Toxicology and Applied Pharmacology 279 (2014) 129–140

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Chrysin alleviates testicular dysfunction in adjuvant arthritic rats via suppression of inflammation and apoptosis: Comparison with celecoxib Hebatallah A. Darwish a, Hany H. Arab a,⁎, Rania M. Abdelsalam b a b

Department of Biochemistry, Faculty of Pharmacy, Cairo University, Cairo 11562, Egypt Department of Pharmacology and Toxicology, Faculty of Pharmacy, Cairo University, Cairo 11562, Egypt

a r t i c l e

i n f o

Article history: Received 11 February 2014 Revised 12 May 2014 Accepted 30 May 2014 Available online 14 June 2014 Keywords: Chrysin Celecoxib Testicular dysfunction Rheumatoid arthritis Apoptosis Inflammation

a b s t r a c t Long standing rheumatoid arthritis (RA) is associated with testicular dysfunction and subfertility. Few studies have addressed the pathogenesis of testicular injury in RA and its modulation by effective agents. Thus, the current study aimed at evaluating the effects of two testosterone boosting agents; chrysin, a natural flavone and celecoxib, a selective COX-2 inhibitor, in testicular impairment in rats with adjuvant arthritis, an experimental model of RA. Chrysin (25 and 50 mg/kg) and celecoxib (5 mg/kg) were orally administered to Wistar rats once daily for 21 days starting 1 h before arthritis induction. Chrysin suppressed paw edema with comparable efficacy to celecoxib. More important, chrysin, dose-dependently and celecoxib attenuated the testicular injury via reversing lowered gonadosomatic index and histopathologic alterations with preservation of spermatogenesis. Both agents upregulated steroidogenic acute regulatory (StAR) mRNA expression and serum testosterone with concomitant restoration of LH and FSH. Furthermore, they suppressed inflammation via abrogation of myeloperoxidase, TNF-α and protein expression of COX-2 and iNOS besides elevation of IL-10. Alleviation of the testicular impairment was accompanied with suppression of oxidative stress via lowering testicular lipid peroxides and nitric oxide. With respect to apoptosis, both agents downregulated FasL mRNA expression and caspase-3 activity in favor of cell survival. For the first time, these findings highlight the protective effects of chrysin and celecoxib against testicular dysfunction in experimental RA which were mediated via boosting testosterone in addition to attenuation of testicular inflammation, oxidative stress and apoptosis. Generally, the 50 mg/kg dose of chrysin exerted comparable protective actions to celecoxib. © 2014 Elsevier Inc. All rights reserved.

Introduction Rheumatoid arthritis (RA) is a chronic systemic inflammatory disease that principally attacks the joints. However, extra-articular manifestations are serious complications that jeopardize patients' quality of life. Among these complications, increased incidence of testicular dysfunction and impaired testosterone production have been reported in men with longstanding RA, particularly, during the eruption of disease activity (Bove, 2013; Bruot and Clemens, 1992; Clemens and Bruot, 1989; Cutolo et al., 2003). A negative correlation has been described between serum androgen levels and RA disease activity (Bove, 2013; Cutolo et al., 2003). The converse is also true; there is an increased incidence of autoimmune diseases including RA in men with untreated Abbreviations: AA, adjuvant-induced arthritis; COX-2, cyclo-oxygenase-2; Fas, apoptosis stimulating fragment; FasL, Fas ligand; FCA, Freund's complete adjuvant; FSH, follicle-stimulating hormone; H&E, hematoxylin and eosin; IL-10, interleukin-10; LH, luteinizing hormone; LPS, lipopolysaccharide; MDA, malondialdehyde; MPO, myeloperoxidase; NO, nitric oxide; NSAID, non-steroidal anti-inflammatory drug; RA, rheumatoid arthritis; ROS, reactive oxygen species; StAR, steroidogenic acute regulatory protein; TNF-α, tumor necrosis factor-α. ⁎ Corresponding author. Fax: +20 223628246. E-mail addresses: [email protected], [email protected] (H.H. Arab).

http://dx.doi.org/10.1016/j.taap.2014.05.018 0041-008X/© 2014 Elsevier Inc. All rights reserved.

