Biomedicine & Pharmacotherapy 95 (2017) 529–535

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Original article

Protective effects of phloridzin against methotrexate-induced liver toxicity in rats

MARK



Mohamed M.A. Khalifaa, Adel G. Bakrb, , Adel T. Osmanb a b

Faculty of Pharmacy, Department of Pharmacology & Toxicology, Minia University, Minia 61511, Egypt Faculty of Pharmacy, Department of Pharmacology & Toxicology, Al-Azhar University, Assiut 71524, Egypt

A R T I C L E I N F O

A B S T R A C T

Keywords: Methotrexate Phloridzin Caspase-3 Hepatotoxicity Oxidative stress Rats

Background: Liver is the largest internal organ concerning with metabolism, hormonal balance and clarifying of the toxins. One of the main complications of methotrexate (MTX) therapy was the hepatic injury. Objective: This study was conducted to elucidate the possible protective effects of phloridzin (PHL) against MTXinduced hepatotoxicity as compared to standard agent N-acetylcysteine (NAC). Materials and methods: Rats were randomly divided into a normal control group, a respective group (PHL 40 mg/ kg/day orally (p.o.) for 10 consecutive days), a hepatotoxicity control group (MTX 20 mg/kg, i.p., once), and three treated groups received NAC (150 mg/kg/day; a reference standard), PHL (40 mg/kg/day) and PHL (80 mg/kg/day) p.o. for 10 consecutive days, at the end of the day 3 of the experiment rats were administered MTX. Assessed biomarkers included serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) as liver function parameters, serum tumor necrosis factor-α (TNF-α) and cyclooxygenase-II (COX-II), as inflammatory biomarkers, hepatic total antioxidant capacity (TAC), thiobarbituric acid reactive substances (TBARS), glutathione reduced (GSH), nitrite (NO2−), catalase (CAT), glutathioneS-transferase (GST) and superoxide dismutase (SOD) as oxidative stress biomarkers. Furthermore, hepatic caspase-3 expression was assessed. Biochemical and molecular estimations reinforced by histopathological findings. Results: Rats pre-treated with PHL significantly reduced hepatic injury, evidenced by significant reductions in ALT, AST and LDH, TNF-α and COX-II levels, significant reductions in hepatic NO2− and TBARS levels, and significant elevations in hepatic TAC, GSH, GST, CAT and SOD levels. Additionally, downregulation of hepatic caspase-3 expression. Finally, histopathological results consistent with our previous findings. Conclusion: PHL protects against hepatic injury in rats mainly through mitigation of oxidative stress, inflammation and apoptosis in hepatic tissues and may be promising to alleviate and early treatment of MTXinduced hepatoxicity in man.

1. Introduction Liver is the main vital organ in the body. However, the drug-induced hepatic injury could be classified as a major problem that challenges the course of the drug therapy and limits its beneficial role [1]. Drugs-induced liver injury through different pathways comprises an immunological reaction, direct toxic effect, and active metabolite formation [2]. Methotrexate (MTX), is a folic acid antagonist widely used for treatment and prophylaxis of several disorders, including autoimmune diseases, malignant tumours, and inflammatory disorders [3,4]. The molecular mechanism by which MTX induces hepatotoxicity is not fully understood, but clinical and experimental studies suggest oxidative stress-mediated injury has a role in this toxicity, mainly by depleting folate species and this enhances several biochemical pathway



Corresponding author. E-mail address: [email protected] (A.G. Bakr).

http://dx.doi.org/10.1016/j.biopha.2017.08.121 Received 2 May 2017; Received in revised form 26 August 2017; Accepted 28 August 2017 0753-3322/ © 2017 Elsevier Masson SAS. All rights reserved.

alterations, including purine metabolism [5]. These biochemical alterations have been mainly reported for both the therapeutic and the toxic effects of MTX mainly through the propagation of the oxidative stress and initiation of inflammatory pathways [6,7]. Furthermore, a reduction in an intracellular NADPH levels, which in consequence depletes reduced glutathione (GSH), which is an important cytosolic antioxidant agent [8]. Finally, MTX disrupts oxidant/antioxidant status and these effects can be attenuated by treatment with scavengers of reactive oxygen species (ROS). It is reported that this approach has promise for preventing and early treatment of MTX-induced liver damage [9]. Phloridzin (PHL) is a dihydrochalcone glycoside, which is a kind of flavonoid glycoside mainly found in apples and strawberries with a chemical structure shown in Fig. 1 [10]. PHL is a competitive inhibitor

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days, at the end of the 3rd day, rats were injected i.p. once with MTX 20 mg/kg bw [15]. 6. Group VІ: Administered PHL 80 mg/kg/day, p.o., for 10 consecutive days, at the end of the 3rd day, rats were injected i.p. once with MTX 20 mg/kg bw [15]. 2.4. Methods 2.4.1. Induction of liver toxicity Administration of single dose of methotrexate to all groups (except control and PHL40) for developing liver injury was done. Each animal received 20 mg/kg methotrexate in normal saline i.p. once [22].

