iNOS regulation during experimental hepatic injury and its mitigation by cloudy apple juice

International Journal of Biological Macromolecules 140 (2019) 1006–1017

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International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac

COX-2/iNOS regulation during experimental hepatic injury and its mitigation by cloudy apple juice Devoshree Mukherjee, Riaz Ahmad ⁎ Section of Genetics, Department of Zoology, Faculty of Life Sciences, Aligarh Muslim University, Aligarh 202002, India

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Article history: Received 29 June 2019 Received in revised form 8 August 2019 Accepted 20 August 2019 Available online 21 August 2019 Keywords: Cloudy apple juice Cyclooxygenase-2 Hepatic injury Diethylnitrosamine Inducible nitric oxide synthase Oxidative damage

a b s t r a c t A number of enzymes and transcription factors have been correlated with disease etiology. In this study, involvement of cyclooxygenase-2 and inducible-nitric oxide synthase is examined during diethylnitrosamine (DEN)-induced hepatic injury and cloudy apple juice (CAJ) supplementation. Liver injury was administered in rats by single dose of DEN (10 ml/kg bwt of 1% DEN), while 10 ml/kg bwt CAJ daily was given after 2 h of latency in DEN-treated animals for two weeks. CAJ was characterized by HPLC and subsequently examined for antioxidant power. During the course of treatment liver function, collagen (hydroxyproline), malondialdehyde, protein oxidation, antioxidant enzymes, ATPases, nitrite levels were investigated along with liver histopathology and electron microscopy. COX-2 and iNOS proteins were also localized in liver specimens. The results demonstrated rich polyphenols and antioxidant activity in CAJ. CAJ supplementation significantly restored liver biochemistry and anatomy as revealed by the refurbished investigated parameters. CAJ treatment also declined COX-2 and iNOS activities in injured animals. Electron microscopy demonstrated rejuvenated hepatocytes, Kupffer cells, RER, mitochondria and nucleus in CAJ supplemented animals. The novel outcomes of this study suggest that CAJ potentiates hepatoprotection by stimulating antioxidant power and regulating the COX-2 and iNOS proteins in the liver during experimental liver injury. © 2019 Elsevier B.V. All rights reserved.

1. Introduction Liver is an extremely important organ that dynamically performs detoxification, protein synthesis, biotransformation of endogenous and exogenous harmful materials. Any inflammation or injury to the liver is initiated as a result of prolong insult by high levels of drugs, toxins, viruses or protozoa which in turn leads to metabolic dysfunctioning and life threatening diseases such as liver fibrosis, cirrhosis ultimately leading to liver carcinoma [1–3]. Liver diseases are regularly rising due to alternate dietary habits, lifestyle and horrifying outcomes of synthetic medication. To minimize the appalling effects of medicines or therapeutics, discovery of novel alternatives with lesser side effects shall be on the forefront. Therefore, these days intake of dietary antioxidants and phytotherapeutics is voluntary part of most of the treatment regime due to their effective and safer nature in disease prevention. Humans are regularly exposed to numerous toxic chemicals, reaching to the system through processed food and environmental pollution. One of them is nitrites which are used in many countries as deliberate food additives. For instance, sodium nitrite aids in the stabilization of red colour and flavor of cured meat, and also provides prevention against the risk of botulism [4–6]. Further, nitrates widely ⁎ Corresponding author. E-mail address: [email protected] (R. Ahmad).

https://doi.org/10.1016/j.ijbiomac.2019.08.180 0141-8130/© 2019 Elsevier B.V. All rights reserved.

existing in spinach, beets, celery and lettuce may also undergo bacterial reduction to produce significant amount of nitrites [6,7]. Here, nitrosamines also need a special mention as they exhibit hepatotoxic and carcinogenic potential. More specifically, diethylnitrosamine (DEN) or N′Nitrosodiethylamine (NDEA) is a carcinogen that may reach to our system through processed meat and fish, cheese, tobacco, smoke and alcoholic beverages [8]. DEN-intoxication is simultaneous with metabolic activation of hepatic microsomal cytochrome P450 (i.e. CYP2E1). CYP450 causes deethylation of DEN which leads to the formation of electrophilic ethyl carbonium ions. These ions are capable to generate adequate DNA-adducts [9]. Enzymes such as cyclooxygenase-2 (COX2) and inducible nitric oxide synthase (iNOS) are reported to be involved in excessive production of pro-inflammatory mediators [10]. The investigation on the assessment of COX-2/iNOS is expected to provide interesting facts on the comprehensive understanding of the molecular mechanism of abatement of liver injury. Some interesting reviews published recently demonstrate chemopreventive, antiinflammatory and modulatory activities of red raspberry and its anthocyanins and a number of dietary flavonoids [11,12]. Further, agrimonalide isolated from Agrimonia pilosa and Sonchus oleraceus Linn have been reported to suppress inflammatory response in LPSstimulated macrophages via COX-2/iNOS down regulation [13,14]. Curcumin is reported to reduce the severity of experimental steatohepatitis in mice via inhibiting NF-κB activation and also prevents

