ADH activity. Each value represents mean S.E., n 6. p < 0.01 compared with the corresponding value for control group. NS non-signicant, *p < 0.05 compared with the corresponding value for ethanol-treated group.

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compared with control group. Diosmin signicantly restores the level of xanthine oxidase (XO) activity by 56.14 and 77.19% at dose D1 and D2 respectively. Only D2 group showed no signicant change as compared to control group. Effect of diosmin on hepatic membrane damage (lipid peroxidation) MDA formation was measured to demonstrate the oxidative damage in ethanol-induced liver injury of Wistar rats. A signicant amplication by 177.1% of ethanol-induced MDA formation was found as compared with control group (25.83 nmol/g 1.15 vs 9.32 nmol/g 1.23, p < 0.001). We have observed that treatment with diosmin leads to the signicant restoration of membrane integrity 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 any signicant difference as compared to control. Diosmin treatment restores the activities of hepatic antioxidants Ethanol treatment was found to diminish hepatic antioxidants GSH, GPx, GR and Catalase by 56.25%, 47.11%, 48.83% and 36.72% as compared to corresponding control group (p < 0.001). Treatment of diosmin signicantly increases the level of GSH, GPX, GR and Catalase 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), which indicates antioxidant property of diosmin against ethanol-induced oxidative stress. Diosmin attenuates ethanol-induced hepatotoxicity Ethanol-treated groups showed signicant increase in the serum 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 compared with the control group. Administration with diosmin was found effective in the normalization in these serum toxicity markers by 44.18, 33.35 and 15.58% at dose D1 and 97.7, 81.1 and 38.32% at dose D2 respectively. Restoration of TNF-a level by diosmin Ethanol-treated groups showed signicant (p < 0.001) elevation of 28.05% in hepatic TNF-a levels as compared with the control group (307.22 12.89 vs 39.9 8.6), while diosmin administration at 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 been shown in Figs. 9e11 respectively. In the ethanol-treated group,

Fig. 5. Inhibition of LPO by diosmin in liver. Representative gure showed amelioration of ethanol-induced hepatic lipid peroxidation by diosmin. Group II (Ethanol group) showed increased lipid peroxidation and groups administered with diosmin in addition to ethanol showed depletion in lipid peroxidation. Each value represents mean S.E., n 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 ethanoltreated group.

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

treatment

groups

Effect of diosmin on body weight and relative liver weight of rats Figs. 1 and 2 shows respective body weight and relative organ weight of rats treated with diosmin (10 and 20 mg/kg b wt) for 4 weeks. Our results demonstrate that there was a slight decrease in the body weight (per week) and liver body weight ratio of rats that ingested alcohol only compared to normal group. Diosmin administration prevented ethanol-induced weight loss. Effect of diosmin on ethanol metabolizing enzymes CYP2E1 induction in hepatic tissues from ethanol-treated group was increased 77.82% compared to control animals (23.42 nmol/ mg 1.15 vs 13.17 nmol/mg 0.68, p < 0.001). Treatment with diosmin at dose D1 and D2 signicantly brought back the level of CYP 2E1 by 44.79% (p < 0.01) and 68.33% (p < 0.001) respectively (Fig. 3). In ethanol-treated groups (Fig. 4), liver tissue showed signicant (p < 0.01) enhancement in ADH activities (9.58 1.02) as compared with control group (7.24 1.24). Only high dose of diosmin (D2) signicantly suppressed the ethanol-induced ADH activity by 31.35%. Effect of diosmin on xanthine oxidase activity Hepatic xanthine oxidase (XO) activity reected a signicant increase of 82.45% in ethanol-treated group (p < 0.001) as

Table 1 Effect of diosmin administration on ethanol mediated depletion of hepatic reduced glutathione, glutathione peroxidase, glutathione reductase, catalase and elevation in xanthine oxidase activity in rats. Groups Reduced glutathione (mmol GSH conjugate/g tissue) 0.64 0.28 0.39 0.44 0.59 0.01 0.02# (56.2%) 0.02NS (17.2%) 0.02** (25%) 0.02 Glutathione peroxidase (nmol NADPH oxidized/ min/mg protein) 447.6 236.7 403.8 459.9 448.6 32.37 24.5# (47.11%) 16.3** (37.3%) 36.87*** (49.86%) 40.92 Glutathione reductase (nmol NADPH oxidized/ min/mg protein) 465.9 238.4 339.2 447.6 458.7 18.8 12.6# (48.83%) 12.6** (21.63%) 16.85***(44.9%) 15.64 Catalase (nmol H2O2 consumed/min/mg protein) 73.8 46.7 70.8 76.6 76.5 4.3 2.4# (36.7%) 4.2** (32.65%) 4.2*** (40.5%) 4.5 Xanthine oxidase (mg of uric acid formed/min/mg protein) 0.57 1.04 0.72 0.60 0.67 0.02 0.07# (82.45%) 0.07** (56.14%) 0.38*** (77.19%) 0.10

Control Ethanol Diosmin D1 ethanol Diosmin D2 Ethanol Only diosmin D2

Each value represents mean S.E., n 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 ethanol-treated group. NS Non signicant compared with the corresponding value for ethanol treated group.