Fig. 1. Chemical structure of PHL [10].

of sodium-glucose linked transporters (SGLTs) [11], a potent antioxidant [12], anti-inflammatory [13], inhibition of platelet activation [14] and recently used in digestive diseases as liver disorders [15]. N-acetylcysteine (NAC) is a well-documented as a powerful antioxidant, anti-inflammatory, hepatoprotective, sulfhydryl group donor and a precursor for GSH synthesis [16]. NAC plays a vital role as cell survival promotion, downregulation of apoptosis and scavenging of ROS [17]. NAC is used clinically in the treatment of cancer [18], heart disease [19], colitis [20] and pancreatitis [21]. Based on the previous background, the aim of this experimental study was to evaluate the possible hepatoprotective effects of PHL against MTX-induced hepatotoxicity when compared to standard agent NAC.

2.4.2. Serum preparation At the end of the experiment, rats were anasthetized with ketamine (50 mg/kg) and xylazine (10 mg/kg) by i.p. injection [24], blood samples were withdrawn by a direct cardiac puncture. Sera were separated and immediately stored at −40 °C till the time of assay. 2.4.3. Preparation of tissue homogenate Liver of each animal was excised being careful to remove adhering fat and connective tissues and washed in ice-cold isotonic saline and divided into three portions. The first portion was stored in 10% neutral buffered formalin solution and was subjected to histopathological examinations. The second portion was homogenized (20%) with (ColeParmer instrument company, USA) in cold phosphate buffered saline (PBS). Tissue homogenates were centrifuged at 3000 rpm for 15 min at 4 °C. The supernatant was collected, divided into aliquots and stored at − 70 °C for evaluation of oxidative stress parameters. The third portion was frozen in liquid nitrogen and was stored separately at −70 °C for subsequent reverse transcriptase-polymerase chain reaction (RT-PCR) analysis.

2. Materials and methods 2.1. Animals Sixty-six healthy male Swiss albino rats (weighing 200 ± 10 g) were obtained from the laboratory animal colony (Faculty of Medicine, Assiut University, Egypt). Rats were divided into 12 cages at a regulated environment (12-h dark/light cycle, 22 ± 2 °C temperature and 50 ± 5% humidity). Rats were fed with standard diet (El-Nasr Company, Abou Zaabal, Cairo, Egypt) with free access to water ad libitum. Experimental ethics and procedure in accordance with the international ethical guidelines for animal care and approved by the ethical committee, Faculty of Pharmacy, Minia University, Egypt.

2.4.4. Assessment of biochemical parameters Using commercially available kits, ALT, AST were analyzed according to the method described by Reitman and Frankel [25]. LDH was analyzed according to the method described by Izquierdo and Dias [26]. Serum and tissue total proteins were determined according to the method described by lowery et al. [27]. Serum COX-II level was determined using ELISA kit according to the manufacturing instruction based on the principle described by Van Weemen and Schuurs [28]. Serum TNF-α was determined using ELISA kit according to the manufacturing instruction based on the principle described by Wolters et al. [29]. Hepatic GSH was assayed according to the method described by Ellman [30]. Hepatic TBARS were assayed according to the method described by Uchiyama and Mihara [31]. NO2− was assayed according to the method described by Montgomery and Dymock [32]. Hepatic TAC was assayed according to the method described by Koracevic et al. [33]. Antioxidant enzyme GST, CAT and SOD were assayed according to the methods described by Keen et al. [34], Claiborne [35] and Marklund [36] respectively.