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alcohol-induced liver disease in rats by regulating NF-κβ activation and its dependent pro-inflammatory genes [15,16]. To study the molecular mechanism of the disease as well as to discover novel therapeutics, biological models are utilized which best imitate the human condition. Hepatic fibrosis in rodents is the extensively utilized disease model to discover novel drugs, therapeutic intervention as well as to comprehend the role of transcription factors or enzymes. In this direction, rodent models have been developed with pathology by nitrosamines (DEN, DMN), CCl4 and ethanol. In this communication, we investigate the role of COX-2 and iNOS proteins during DENinduced liver injury and supplementation with Cloudy Apple Juice (CAJ). Apple (Malus domestica) juice is known for various pharmacological properties ranging from hepatoprotection [17], anti-cancerous [18] to obesity [19]. Further, apple juice also possesses antioxidant [20] and anti-allergic potential [21]. However, published literature indicates that diphenylamine and few other pesticides are in practice to increase the yield of apples [22,23]. Through direct consumption of apples, these noxious nitrogenous compounds reach the human system and may cause severe irreparable effects [22]. We encountered only a limited number of reports on the in vivo hepatoprotective potential of apple [17,24]. However, attempts to discourse efficacy of fruit juices in terms of their action at molecular level specifying the role of COX-2 and iNOS are yet to be seriously investigated. 2. Materials and methods 2.1. Chemicals and reagents Acrylamide, Adenosine 5′-triphosphate (ATP) disodium salt hydrate, Ammonium per sulfate (APS), Bis-acrylamide, CBBR-250, Diethylnitrosamine (DEN), Sodium dodecyl sulfate (SDS), 2Thiobarbituric acid (TBA), Tetramethylethylenediamine (TEMED) were purchased from Sigma. Bovine serum albumin (BSA) and Ethylenediaminetetraacetic acid (EDTA) were procured from SRL (Mumbai, India). Polyclonal COX-2 and iNOS antibodies were purchased from Trends Bioproduct, India and Genetix Biotech Pvt. Ltd., India respectively. LFT assay kits were from Autozyme (Accurex Biomedical Pvt. Ltd, Mumbai, India), Erba Diagnostics Mannheim Gmbh (Mumbai, India). All other chemicals, reagents and salts used were of AR grade. 2.2. Experimental animals and diet Rattus norvegicus, Wistar strain male 5–6 weeks of age (110 ± 10 g) were brought to the laboratory and acclimatized for about a week. These rats were kept in cages under hygienic conditions at a maintained temperature and relative humidity. The animals were regularly fed with diet and water ad libitum throughout the tenure of the experiment. Experiments, handling and care of animals were performed according to the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), India. The study synopsis was approved by the institutional ethical committee (Reg. No:714/GO/ RE/SO2/CPCSEA/4.02.16). 2.3. Preparation of cloudy apple juice (CAJ) Garden-fresh Kashmiri apples (Malus domestica), about 1 kg were brought to the laboratory. These apples were washed thoroughly in running tap water and dried by tissue towel. Apple peels and seeds were carefully removed and the pulp was used to prepare juice in commercial blender at room temperature. The yield of fresh crude apple juice was about ~400 ml. Crude viscous apple juice was passed through a clean plastic strainer, and the obtained filtrate was centrifuged at 1000 rpm for 10 min at 4 °C. The so-called cloudy apple juice (CAJ) was obtained as supernatant and used in the experiments. For future experiments, the unused juice was stored in air tight microfuge tubes in aliquots of 2 ml at −20 °C for a period of not exceeding two weeks.

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2.4. High performance liquid chromatography (HPLC) of CAJ The cloudy apple juice was dissolved in 70% aqueous methanol (1:1 v/v). The sample was initially filtered through a Whatman filter paper (#1) under vacuum followed by filtration through a 0.45 μm filter for HPLC analysis. HPLC system (LC-2010, Shimadzu, Japan) equipped with a variable wavelength UV–Visible detector fixed at 280 nm was deployed for characterization and identification of various ingredients in CAJ. For separation of polyphenolic compounds, HPLC column of Kinetex Polar (C18, 5 μm, 4.6 × 250 mm) was used. Binary mobile phase consists of a 6% acetic acid in 2 mM sodium acetate buffer (solvent A, pH 2.55, v/v) and acetonitrile (solvent B), with the following gradient conditions: 0% B to 10% B in 10 min, 10% B to 20% B in 10 min, 20% B to 50% B in 20 min, and 50% B to 100% B in 30 min. For reconditioning, about 10 min post-run was done to return back to its starting condition. The flow rate was 1.0 ml/min for a total run time of 70 min. About 20 μl of CAJ sample was injected into the column using a syringe. All polyphenols were identified using only few external standards [25,26]. Data acquisition was done by Shimadzu Lab Solutions software. 2.5. Treatment schedule During the experiment, healthy animals were randomly divided into four groups comprising five rats each. Group-1, animals were administered normal saline (0.9% NaCl) once in a week for two weeks (Saline Control); Group-2, animals received CAJ (10 ml/kg b.wt.) orally daily for two weeks (CAJ Control); Group-3, rats treated with DEN (10 ml/kg bwt of 1% DEN, i.p.) once during the entire course of experiment (DEN-treated); Group-4 animals were administered DEN and supplemented with CAJ in the prescribed doses after 2 h of first DEN dose. Subsequent CAJ doses were given on daily basis for the total fourteen days (DEN+CAJ). These animals were properly anaesthetized and sacrificed after two weeks to collect blood and liver. 2.6. Evaluation of in vitro antioxidant potential of CAJ 2.6.1. DPPH assay The free radical scavenging activity of apple juice was measured as already described [27,28]. Briefly, 0.1 ml of DPPH (0.2mM stock) solution was taken and added to 0.1 ml CAJ. The content were gently mixed with constant shaking and later incubated for 30min at 18 °C in the dark. Taking known concentrations of ascorbic acid as standard, the percentage of antioxidant activity in CAJ was calculated as follows: % Antioxidant Absorbance activity = AB − AS / AB × 100; where A = absorbance, B = blank; S = Standard or Sample. 2.6.2. FRAP assay The FRAP assay of CAJ was carried out as per the protocol of Benzie and Strain [29]. To 100 μl CAJ sample, 33.33 μl of standard ascorbic acid was mixed with 966.7 μl of FRAP reagent (acetate buffer 300 mM, pH 3.6; FeCl3, 20 mM and TPTZ, 10 mM) and incubated at 37 °C for 10 min. Following vigorous shaking, absorbance of the mixture was read at 593 nm. Known amounts of ascorbic acid (50–1000 μM) were used as standard and FRAP values were extrapolated as μM equivalents of ascorbic acid. 2.6.3. Total phenolics in CAJ The established protocol of Singleton and Rossi [30] was used to estimate the total phenolics in CAJ. To approximately 0.02 ml of CAJ, 1.58 ml of distilled water was added. To this, Folin's Ciocalteu reagent and sodium carbonate solution were added in 0.1 and 0.3 ml respectively. Following shaking, the reaction mixture was incubated in dark for 2 h at room temperature. Finally, the optical density was read at 765 nm and the obtained values presented as gallic acid equivalent (mg GAE/l of CAJ).