hypogonadism (Jimenez-Balderas et al., 2001). It has been suggested that the severe presentation of RA symptoms in older men is probably secondary to the age-related deficiency of testosterone in those patients (Bove, 2013). Clinically, the low testosterone levels have been associated with increased risk of cardiovascular events, osteoporosis, obesity, cognitive impairment and depression (Buvat et al., 2013). The hallmark of RA is persistent activation of the immune system which impacts diverse body functions, including the endocrine system. The chronic inflammation and excessive generation of proinflammatory cytokines have been reported to downregulate testicular androgen production with consequent decline of serum bioavailable testosterone. Generally, testosterone has been regarded as a natural antiinflammatory agent that can suppress hormonal and cellular immune responses (Bove, 2013; Cutolo et al., 2002). Thus, the deficiency of endogenous testosterone to control the immune system has been implicated in the incidence of RA flares (Tengstrand et al., 2002). Adjuvant-induced arthritis (AA), an experimental model of human RA, is commonly used to investigate the mechanisms of immune system modulation of the reproductive system (Bruot and Clemens, 1992; Clemens and Bruot, 1989; Jurcovicova et al., 2009). In male rats with AA, testicular impairment was evident with lowered serum testosterone and elevated luteinizing hormone (LH) levels (Clemens and Bruot,

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1989). During the clinically active phase of AA, testicular macrophages have been reported to secrete a proinflammatory factor that diminishes the production of testosterone (Bruot and Clemens, 1992). Attempts have been made towards the management of testicular dysfunction and associated hypogonadism in RA. Some studies have demonstrated the benefits of testosterone therapy in improving the clinical and chemical markers of immune response in men with RA (Bove, 2013; Cutolo et al., 2002). However, pharmacological doses of testosterone might be associated with adverse effects and risk of developing prostate cancer (Bain, 2007). A previous study has indicated the ameliorative actions of methotrexate on testicular suppression in rats with AA (Jurcovicova et al., 2009). Yet, the risk of genotoxicity and chromosomal aberrations is a drawback in the use of methotrexate therapy (Karagiannis and Harsoulis, 2005). Thus, the search for effective agents that can halt the progression of RA disease activity along with associated testicular impairment in RA seems imperative. Emerging evidence has indicated the central role of the steroidogenic acute regulatory (StAR) protein in the biosynthesis of testosterone within Leydig cells (Wang et al., 2005, 2009). Chrysin, a natural flavone abundant in honey and bee propolis, has been reported to upregulate the expression of StAR gene with concomitant increase of testosterone production (Jana et al., 2008). It also boosts testosterone via suppression of aromatase, an enzyme that converts testosterone to estradiol (Dhawan et al., 2002; Wang et al., 2009). Chrysin displayed beneficial actions on male reproductive system in normal adult rats (Ciftci et al., 2012) and halted the decline of testosterone in aging rats (Dhawan et al., 2002). In the same context, Wang et al. (2005) have reported the favorable actions of cyclo-oxygenase-2 (COX-2) inhibition approach for reversing age-related decrease of StAR gene expression and enhancing testosterone biosynthesis. Interestingly, celecoxib, a selective COX-2 inhibitor, has been reported to mitigate lipopolysaccharide (LPS)-induced testicular injury (Winnall et al., 2009). Together, in the current study, we aimed to investigate the protective potential of chrysin and celecoxib, two testosterone boosting agents, and the implicated mechanisms against testicular dysfunction in rats with adjuvant arthritis. Materials and methods Animals All procedures relating to animal care and treatments strictly adhered to the recommendations in the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH publication No. 85-23, revised 1996). The protocol was approved by the Committee of Animal Care and Use of Faculty of Pharmacy, Cairo University. All efforts were made to minimize animal suffering. Adult male Wistar rats (200 ± 20 g) were obtained from Faculty of Pharmacy, Cairo University, Cairo, Egypt. Housing conditions were kept the same throughout the whole study. The animals were housed 4 per cage in clean sanitized standard cages (42 × 26 × 15 cm) under controlled environmental conditions at constant temperature (23 ± 2 °C), humidity (60 ± 10%), and a 12/12 h light/dark cycle. Frequent change of bedding and proper cleaning and disinfection of cages with appropriate ventilation of cages and removal of ammonia and odors were adopted to minimize the incidence of infection to animals. The rats were acclimatized for one week before any experimental procedures and were allowed standard rat chow (El-Nasr, Abu Zaabal, Egypt; 15% of protein, 3.5% of fat, 6.5% of crude fibers plus vitamins/minerals mixture) and water ad libitum. Reagents and drugs Freund complete adjuvant (FCA), chrysin and celecoxib were purchased from Sigma-Aldrich, St. Louis, MD, USA. ELISA kits for determination of TNF-α and IL-10 and caspase-3 colorimetric kit were