2.2. Drugs, chemicals and reagent kits Methotrexate was purchased from T3A Company (Cairo, Egypt), phloridzin dihydrate was purchased from Sigma-Aldrich chemical company (St Louis, MO, USA). NAC was obtained from SEDICO (6th October, Giza, Egypt). Thiobarbituric acid, GSH, Pyrogallol, N-(1Naphthyl) ethylenediamine dihydrochloride and 5,5’-dithio-bis-(2- nitrobenzoic acid; DTNB) were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other chemicals were obtained from local sources with highest analytical grade. 2.3. Experimental design

2.4.5. Determination of hepatic Caspase-3 expression level by reverse transcriptase-polymerase chain reaction Total RNA was extracted using Thermoscript™ reverse transcriptasepolymerase chain reaction (RT-PCR) system (Promega, Madison, WI, USA) according to the manufacturer’s protocol, cDNA synthesis was done by reverse transcriptase (RT) reaction using 1 μg of total RNA according to manufacture instruction with random primers. RT-PCR technique using available kits and according to the method described by Dahiya et al. [37], using specifically designed primers as follow: The caspase-3 gene was analyzed with the primers 5′-CGGCAGGCCTGATGAAG-3′(sense) and 5′GGACAGCAGTTCAAAATGGATTA-3′ (antisense). The β-actin gene was analyzed with the primers 5′-CCACCATGTACCCAGGCATT-3′ (sense) and 5′ACGCAGCTCAGTAACAGTCC-3′ (antisense).

After the acclimatization period, sixty-six male Swiss albino rats were divided into six groups; 11 rats in each group as follows: 1. Group I: control group which received normal saline only. 2. Group ІІ: Administered PHL 40 mg/kg/day, p.o., for 10 consecutive days, at the end of the 3rd day, rats were injected with normal saline i.p. once. 3. Group ІІІ: Injected i.p. once with MTX 20 mg/kg bw [22]. 4. Group ІV: Administered NAC 150 mg/kg/day, p.o., for 10 consecutive days, at the end of the 3rd day, rats were injected i.p. once with MTX 20 mg/kg bw [23]. 5. Group V: Administered PHL 40 mg/kg/day, p.o., for 10 consecutive 530

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The amplification products of caspase-3 and β-actin were detected at 393 bp and 243 bp respectively. The amplification reaction was performed in a total volume of 50 μl in the presence of 1.5 μl of each primer, 33.5 μl water nuclease free, 9.5 μl of Master Mix and 1.5 μl of cDNA. PCR procedure was performed in a thermal cycle (Crocodile III, Appligene, France), with an initial denaturation step of 5 min at 95 °C, followed by annealing and elongation steps (40 s at 95 °C and 40 s at 53 °C) for caspase-3 and (40 s at 95 °C and 40 s at 56 °C) for β-actin of total 40 cycles of each, followed by extension step at 72 °C for 1 min and terminated by one cycle of Final extension step at 72 °C for 10 min. All samples were amplified and RT-PCR products were analyzed using gel (2% agarose containing 0.5 mg/ml ethidium bromide) electrophoresis technique. Electrophoresis was done using a constant volt (40 V) for10 min and (80 V) for 30 min using a PCR marker (Promega Madison, Code No. G316A), photographed using a digital camera and quantification of bands intensity using Image J software and expressed as a β-actin ratio.

3.2. Effect of NAC, PHL40 and PHL80 administration on inflammatory biomarkers of MTX- treated rats In our study, COX-2 and TNF-α levels were significantly increased in rats administrated with MTX to 605% and 312%, respectively, when compared to control group. Rats pre-treated with NAC, PHL40 and PHL80 for 10 consecutive days significantly reduced serum COX-2 levels to 39%, 60% and 39%, respectively, and significantly reduced serum TNF-α levels to 45%, 58% and 43%, respectively, when compared to MTX-treated group (Table 2). 3.3. Effect of NAC, PHL40 and PHL80 administration on oxidative stress biomarkers of MTX- treated rats Hepatic tissue GSH content and TAC were significantly decreased in rats administrated with MTX to 37% and 47%, respectively, when compared to control group. Rats pre-treated with NAC, PHL40 and PHL80 for 10 consecutive days significantly increased hepatic GSH content to 292%, 185% and 225%, respectively, and significantly increased hepatic TAC to 191%, 159% and 176%, respectively, when compared to MTX-treated group. Hepatic tissue TBARS and NO2− contents significantly increased in rats administrated with MTX to 255% and 250%, respectively, when compared to control group. Rats pre-treated with NAC, PHL40 and PHL80 for 10 consecutive days significantly reduced hepatic TBARS content to 46%, 64% and 50%, respectively, and significantly reduced hepatic NO2− content to 55%, 70% and 53%, respectively, when compared to MTX-treated group. Hepatic tissue SOD, CAT and GST enzymes activities significantly decreased in rats administrated with MTX to 50%, 7% and 34%, respectively, when compared to control group. Rats pre-treated with NAC, PHL40 and PHL80 for 10 consecutive days significantly raised hepatic SOD activity to 182%, 142% and 164%, respectively, and significantly raised hepatic catalase enzyme activity to 811%, 622% and 784%, respectively. Furthermore, these groups significantly increased hepatic GST enzyme activity to 198%, 151% and 192%, respectively, when compared to MTX-treated group (Table 3).