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2.7. Serum collection, tissue preparation and protein estimation Freshly drawn blood from the heart of animal was kept undisturbed to ooze out the sera. The pale yellow colored serum was separated from the clot by low-speed centrifugation in cooling [31]. The animal liver was excised carefully in sterilized condition avoiding mixing of adjacent tissues. The collected tissue was washed thrice in chilled PBS (50 mM, pH 7.1). Tissue homogenate were prepared according to the routine method of Ahmad and Ahmad [32] for biochemical assays. A separate portion of liver from each animal was also fixed and stored in 10% formalin for histopathology. Bovine serum albumin (BSA) was used as the standard to find protein concentrations in unknown sera and liver samples. Protein concentrations of all the samples were estimated using the protocol of Lowry et al. [33]. Absorbance of the samples, following incubation for 20–25 min was read at 660 nm. The unknown protein concentrations were extrapolated using mathematical equation conceived from a standard graph. 2.8. Liver biochemistry 2.8.1. Examination of hepatic ALT, AST, ALP and T.Bil Serum level of enzymes such as ALT, AST, ALP, direct and total bilirubin were investigated using commercial diagnostic kits (Erba Diagnostic kits GmBH, Mumbai and Autozyme Accurex Biomedical Private Limited, Mumbai). The values of ALT, AST, ALP, direct and total bilirubin were expressed as units/mg protein. All the examinations were conducted in triplicates. 2.8.2. Lipid peroxides estimation Hepatic lipid peroxides were measured according to the protocol of Ohkawa et al. [34]. The reaction of lipid peroxides with thiobarbituric acid (TBA) is a sensitive assay to determine lipid peroxidation in animal tissues. Lipid peroxides are measured in terms of malondialdehyde formation of the organic layer (upper layer) in the reaction mixture centrifuged at 4000 rpm for 10 mins. It was read at 532 nm and the levels of malondialdehyde were calculated using extinction coefficient of 1.56 × 105 M−1 cm−1.

Table 1 List of polyphenols identified in cloudy apple juice by High Performance Liquid Chromatography (HPLC). Peak #

tr (retention time)

Identified ingredients

1 2 3 4 5 6 7 8 9 10 11 12 13 14

14.150 15.700 19.690 27.472 31.316 35.830 36.528 38.541 41.462 42.029 43.408 44.116 46.445 57.661

[+]-Catechina,b Chlorogenic acida,b Cyanidin 3-galactosideb p-Coumaroylquinicacidb Hyperin (quercetin-3-O-galactoside)a Isoquercitrin (quercetin-3-O-glucoside)a Reynoutrin (quercetin-3-O-xyloside)a Quercitrin (quercetin-3-O-rhamnoside)a Phloretin-2-O-xyloglucosidea 3-hydroxyphloretin 2′-xyloglucosideb Quercetin 3-galactosideb Quercetin-3-glucosideb Quercetin 3-xyloside b Phloridzinb

2.8.4. Estimation of glutathione-s-transferase Glutathione-S-transferase (GST) assay was performed in liver homogenates using 10 mM of 1-chloro-2, 6-dinitrobenzene (CDNB) [37]. The enzyme kinetics was studied for 3 min at 340 nm. The activity of enzyme was determined in terms of μM GSH-CDNB conjugate/min. 2.8.5. Hepatic catalase assay Activity of catalase in samples were measured by taking 0.1 ml of liver homogenate, 1.9 ml of phosphate buffer (50 mM; pH, 7.0) and 1.0 ml of freshly prepared H2O2 (30 mM) [38]. Decomposition of H2O2 was correlated with the decline in absorbance which was read at 240 nm. Absorbance difference (ΔA) per unit time was taken as the quantitative measurement of catalase activity. 2.8.6. Estimation of ATPases Activity of ATPases (Ca2+, Na+/K+, Mg2+) was determined in the liver as per the already described protocol [39]. The colour was developed by the addition of 0.5 ml of ammonium molybdate and 0.25 ml of 1-amino-2-naphthol-4-sulfonic acid (ANSA). After incubation at 25 °C for 20 min, ATPase levels were measured in terms of inorganic

2.8.3. Superoxide dismutase (SOD) assay SOD was estimated in liver of all groups of animals following the procedure of Marklund and Marklund [35]. The kinetics of the enzyme was performed at 420 nm for 5 min with an interval of 1 min. SOD activity was examined by 50% inhibition in auto-oxidation of Pyragallol. Simultaneously, reagent blank and sample blank were also run with the tissue sample. The changes in the levels of SOD were determined according to the protocol of Nandi and Chatterjee [36].