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Fig. 6. Serum markers of liver toxicity AST and ALT. Representative gure showed elevation in ethanol-induced AST and ALT levels. Enhanced levels of AST and ALT are the key serum markers of liver toxicity. Diosmin administration signicantly restores the levels of ethanol-induced AST and ALT. Each value represents mean S.E., n 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.

Fig. 8. Inhibition of TNF-a by diosmin in liver. Representative gure showed the effect of ethanol and diosmin treatment on TNF-a. Each value represents mean S.E., n 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.

Discussion Alcohol is one of the main causes of end-stage liver diseases. From our previous work (Tahir & Sultana, 2011), in fact it is clear that sequential increase in ethanol dose (5e12 g/kg b wt) induces maximum tissue damage and also over comes the tolerance produced by the ethanol consumed at the same dose. In the present study, it has been observed, that the per week sequential increase in the dose of ethanol administration, there is a reduction in the rate of body weight gain and relative liver weights in the animals fed with ethanol only. This reduction in the rate of body weight gain and relative liver weight has also been reported by Aruna, Rukkumani, Varma, and Menon (2005). This decrease in body weight gain has been attributed to the reduction in the adipose tissue content due to ethanol consumption (Aruna et al., 2005). In contrast, diosmin administration prevented the ethanol-induced weight loss and relative liver weights signicantly. The generation of oxygen metabolites such as superoxide (O 2 ), hydrogen peroxide (H2O2) and hydroxyl radical (OH) during ethanol metabolism is believed to be the main cause in the pathogenesis of alcoholic liver injury (Zima et al., 2001). Increased generation of free radicals results in the loss of membrane integrity and function via lipid peroxidation. Oxidation of ethanol via ADH and CYP 2E1 leads to the generation of NADH (Lieber, 2004); that in turn elevates the xanthine oxidase activity (Zima et al., 2001). Generation of NADH in this pathway of ethanol oxidation is concomitant with the utilization of NADPH that suppresses the reduction of oxidized glutathione by glutathione reductase and subsequently depletes the reduced glutathione content, thereby depleting other glutathione dependent enzymes. Depletion of reduced glutathione cannot only impair the cellular defense but also result in the enhanced ethanolinduced oxidative stress and oxidative injury (Hayes & Pulford, 1995). In the present study, our ndings have been in complete conformity with the above facts related to ethanol-induced hepatic toxicity. Our results also demonstrated that ethanol consumption is associated with the depletion in glutathione and dependent enzymes leading to the imbalance in the oxidant: antioxidant status in the cells and diosmin administration suppresses the activities of ethanol metabolizing enzymes (ADH, CYP 2E1), thereby reducing the ethanol-induced hepatic toxicity. Ethanol consumption is notably associated with hepatic damage and the prominent sign of hepatic injury is the leakage of cellular enzymes (AST, ALT and LDH) in to the serum (Sehrawat & Sultana, 2006). Ethanol-treated rats show higher levels of these toxicity marker enzymes, indicating increased membrane permeability, cellular damage and/or necrosis of hepatocytes (Baldi, Burra,

there was higher number of cells showing expression of these proteins as indicated by the brown stains. Expression of these proteins (NF-kB, COX-2 and iNOS) is markedly suppressed in the diosmin-treated groups. For immunohistochemical analysis, brown color indicates specic immune staining of these proteins and light blue color indicates hematoxylin staining (original magnication: 400). Histopathology of liver tissue Analysis of tissue sections of animals from different treatment groups under microscope (400 magnication) revealed marked changes when compared with control group animals (Fig. 12). In the ethanol-treated animals, there was an evident of vacuolar degeneration and pronounced necrosis around the central vein. Moreover ethanol causes apparent inammatory response around the central vein in terms of inltration of inammatory cells (Fig. 12B) in liver tissue. In contrast, diosmin administration at both the doses (10 and 20 mg/kg b wt) protected the liver histology against ethanolinduced alterations (Fig. 12C and D). Only diosmin administration (20 mg/kg b wt) does not show any alterations from normal liver histology (Fig. 12E).

Fig. 7. Effect of administration of diosmin on cytotoxicity marker LDH. Representative gure showed the lactate dehydrogenase (LDH) activity in serum. LDH is an important cytotoxicity marker. Ethanol administration leads to the cellular toxicity and resulted in the release of LDH into the circulation. Hence estimation of LDH activity is indicator of cytotoxicity. Diosmin at both doses (D1 and D2) showed depleted LDH activity as compared with the only ethanol-treated group. Each value represents mean S.E., n 6. #p < 0.001 compared with the corresponding value for control group.*p < 0.05, **p < 0.01 compared with the corresponding value for ethanoltreated group.