2.4.6. Histopathological examination The technique was performed by immediately immersion of a portion of the isolated livers in 10% buffered formalin solution in normal saline for 24 h, followed by washing with distilled water, dehydration using serial dilutions of alcohol, dehydration using xylene and embedded in paraffin for 24 h in a hot air oven at 56 °C respectively. Tissue blocks were made using paraffin beeswax by sledge microtome, followed by deparaffination and staining by hematoxylin and eosin [38]. 2.4.7. Statistical analysis Statistical analyses of the data were carried out using GraphPad prism version 5.0. Data comparisons were performed using analysis of variance (ANOVA) followed by Tukey-Kramer multiple comparisons test for post hoc analysis. The levels of significance were accepted with p < 0.05 and all relevant results were graphically displayed as mean ± SEM. Survival curve was generated using the Kaplan-Meier method by GraphPad prism version 5.0 (Graph pad software San Diego, USA).

3.4. Effect of NAC, PHL40 and PHL80 administration on caspase-3 expression in hepatic tissue estimated by RT-PCR of MTX- treated rats

3. Results

As shown in Fig. 2, administration of MTX resulted in a marked increase in the expression of the mRNA for caspase-3 as detected by RTPCR. However, PHL40 group showed a moderate reduction in caspase-3 mRNA expression. These reductions in caspase-3 mRNA expression were more prominent in NAC and PHL80 groups when compared to MTX-treated group.

3.1. Effect of NAC, PHL40 and PHL80 administration on liver function biomarkers of MTX- treated rats In our study, ALT, AST and LDH activities significantly increased in rats administrated with MTX to 418%, 231% and 377%, respectively, when compared to control group. Rats pre-treated with NAC, PHL40 and PHL80 for 10 consecutive days significantly reduced serum levels of ALT to 64%, 89% and 67%, respectively, and significantly reduced AST serum levels to 68%, 85% and 70%, respectively. Furthermore, LDH levels significantly reduced to 70%, 80% and 68%, respectively, when compared to MTX-treated group (Table 1).

3.5. Effect of NAC, PHL40 and PHL80 administration on survivability of MTX- treated rats At the time of our initial experiment, survival analysis was

Table 1 Effect of NAC, PHL40 and PHL80 administration on serum liver function biomarkers of MTX-treated rats. Parameters ALT (U/ml) AST (U/ml) LDH (U/L)

Control 53 ± 2.5 114 ± 5.7 1200 ± 53

PHL40 58 ± 3.4 115 ± 5.4 1267 ± 49

MTX

MTX + NAC a

222 ± 4.4 263 ± 4.7a 4527 ± 119a

a,b

141 ± 3.2 179 ± 7.4a,b 3150 ± 132a,b

MTX + PHL40 a,b,c

197 ± 4.3 223 ± 8.5a,b,c 3600 ± 115a,b,c

MTX + PHL80 149 ± 3.6a,b 185 ± 6.4a,b 3087 ± 127a,b

(MTX; methotrexate, PHL40; phloridzin40, PHL80; phloridzin80, NAC; N- acetylcysteine, ALT; alanine aminotransferase, AST; aspartate aminotransferase and LDH; lactate dehydrogenase). −Data were expressed as mean ± SEM (n = 8). −Multiple comparisons were done using one-way ANOVA followed by Tukey-Kramer as a post ANOVA test. a Significantly different from control group at P < 0.05. b Significantly different from MTX group at P < 0.05. c Significantly different from MTX + NAC group at P < 0.05.