Fig. 1. HPLCs Chromatogram of Cloudy apple juice derived from pulp recorded at wavelength of 280 nm. (a,b = 25-26)

Fig. 2. Merged bar-graphs showing relative changes in the liver function parameters as deterioration phase (DEN-intoxicated) and recovery phase (CAJ administration) in male rats. Symbols are: AST = Aspartate aminotransferase (IU/L), ALT = Alanine transaminase (IU/L), ALP = Alkaline phosphatase (IU/L), B = Bilirubin (mg/dL). Data are expressed Mean ± SEM (n = 5).

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phosphate liberation [40]. The samples were read in visible range (640 nm) on spectrophotometer. 2.8.7. Assessment of protein oxidation To determine protein oxidation, hepatic protein carbonyl levels were estimated [41]. The reaction was started by adding 2 ml of 2,4dinitrophenylhydrazine (DNPH) (mixed in 2.5 M HCl) in 0.5 ml of tissue homogenate. During incubation for 1 h at room temperature, the reaction mixture was repeatedly vortexed after every 10–15 min. In the tube, protein was precipitated by adding 2 ml of 20% trichloroacetic acid and the solution was washed thrice with 2 ml of ethanol:ethyl acetate (1:1). The contents were centrifuged at 10,000 g for 3 min. The precipitate so obtained was solubilized in 1 ml of 6 M guanidine HCl (20 mM potassium phosphate, pH 6.5). Absorbance of the solution was read at 380 nm. The total protein carbonyl content was calculated using a molar absorption coefficient of 22,000 M−1 cm −1 and measured as nanomole carbonyl mg −1 protein. 2.8.8. Nitrite estimation We followed the protocol of Green et al. [42] to estimate nitrite levels in serum and liver of all the experimental groups. The reaction

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was initiated by adding 50 μl of Griess reagent (1:1 solution of 1% sulfanilamide, 5% phosphoric acid and 0.1% naphthylamine hydrochloric acid) to 100 μl of tissue supernatant. The contents were incubated for 10 min in dark and absorbance was read at 546 nm. Concentration of nitrite was extrapolated against sodium nitrite standard. 2.8.9. Evaluation of hydroxyproline Hydroxyproline assay was performed in liver samples to determine collagen content in animals [43]. Optical density of the solution was read at 560 nm. Collagen levels in the liver were calculated multiplying the obtained absorbance value by a factor of 7.46 [44]. 2.9. Assessment of liver pathology Small portions of liver were fixed in 10% formalin and processed for histopathology as per the routine procedure. Tissue sections of 5 μm thickness were serially cut and prepared for Hematoxylin-Eosin staining for observing general changes while Masson's Trichrome staining and Picrosirius red for specific collagen staining in liver. Picrosirius red stained sections were visualized under fluorescence at 475–500 nm.

Fig. 3. Row graphs showing the effect of CAJ supplementation on the biochemical and antioxidant parameters during the DEN-induced liver injury. Lipid Peroxidation (MDA, nanomoles/g of tissue); SOD (Superoxide Dismutase, U/mg of protein/min); Catalase (U/mg protein/min); Serum nitrite (μmoles of nitrite/ml); Na-ATPase (μmoles of Pi liberated/mg of protein/min); Mg-ATPase (μmoles of Pi liberated/mg of Protein/min); Ca-ATPase (μmoles of Pi liberated/mg of Protein/min); Hepatic nitrite (μmoles of nitrite/mg of protein); GST (Glutathione-STransferase, μmol CDNB-GSH conjugate/min); Collagen (mg/g tissue); Hydroxyproline levels (mg/g of tissue); Protein Carbonyl (nmol carbonyl/mg of protein). Each data represent the mean ± SEM value (n = 5) of experiments performed in triplicates (⁎P b 0.05; ⁎⁎P b 0.01; #P b 0.001).

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Photographs were taken on Nikon (Model: 80i) with LCD attachment and Zeiss Axioscope A1 attached with JenoptikProg Res C5 camera.

2.10. Identification of COX-2 and iNOS activities by immunohistochemistry Deparaffinised liver specimens of 5 μm size were incubated with 3% H2O2 for 15 min to quench endogenous peroxidase activity. Tissue specimens were washed with PBS for 30 min and then incubated in humid chamber with primary COX-2 mouse (1:400) and iNOS polyclonal antibodies (1:100) respectively overnight. These tissue specimens were carefully rinsed with PBS thrice and then incubated with HRPconjugated goat anti-mouse IgG (secondary antibody) for 2–3 h. Following washing with PBS for 10 min, the tissue sections were stained in 3, 3′-diaminobenzidine tetrachloride hydrate (DAB) for 30–50 min. The counter staining of these sections was performed in Mayer's Hematoxylin for 10 s. The slides were finally dehydrated and mounted with DPX.