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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): only diosmin D2-treated group.

Plebani, & Salvagnini, 1993; Tahir & Sultana, 2011). Diosmin administration to rats showed depression in these liver injury marker enzymes, thus suggesting its cell membrane stabilizing property and hepatoprotective efcacy against ethanol-induced liver damage. Numerous reports have demonstrated that TNF-a plays a pivotal role in the ethanol-induced liver pathology (Honchel et al., 1992; Ji, Deng, & Kaplowitz, 2004). Activation of transcription factor NF-kB by TNF-a is one of the myriad actions of TNF-a that causes genes to generate potentially cell damaging oxidative enzymes such as NADP oxidase, cyclooxygenase (COX-2) and iNOS as well as further

release of TNF-a and other pro-inammatory cytokines (Nanji et al., 2003). In the present strategy of inducing hepatic injury by chronic ethanol administration, our results are in complete accordance with the above reports that the levels of necrosis and inammatory markers (TNF-a, NF-kB, iNOS and COX-2) increased in the rats treated with ethanol only. Furthermore, diosmin administration in the present study has been found to restore these elevated levels of inammatory cytokines and cell necrosis markers. Our results directly show that diosmin alters the ethanol metabolizing enzymes thereby inhibiting the production

Fig. 10. Expression of COX-2 in liver tissue by immunohistochemistry. Immunohistochemistry of COX-2 in the liver of diosmin-treated rats. Positively stained COX-2 staining yielded a brown-colored product (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) plus ethanol treated, 400; (E): only diosmin (20 mg/kg b wt) treated, 400. (For interpretation of the references to color in this gure legend, the reader is referred to the web version of this article.)

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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-colored product (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) plus ethanol treated, 400; (E): only diosmin (20 mg/kg b wt) treated, 400. (For interpretation of the references to color in this gure legend, the reader is referred to the web version of this article.)

of ethanol mediated ROS, inammatory cytokines, which thereby conrms its signicant efcacy against ethanol-induced oxidative stress. The above mentioned ndings corroborated with the histological data which exhibited the protective effects of diosmin against ethanol-induced toxicity. Clinically, ethanol-induced histopathological changes in liver tissue vary depending on the extent and state of injury and additionally due to concentration and duration of the exposure. In humans, in addition to fat accumulation in liver tissue, other pathological changes may be observed which include lobular inammation, periportal brosis, and nuclear vacuolization etc. Most of these symptoms are

reported to mainly arise in heavy drinkers. In the present study, we also found that ethanol causes apparent inammatory response around the central vein in terms of inltration of inammatory cells and portal expansion in rat liver tissue. In addition to the histopathological changes, all animal models established so far, depict that ethanol leads to the induction of ethanol metabolizing enzymes, oxidative stress and suppression in the natural cellular antioxidant defense system. Our model of ethanol-induced liver injury is also in complete conformity with these alterations occurred during ethanol-induced liver toxicity. Although there is no single animal model that completely mimics alcoholic liver disease as it occurs in humans (Nanji & French,

Fig. 12. Effects of diosmin on ethanol-induced histological changes in the livers of rats. (A): liver section of rat control group showing structural intactness and normal architecture (400). (B): ethanol-treated group showing inammatory response around central vein (arrow) with vacuolar degeneration and necrosis (400). (C): co-administration of diosmin (at a dose of 10 mg/kg b wt) with ethanol group showing lesser vacuolar degeneration and lesser inammatory response around central vein (400). (D): co-administration of diosmin (dose 20 mg/kg b wt) showing almost normal architecture (400). (E): slide showing liver histology of rats treated with only diosmin at a dose of 20 mg/kg b wt (400).

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2003). In order to simulate the ethanol toxicity from preclinical to clinical aspects, researchers have used various animal models to separately address specic questions about alcohol liver disease. Our present work suggested likely changes in humans when exposed to alcohol. In the present study, several mechanisms by which ethanol lead to hepatotoxicity were evaluated. Among them, generation of free radicals, imbalance in redox state, damage to mitochondria, peroxidation of membrane lipids and induction of TNF-a and activation of the NF-kB and its translocation to nucleus are the major mechanism revealing ethanol inducing liver toxicity. All these mechanisms lead to cell death and require ethanol metabolism to acetaldehyde. Findings from the present studies permit us to conclude that diosmin alleviates alcoholic liver injury via modulating ethanol metabolizing pathway, inhibition of oxidative stress and repression of inammation. This may represent a novel protective strategy against ethanol-induced liver diseases. References
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