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Table 2 Effect of NAC, PHL40 and PHL80 on inflammatory biomarkers of MTX treated rats. Parameter

Control

PHL40

MTX

MTX ± NAC

MTX ± PHL40

MTX ± PHL80

COX-2 (ng/mg protein) TNF-α (ng/mg protein)

76 ± 2.9 204 ± 12

80 ± 1.8 202 ± 10

460 ± 20a 636 ± 17a

178 ± 4.6a,b 286 ± 21a,b

277 ± 8a,b,c 366 ± 17a,b,c

180 ± 3.2a,b 275 ± 14a,b

(MTX; methotrexate, PHL40; phloridzin40, PHL80; phloridzin80, NAC; N- acetylcysteine and COX-2; cyclooxygenase-2 and TNF-α; tumor necrosis factor-alpha). − Data were expressed as mean ± SEM (n = 8). −Multiple comparisons were done using one-way ANOVA followed by Tukey-Kramer as a post ANOVA test. a Significantly different from control group at P < 0.05. b Significantly different from MTX group at P < 0.05. c Significantly different from MTX + NAC group at P < 0.05.

pointer of hepatocellular injuries [43,44]. GSH is a very important powerful reducing agent [45]. TBARS is an important marker for the degree of lipid peroxidation [4]. NO plays vital roles in pathological conditions and inflammation [46]. SOD, GST and CAT, whose primary role within the antioxidant defence mechanism, reduce or completely eliminate the hazardous effect of ROS [34,47]. TAC is an important marker for whole redox status evaluation [48]. COX-2 is an important inflammatory mediator which strongly participates in the progression of inflammation and cancer [49]. TNF is a fundamental mediator for both acute and chronic systemic inflammatory processes [50]. Caspases are important signalling molecules of apoptosis, detection of cleaved caspases indicative for an early stage of apoptosis [51]. Caspase-3 is one of the most important proteases that initiate both the extrinsic and intrinsic apoptosis pathway and, also an indicator of the irreversible point of the apoptosis [52]. The present investigation was conducted to illustrate the possible protective effects of PHL with strongly promising backgrounds, against liver injury induced experimentally by MTX in adult male rats. In our study, acute single administration of MTX was shown to cause deterioration in liver function biomarkers evidenced by a significant increase in serum levels of ALT, AST and LDH as compared to control group. Additionally, MTX caused significant elevations in inflammatory mediators TNF-α and COX-2 as compared to control group, which confirmed a link between MTX injury and inflammation. Moreover, MTX was demonstrated to initiate oxidative and nitrosative biomarkers evidenced by a significant reduction in hepatic TAC, a significant reduction in hepatic GSH content, significantly reduced hepatic antioxidant enzymes GST, CAT and SOD activities, and significantly elevated hepatic TBARS and NO2− contents. Additionally, MTX was shown to increase cell apoptosis, which manifested by a significant upregulation of hepatic mRNA caspase-3 expression. Furthermore, a reduction in survival rate, which manifested by acute liver failure caused by MTX [3]. Finally, a progression of histopathological changes with inflammatory cell infiltration in the liver tissue compared to control group, which strongly coherent with biochemical

performed by monitoring mortality of 66 rats divided into six groups for 10 consecutive days. In our study, MTX-treated group showed survivability rates 91%, 81% and 73% at the 7th, 8th and 10th days respectively. Additionally, Rats pre-treated with PHL40 for 10 consecutive days showed the same survivability rates of MTX-treated group. While, rats pre-treated with NAC and PHL80 for 10 consecutive days showed survival rates 91% and 82% at the 8th and 10th days respectively, as shown in Kaplan-Meier plots Fig. 3. 3.6. Effect of NAC, PHL40 and PHL80 administration on histopathological alteration of MTX-treated rats Results of the histopathological study, showed that administration of MTX caused severe changes confined as vacuolation of hepatocytes, focal hepatic necrosis associated with inflammatory cell infiltration, congestion of central vein and dilatation of hepatic sinusoids, cholangitis and the appearance of newly formed bile ductules. Individual administrations of PHL40, PHL80 and NAC for 10 consecutive days were able, to some extent, to reduce MTX-induced pathological alterations in the liver tissue architecture as shown in Fig. 4 (photo A–H). 4. Discussion The mechanism by which liver injury predominantly caused by MTX was completely illustrated, it has been reported that chronic use of MTX was established to be one of the aggravating factors of hepatotoxicity [39]. Additionally, a high dose administration was considered as a risk factor for inducing liver injury [40], which manifested by steatosis, cholestasis, fibrosis and cirrhosis [41]. The postulated mechanism of MTX-induced hepatotoxicity mainly investigated by two routes, one of them is a polyglutamate accumulation inside the liver cells, which consequently deplete folate pool [42], and the other; initiation of oxidative stress pathways [39]. Serum ALT, AST and LDH are the most often used and particular Table 3 Effect of NAC, PHL40 and PHL80 on oxidative stress biomarkers of MTX-treated rats. Parameters GSH (μmol/mg protein) TBARS (nmol/mg protein) NO2− (μmol/mg protein) TAC (mmol/mg protein) SOD (U/μg protein) CAT (U/μg protein) GST (U/μg protein)