2.11. Scanning electron microscopy of liver The fresh tissue was sliced (1 × 1 × 5 mm3) and rinsed in chilled phosphate buffer thrice. Fresh tissue was fixed in 2.5% glutaraldehyde prepared in 0.1 M sodium phosphate buffer (pH, 7.4) for 12 h. Then the tissue was stained in 1% osmium tetraoxide prepared in phosphate buffer. Afterward, the tissues were thoroughly washed in distilled water, dehydrated in a graded series of ethanol and finally dried using critical-point drying. The tissue was trimmed, mounted on aluminum stubs, samples were sputter coated with gold and examined under the scanning electron microscope (FE-SEM, JEOL JSM6510LV) at 10 kV. 2.12. Transmission electron microscopy of liver Liver tissue were trimmed in 1mm3 size and then quickly fixed in 2.5% Glutaraldehyde for overnight followed by subsequent washing in phosphate buffer saline (pH, 7.4) for thrice. To provide density and better contrast to the sample they were finally fixed in secondary fixative

Fig. 4. H&E and Masson's trichrome stainings of rat liver specimens of all the groups. (A, A′) Normal liver architecture observed in animals of control group (40×); (B, B′) CAJ control showing normal lobular architecture with central vein and normal sinusoidal spaces (20×); (C,C′) Day-14, DEN treated showing severe hemorrhage, neutrophilic infiltration, distorted hepatocytes, fused nuclei, bridging fibrous (inset, 20×) (40×); (D, D′) Day-14, DEN+CAJ treated rat liver specimens depicting restoration of normal lobular structure, central vein and lesser hemorrhage (10×); (E, E′) Control group showing normal liver and lobular architecture and absence of collagen fibers in rats (20×); (F, F′) CAJ administered control group of rats exhibiting liver section exhibiting normal liver architecture without collagen fibers (40×); (G, G′) DEN-intoxicatedday-14, liver specimen demonstrated abnormal cellular and lobular architecture well characterized by liver inflammation and amassing of collagen fibers (blue) around the central vein (40×); (H, H′) DEN+CAJ,day-14 liver specimens showing restitution of liver architecture with scarce collagen fibers (20×). CV = central vein, SS = sinusoidal space, HR = hemorrhage, NI = neutrophilic infiltration, COL = collagen.

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Fig. 5. Bright and light field microscopy of rat liver sections stained with picrosirius red. (A&E) Control (normal saline) group with normal cellular and lobular architecture (20×); (B&F) CAJ control rat liver sections showing normal liver architecture with prominent nuclei (40× & 20×); (C&G) DEN, day-14 exhibiting extensive and amassing of collagen bundle (10× & 40×); (D&H) Liver sections supplemented by DEN+CAJ show restored liver structure with mild collagen fibers (40× & 20×). COL = collagen.

i.e., 1% osmium tetraoxide for about 1 h at 4 °C. Subsequently, sequential steps of trimming, dehydrating in graded series of alcohol and embedding in epoxy were done. Section was then mounted on copper grids

followed by staining with lead and uranyl salts. The grids containing the liver sample were then viewed under a transmission electron microscope (JEOL JEM-2100) at 100 kV.

Fig. 6. Confocal microscopy of rat liver sections stained with picrosirius red. (A) Control (normal saline) group without collagen accumulation (20×); (B) CAJ control rat liver sections showing normal liver architecture with no sign of collagen (20×); (C) DEN, day-14 exhibiting amassing of collagen bundle and fibers (40×); (D) DEN+CAJ supplemented liver specimen showed almost liver architecture with slight collagen fibers (20×). COL = collagen.

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2.13. Statistical analysis

3.2. Changes in liver biochemistry

Statistical analyses between biochemical parameters among different groups were carried out by using one-way ANOVA and post-hoc Tukey's test (Graph Pad Insta 3.0). Statistically significant level was considered at p b 0.05, 0.01, 0.001. Results were expressed as mean ± SEM of all triplicates run under similar conditions.

Status of liver enzymes and the quantity of bilirubin were taken as the first line of investigation. DEN-treated rats indicated marked increase in the liver function enzymes such as ALT, AST and ALP as compared to control groups. Administration of CAJ to rats in prescribed doses noted to significantly decline the levels of liver biomarkers compared to DEN-intoxicated group. The recovery of hepatic enzymes by CAJ signifies its protective role against DEN-induced hepatic inflammation in rats (Fig. 2). Malondialdehyde (MDA) generation is an assessment of lipid peroxides and hence is regarded as one of the basic criterion of evaluating cellular damage triggered by oxyradicals. DEN-administration leads to the significant elevation in liver MDA (~78%, b0.001) in contrast to control groups. Further, treatment of CAJ showed a decline in MDA levels of about ~44% (p b 0.001) in comparison to DEN-intoxicated rats. DENadministered rats showed considerably depleted levels of hepatic SOD (~68%, p b 0.001) due to increased production of free radicals in response to hepatic injury. In contrast, CAJ administered group showed recovery in SOD activity (~64%) indicating that CAJ stimulates the antioxidant system. Protein carbonyls, an indicator of oxidative damage, showed significantly higher levels in DEN-treated groups in comparison to controls. Further, decline in the levels of hepatic total carbonyl content (~32%) by CAJ signify its antioxidant potential. Glutathione-STransferase belongs to the major class of detoxifying enzymes for endogenous and exogenous compounds. GST also showed marked reduction in its level in DEN-treated groups to about ~75% as compared to animals belonging to control. While animals received CAJ showed recovery in GST levels of ~62% in comparison to DEN-treated groups. We also assessed the levels of catalase which is an important enzyme