Control 3.26 ± 0.16 11 ± 0.34 8 ± 0.6 0.73 ± 0.030 0.11 ± 0.009 0.05 ± 0.001 2.5 ± 0.05

PHL40 3.7 ± 0.08 10 ± 0.2 7.6 ± 0.6 0.80 ± 0.025 0.120 ± 0.008 0.06 ± 0.0006 2.1 ± 0.08

MTX

MTX ± NAC a

1.2 ± 0.09 28 ± 1.5a 20 ± 1.5a 0.34 ± 0.026a 0.055 ± 0.003a 0.0037 ± 0.0002a 0.86 ± 0.02a

b

3.5 ± 0.14 13 ± 0.32b 11 ± 0.8b 0.65 ± 0.046b 0.10 ± 0.0004b 0.030 ± 0.0008a,b 1.7 ± 0.08a,b

MTX ± PHL40 a,b

2.22 ± 0.19 18 ± 0.50a,b,c 14 ± 1.1a,b 0.54 ± 0.026a,b 0.078 ± 0.004a,b,c 0.023 ± 0.001a,b,c 1.3 ± 0.05a,b,c

MTX ± PHL80 2.7 ± 0.16b 14 ± 0.33b 10.5 ± 0.8b 0.60 ± 0.031b 0.09 ± 0.006b 0.029 ± 0.0017a,b 1.65 ± 0.014a,b

(MTX; methotrexate, PHL40; phloridzin40, PHL80; phloridzin80, NAC; N- acetylcysteine, GSH; reduced glutathione, TBARS; thiobarbituric acid reactive substance, NO2−; nitrite, TAC; total antioxidant capacity, SOD; superoxide dismutase, CAT; catalase and GST; glutathione-S-transferase). −Data were expressed as mean ± SEM (n = 8). −Multiple comparisons were done using one-way ANOVA followed by Tukey-Kramer as a post ANOVA test. a Significantly different from control group at P < 0.05. b Significantly different from MTX group at P < 0.05. c Significantly different from MTX + NAC group at P < 0.05.

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Fig. 2. Effect of NAC, PHL40 and PHL80 administration on caspase-3 expression in hepatic tissue estimated by RT-PCR of MTX-treated rats. Effect of NAC, PHL40 and PHL80 on caspase-3 expression in hepatic tissue estimated by RT-PCR. Detection of mRNA fragments of β-actin was achieved in parallel as an internal control. The lower panels represent the corresponding quantification of bands intensity using Image J software and expressed as a β-actin ratio. Data were expressed as mean ± SEM. a Significantly different from control group at P < 0.05. b Significantly different from MTX group at P < 0.05. c Significantly different from MTX + NAC group at P < 0.05.

phosphorylated IkK loses its activity which may lead to KB attachment to DNA sequences which consequently reduce inflammatory mediators expression, and manifested by significantly decreased serum levels of COX-2 and TNFα. In agreement with Chang et al. who reported that phloretin reduces inflammatory mediators in lipopolysaccharide-stimulated mouse macrophages [13]. More recently, Aliomrani et al. concluded that phloretin ameliorates inflammation in a rat model of cecal ligation and puncture-induced sepsis [56]. PHL showed strong antioxidant capacity, which explained by its ability to inhibit xanthine oxidase enzyme which, consequently inhibits free radical formation as well as inhibiting the breakdown of a potent antioxidant uric acid [57]. Additionally, stabilization of the radical after hydrogen abstracting by keto-enol tautomerization state which strengthened by intramolecular hydrogen bond [58], and demonstrated by a significant raising in hepatic TAC, a significant elevation in hepatic content of GSH, and significantly reduced hepatic TBARS and NO2− contents, which confirmed by Serino et al. in streptozocin-induced diabetic in rat [59]. Moreover, significantly elevated hepatic antioxidant enzymes GST, CAT and SOD activities. In addition, PHL showed anti-apoptotic activity in the liver tissue, which explained by induction of heme oxygenase (HO)-1 expression by nuclear factor erythroid 2 (NrF2) and c-Jun N-terminal kinase (JNK) pathways activation, which contribute to cell survival and inhibit cell progression to apoptosis, and demonstrated by a significant downregulation of hepatic mRNA caspase-3 expression, came in accordance with Choi et al. who concluded that phloretin reduces cisplatin-induced apoptosis in auditory cells [60]. Furthermore, pre-treatment with PHL showed a reduction in the mortality rate of MTX-treated rats demonstrated by the ability of PHL to ameliorate liver injury caused by MTX [15]. Finally, PHL showed alleviation in histopathological changes which strongly consistent with biochemical findings. According to our study, PHL exerts its protective action on hepatic tissue as compared to MTX control group, which explained by free radical scavenger activity and alleviation of injury caused by ROS production and have the potential as an anti-inflammatory agent as well as reduction of apoptotic changes induced by MTX.