3. Results 3.1. Characterization and antioxidant activity of CAJ HPLC profile showed the presence of at least fourteen major polyphenols in CAJ (Fig. 1). These include [+]-Catechin, Chlorogenic acid, Cyanidin 3-galactoside, p-Coumaroylquinicacid, Hyperin (quercetin-3O-galactoside), Isoquercitrin (quercetin-3-O-glucoside), Reynoutrin (quercetin-3-O-xyloside), Quercitrin (quercetin-3-O-rhamnoside), Phloretin-2-O-xyloglucoside, 3-hydroxyphloretin-2′-xyloglucoside, Quercetin-3-galactoside, Quercetin-3-glucoside, Quercetin-3-xyloside and Phloridzin. The major polyphenolic peaks observed in chromatogram are Hyperin (quercetin-3-O-galactoside), Isoquercitrin (quercetin-3-O-glucoside), Reynoutrin (quercetin-3-O-xyloside), Quercitrin (quercetin-3-O-rhamnoside), 3-hydroxyphloretin 2′-xyloglucoside, Quercetin-3-xyloside and Phloridzin respectively (Table 1). Antioxidant activity of CAJ was investigated using DPPH, total phenolics and FRAP assays. DPPH activity of CAJ was evaluated as the percentage antioxidant capacity, which was ~40%. The total phenolic content in CAJ is estimated to be ~280 ± 23 mg of gallic acid equivalents/l. Besides, FRAP assay demonstrate a value of about 1127 μM equivalents of ascorbic acid in CAJ.

Fig. 7. Scanning electron micrographs (SEM) of rat liver.(A) Control (normal saline) rat liver showing intact liver architecture with normal shaped Kupffer cells and leukocytes (1000×, 50 μm); (B) CAJ control rat liver showing smooth cellular architecture with properly shaped Kupffer cells and leukocytes (500×, 50 μm); (C) Day-14, DEN treated section exhibiting rough outer surface, distorted cells with irregularly shaped RBCs and activated Kupffer cells (1000×, 10 μm); (D) Day-14, DEN+CAJ showing restoration of normal cellular structure with lesser number of distorted Kupffer cells and leukocytes (7000×, 2 μm). Arrows show: Kc = Kupffer cells, LK = Leukocytes and RBCs = Red blood corpuscles.

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involved in protecting the cell from severe oxidative stress. Administration of DEN in rats leads to remarkable fall in catalase levels indicating imbalance in the defensive potential and antioxidant status. In CAJtreated animals, significant increase in the levels of catalase was noticed indicating strong antioxidant potential of fruit juice that resulted in abatement of liver injury via encouraging antioxidant system (Fig. 3). Nitrite levels were also examined in the serum and liver of all the animals. Nitrite levels basically represent the generation of NO species, which in our case exhibited a significant increase in DEN-treated animals compared to controls (Fig. 3). However, CAJ-supplement demonstrated a noticeable decline in hepatic and serum nitrite levels clearly implying CAJ to be strong hepatoprotectant. Further, DEN-injured group showed declined levels of membranebound Na+/K+, Mg2+and Ca2+-ATPases in contrast to control groups indicating fragile transport across the biomembranes. Prominent restoration in ATPases designate proper molecular transport across the membranes as observed in CAJ supplemented animals. Hydroxyproline assay in tissue hydrolysate is a direct measurement of the amount of collagen content. We observed an increase in hydroxyproline contents in animals received DEN for two weeks, signifying elevated collagen levels in this group. Post-treatment with CAJ significantly decreases

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the level of collagen as compared to DEN treated animals (p b 0.05) (Fig. 3). Collagen content in treated and control group were further confirmed by the histopathology of liver by Masson's trichrome and Picrosirius red stainings. 3.3. Histopathological and ultrastructural changes in liver Histological assessment was carried out to examine the changes in the liver architecture during progression of liver fibrosis as well as treatment with CAJ. Liver sections of control animals showed normal cellular architecture (Fig. 4A–B). Further, the lobules were intact with a centrally located vein. Liver specimens of DEN-treated group displayed marked liver damage characterized by severe hepatocellular degeneration, neutrophilic infiltration, sinusoidal congestion, marked hemorrhage and bridging fibrosis (Fig. 4C). CAJ treatment showed rectification of damage which was well marked by intact central vein, less congested sinusoidal spaces (Fig. 4D). These results thus, clearly indicate that CAJ acts as hepatoprotectant against DEN-induced hepatotoxicity in rats. Rats treated with DEN showed significant amount of collagen accumulation around central vein in Masson's trichrome stained sections

Fig. 8. Transmission electron microscopic (TEM) examination of rats liver specimen. (A) Control (normal saline) showing normal liver structure with proper nuclei surrounded by rough endoplasmic reticulum and mitochondria (3000×, 500 nm); (B) CAJ control liver sections exhibiting typical cellular architecture with electron dense prominent nucleus and two nucleolus surrounded by stacked rough endoplasmic reticulum (21,000×, 500 nm); (C) Day-14, DEN- administered rat liver showing distorted and activated Kupffer cells with multiple heterolysomes, irregularly shaped nuclei surrounded by shortened rough endoplasmic reticulum in the cell near the disintegrated cell membrane (3000×, 50 nm); (D) Day-14, DEN +CAJ restitute normal cellular structure with almost proper shaped nucleus, well shaped rough endoplasmic reticulum and glycogen granules (3000×, 500 nm). Arrows depicts as follows: Kc = Kupffercells, Ly = heterolysomes, M = mitochondria, N=Nucleus, Ns = Nucleolus and RER = Rough endoplasmic reticulum.