Fig. 3. Effect of NAC, PHL40 and PHL80 administration on survivability of MTX treated rats. Kaplan–Meier curve showing the effects of NAC, PHL40 and PHL80 on rats survivability of MTX-treated groups. The y axis shows the percentage of MTX-treated rats still alive at any point in time. The x axis shows the number of days since starting therapy. (MTX; methotrexate, PHL40; phloridzin40, PHL80; phloridzin80, NAC; N- acetylcysteine).

findings. Thus, these present results confirmed earlier findings [22,53]. In our study, results of PHL administration depend mainly on its metabolite form Phloretin [54]. Pre-treatment with PHL showed corrections in liver function biomarkers, which manifested by significantly reduced serum levels of ALT, AST. In accordance, Deng et al. showed a harmony with our results in CCL4-induced liver toxicity [15], as well as a significant reduction in serum LDH level. In agreement, Ataka et al. reported that PHL administration resulted in a decrease in LDH level in physical fatigue [55]. In our study, pre-treatment with PHL showed hepatoprotective potential evidenced by the ability of PHL to ameliorate the progression of the inflammation, which explained by inhibition of pro-inflammatory cytokines and blocking activation of mitogen-activated protein kinase (MAPK) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) pathways. PHL is responsible for NF-kB deactivation through the I kappa B kinase (IkK) phosphorylation. The 533

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Fig. 4. Effect of NAC, PHL40 and PHL80 administration on histopathological study of MTX-treated rats. Representative photomicrographs of hepatic sections stained with (H & E X 400). (A) Control normal group showed normal histological structure of the hepatic lobule. (B) PHL40 group showed hydropic degeneration of hepatocytes (black arrow). (C) MTX-treated group showed vacuolation of hepatocytes and focal hepatic necrosis associated with inflammatory cell infiltration (black arrow). (D) MTX-treated group showed cholangitis and an appearance of newly formed bile ductules (black arrows). (E) MTX + NAC group showed congestion of central vein and hydropic degeneration of hepatocytes (black arrows). (F) MTX + PHL40 group showed Kupffer cell activation (black arrows). (G) MTX + PHL40 group section shows hydropic degeneration of hepatocytes and mononuclear inflammatory cell infiltration (black arrows). (H) MTX + PHL80 group showed Kupffer cell activation (black arrow) and hydropic degeneration of hepatocytes (black star). VC: Vena centralis; H & E: Hematoxylin-eosin; MTX: Methotrexate; NAC: N-acetyl cysteine; PHL: Phloridzin.

References

5. Conclusion Our study aimed to investigate the effectiveness of PHL on MTXinduced hepatotoxicity in terms of histopathological variables, biochemical parameters and molecular estimation as compared to NAC as a standard agent. Administration of a single dose of MTX (20 mg/kg, i.p.) caused toxic effects on liver tissues and PHL alleviated these effects. PHL has been investigated to ameliorate MTX-toxicity by mitigation of oxidative stress, inflammation and apoptosis in hepatic tissues. These results are promising for further clinical trials to demonstrate the effectiveness of PHL to prevent and early treatment of MTX- induced hepatotoxicity in man.

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Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biopha.2017.08.121. 534

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Protective effects of phloridzin against methotrexate-induced liver toxicity in rats.

Liver is the largest internal organ concerning with metabolism, hormonal balance and clarifying of the toxins. One of the main complications of methot...
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