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(Fig. 4E). Picrosirius red staining also exhibited variability in collagen deposition in all the groups. However, the extent of collagen deposition was much greater in the DEN administered animals that clearly indicate compromised liver function and injury (Fig. 5C&G). CAJ administration for two weeks refurbished hepatic architecture in a time and dosedependent manner. A marked decline in the liver damage was demonstrated by scarce level of collagen in CAJ-supplemented rats (Fig. 5D & H), confirming hepatoprotective potential of CAJ against chemicallyinduced hepatic injury (Fig. 6). SEM analysis of control liver showed normal hepatocytes, RBCs, Kupffer cells and leukocytes (Fig. 7A & B). Day-14 DEN-treated fibrotic liver samples demonstrated significant damage which was characterized by distorted RBCs, damaged hepatocytes, activated Kupffer cells (Fig. 7C). TEM observations reveal asymmetrical Kupffer cells, multiple heterolysosomes, degenerated nucleus surrounded by dilated and varied size of rough endoplasmic reticulum (Fig. 8C). Post-treatment with CAJ displayed regenerated hepatocytes, reduced number of abnormal Kupffer cells and leukocytes (Fig. 8D). These findings indicate significant recovery in the liver architecture by CAJ supplementation in rats (Figs. 8–9).

3.4. Changes in COX-2 and iNOS expression in the liver Immunohistochemical stained liver section revealed absence of COX-2 and iNOS positive cells in animals belonging to control groups. A significant increase in the expression of hepatic COX-2 and iNOS was observed in DEN-induced fibrotic group. CAJ-supplement down regulates expression of hepatic COX-2 and iNOS during DEN-induced

liver fibrogenesis in experimental rats (Fig. 9-10). The obtained results are suggestive of CAJ as the powerful anti-inflammatory phytomedicine. 4. Discussion The data generated during this investigation strongly support therapeutic efficacy of cloudy apple juice (CAJ) that exerts hepatoprotection during diethylnitrosamine (DEN)-induced hepatic injury in rats via regulating COX-2 and iNOS. CAJ refurbished normal tissue anatomy through its antioxidant and anti-inflammatory actions. Published literature reveals a direct correlation between regular consumption of fruits/ phytoextracts and prevention of diseases [12,45–47]. Interestingly, apple is one such fruit which is rich source of flavonoids, polyphenols and antioxidants. CAJ possess radical-scavenging and antioxidant activities due to the presence of ascorbic acid, polyphenols, procyanidins and pectin; and hence is suggested beneficial for human health [47,48]. In this study, we have evaluated antioxidant power of CAJ in terms of its total phenolics, radical scavenging/DPPH and Ferric Reducing Ability of Plasma (FRAP) activity. The examined levels of these selected parameters of CAJ corroborate with the previously published work [25,26,49–51] and signify a remarkable antioxidant potential of apple juice. These findings are further supported by our HPLC data which demonstrated the presence of rich polyphenolic milieu in CAJ (Table 1). Silva et al. [52] observed that epicatechin, catechin and chlorogenic acid possess strong antioxidant activity and inhibit the oxidation of low density lipoprotein (LDL) and alkyl peroxyl radical (ROO•) scavenging activity in vitro. Our HPLC results reveal the presence of these constituents in CAJ confirming its high antioxidant potential.

Fig. 9. Immunohistochemical staining of cyclooxygenase-2 (COX-2) in liver specimen of all experimental groups (A) Control (normal saline) liver slides showed negative COX-2 hepatic cells (20×); (B) CAJ (positive control) liver specimen showing absence of COX-2 positive cells (40×); (C) DEN-treated group showing increase COX-2 positive cell in damaged area (20×); (D) DEN+CAJ supplemented group showed slight positive staining of COX-2 around the portal vein (20×).

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Any injury to the liver consequently results in hepatocyte damage altering the transport across the membrane. This may result in leakage of enzymes from the cells into the plasma [53–55]. For this reason, serum liver function enzymes have been used as sensitive biomarkers of liver injury [4,56]. ALT, abundantly present in hepatocytes, increases during DEN-intoxication signifying liver damage. While AST, present in liver parenchymal cells also rises in DEN-administered rats [54–57]. Besides, ALP that catalyzes the hydrolysis of phosphomonoesters in liver also showed significant elevation in DEN-induced fibrotic animals. Similar increase in the levels of these enzymes has been reported in case of liver injury, either caused by chemicals or viruses [45,53,57,58]. Further, bilirubin (an end product of RBC breakdown) exhibited elevated levels in rats intoxicated with DEN. Bilirubin is known to bind reversibly to albumin which subsequently transported to liver. This bilirubin is conjugated with glucuronic acid and finally excreted out in the bile. Published reports suggest that bilirubin levels in the serum provide a reflection of liver pathophysiology, similar to our case where DEN induces severe damage to the liver [53,59,60]. However, CAJ supplement significantly declined the levels of liver function biomarkers in injured rats suggesting its strong hepatoprotective potential [17,24] It has been reported that electrophilic reactive products, act as free radical initiators, are generated in the liver as a result of DEN metabolism by cytochrome P-450 [9,57]. The increase free radical load leads to rise in lipid peroxides and altered ATPase function which may severely damage cell membranes, hamper molecular transport, inhibits

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various enzymes and reduces cellular functions [61]. Elevated levels of LPO, protein carbonyls, hepatic and serum nitrite levels favored ROS generation conferring oxidative stress in DEN-treated animals. The major ROS present in mammalian system include superoxide anion − (O− 2 •) and hydroxyl anions (HO ) [62,63]. Literature reveals that these ROS are cytotoxic and are endogenously removed by antioxidant machinery comprised of GSH, SOD, CAT and GPx [63,64]. It was observed that DEN treatment remarkably decreases SOD, GST and CAT levels endowing strong evidence of reduced radical-scavenging activity and weaken antioxidant system in experimental animals. Marked hepatic damage was further documented by histopathology of liver tissue. H&E stained biopsies depicts abnormal lobular architecture, neutrophilic infiltration, severe hemorrhage and congested sinusoidal spaces in injured rats. The inflammatory immune cells activate HSCs which become proliferative and fibrogenic to synthesize collagen [65,66]. Further, inflated hydroxyproline levels also support collagen amassing in DEN-treated rats. Moreover, SEM and TEM based analyses demonstrated gross level alteration in liver anatomy characterized by the presence of activated Kupffer cells, abnormal shaped leukocytes, short RER, distorted RBCs in injured rats. However, CAJ supplementation significantly declined the malondialdehyde (LPO), protein carbonyls, hepatic/serum nitrite and hydroxyproline levels in animals within two weeks. Further, CAJ elevates SOD, GST, CAT activities and regulate ATPases to strengthen antioxidant power in injured animals. It is inferred that antioxidants present in CAJ probably act in their own ways

Fig. 10. Immunohistochemical stained liver slides of Nitric oxide synthase (iNOS). (A) Normal liver showing iNOS negative staining (20×); (B) CAJ control liver specimen without iNOS positive cells (20×); (C) Day-14, DEN-treated cell showing excessive accumulation of iNOS positive cell (20×); (D) Day-14, DEN+CAJ supplemented slides showed scattered and decreased positive staining of iNOS with mild liver damage (20×). Arrow shows positive immune cells.

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as reducing agents (donate hydrogen), quenchers of singlet oxygen, chelators and trappers of free radicals at different stages of prevention and repair [17,48,67]. However, CAJ performs antifibrotic action by increasing the levels of hepatic phase-II enzymes, depleting free radicals, discouraging collagenesis via declining HSC activation. Literature reveals that acute inflammation causes an increase in the expression of cytokines TNF-α, IL-6 and IL-10 along with COX-2 and iNOS [68], implying their significant role in numerous human pathologies [13,69]. Hence, these proteins can be considered as prospective targets for therapeutic intervention. Published reports demonstrated upregulation of COX-2 during hepatocyte dedifferentiation or proliferation, suggesting involvement of prostaglandins in the liver pathologies [70,71]. Concurrently, nitric oxide (NO) radical is also considered as a key regulator of various pathophysiological and biological systems [72,73]. The concentration of nitric oxide has been estimated in cultured (RAW264.7) cells either treated with LPS or with phytoextract [74]. These authors seeded 5 × 105 cells/well in 96-well plates, incubated them for 24 h at 37 °C, treated with LPS or C. annuum L stalk extract for 18 h and finally mixed with Griess reagent to measure NO production. LPS-induced injury reportedly activates iNOS in Kupffer cells, endothelial cells and hepatocytes in order to initiate NO production in liver in vitro [75]. In the present study, the expression of COX-2/iNOS was examined during DEN-induced hepatic injury and CAJ supplementation. Animals belonging to controls showed absence of COX-2/iNOS positive cells while DEN-treated group revealed excessive presence of both the proteins indicating liver inflammation. COX-2 up-regulation in the liver of DEN-administered animals specify increased prostaglandin synthesis during fibrogenesis, as also reported previously [76]. Further elevated levels of iNOS specify enhanced NO production, which is also observed during the present investigations. However, a significant decline in the expression of COX-2/iNOS activity in CAJ-supplemented group is suggestive of anti-inflammatory activity of CAJ. These findings, therefore, suggest iNOS and COX-2 as the strong targets of CAJ action in regressing DEN-induced hepatic injury. Recently, we have proposed ten protein signatures with their novel roles during nitrosodimethylamineinduced liver fibrosis and mitigation by resveratrol [77], while in another reports iNOS, COX-2 and Nrf2 proteins have also been specifically worked out as potential therapeutic targets to abrogate liver injury [57,78,79]. 5. Conclusion In conclusion, the present study convincingly demonstrates role of COX-2 and iNOS during therapeutic intervention by CAJ in rodent disease model. The presence of antioxidants (polyphenols) in the CAJ offers remarkable protection against the disease within two weeks. It is envisaged that CAJ-mediated anti-inflammatory action and detoxification occurs via free radical scavenging and triggering the synthesis of hepatic phase-II enzymes along with down-regulation of COX-2/iNOS. Thus, this study discovers a novel mechanism of mitigation by CAJ and suggests CAJ as the potential phytomedicine against DEN-induced hepatic injury in rats. Acknowledgements Authors sincerely express gratitude to the Chairman, Department of Zoology, Aligarh Muslim University, Aligarh for providing necessary facilities. DM is thankful to USIF, AMU (under DST-PURSE program) and AIRF, JNU for electron microscopy facilities. Special thanks are due to Dr. Maryam Sarwat, Amity University for extending HPLC facility. Financial assistance to DM (Non-NET, University Grants Commission, New Delhi) is thankfully acknowledged. Declaration of competing interest Authors confirm that there is no conflict of interest.

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