obtained from R &D systems, MN, USA. All Other chemicals were of the highest purity and analytical grade. Experimental design and treatment protocol In the current study, 40 rats were randomly divided into five groups (8 rats per group). Group I (Control gp): normal rats which received oral vehicle. Group II (AA gp): arthritic rats which received oral vehicle. Group III (AA + CHR 25 gp): arthritic rats treated with oral chrysin (25 mg/kg). Group IV (AA + CHR 50 gp): arthritic rats treated with oral chrysin (50 mg/kg). Group V (AA + CLX): arthritic rats treated with oral celecoxib (5 mg/kg). Chrysin (dissolved in corn oil vehicle) and celecoxib were administered by oral gavage starting 1 h prior to arthritis induction and were continued till the 20th day post FCA inoculation. Arthritis was induced in rats by injecting 0.2 mg of heat-killed Mycobacterium butyricum (FCA) subcutaneously into rat hind paw as previously described (Agha and Gad, 1995). The selected doses of chrysin were based on its previously reported beneficial actions on the reproductive system of normal male rats (Ciftci et al., 2012) and its proven anti-inflammatory effects in animal models of colon (Khan et al., 2012) and hepatic (Pushpavalli et al., 2010) toxicities. Celecoxib was selected as the reference antiinflammatory owing to its wide use as a NSAID in inflammatory conditions including rheumatoid arthritis (Steinbach et al., 2000) with proven anti-arthritic (Khayyal et al., 2005) and alleviating actions against LPS-induced testicular inflammation (Winnall et al., 2009). The selected dose of celecoxib was consistent with previous literature (Khayyal et al., 2005). The chosen regimen is in accordance with previous reports (Arab and El-Sawalhi, 2013; Bruot and Clemens, 1992; Clemens and Bruot, 1989; Jurcovicova et al., 2009). Blood and testes separation At the end of the experimental period (21st day), rats were weighed, slightly anesthetized and the blood samples were collected from the retro-orbital vein for separation of serum. Then, animals were euthanized under deep ether anesthesia and laparotomy was immediately performed. The testes were excised, washed with ice-cold saline, blotted dry and weighed. One testis was cut into 2 halves and fixed in 10% buffered formol saline for the histopathological and immunohistochemical analysis (COX-2 and iNOS protein expression) while the other was decapsulated, divided into 4 parts and stored at − 80 °C. One part was homogenized in 10 volumes of lysis buffer (25 mM HEPES, 5 mM MgCl2, 1 mM EGTA, 0.5% Triton × 100, 5 mM DTT, 1 mM pefablock, pH 7.4) for the estimation of caspase-3 activity. The second part was homogenized in 10 volumes of ice-cold saline for determination of oxidative stress markers (MDA and NO). The third part was homogenized in 50 mM potassium phosphate buffer (pH 6) containing 0.5% of hexadecyltrimethylammonium bromide for the estimation of MPO activity. The last part was used for the separation of RNA to assess the mRNA expression of StAR and FasL genes. Serum was used for the determination of testosterone, LH and follicle stimulating hormone (FSH) in addition to TNF-α and IL-10 inflammatory cytokines. Measured parameters Increase in paw volume. The volume of the injected paw was measured by means of a digital plethysmometer (Ugo Basile, Italy) on day 0 and then on the 4th, 7th, 14th and 21st day as described (Jurcovicova et al., 2009). The pre-injection values of paw volumes were measured just prior to FCA inoculation for each rat and were regarded as the baseline. The increase in paw volume was calculated and expressed in milliliters (ml). Gonadosomatic index. On the last day of experiment and just before euthanization, the body weights of animals were recorded. Immediately

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following laparotomy, testes were weighed and the gonadosomatic index was calculated as follows: (testes weight / body weight) × 100 (Breikaa et al., 2014). Histopathological examination. Full thickness testicular biopsy specimens were fixed in 10% buffered formol saline for 24 h. The specimens were washed, dehydrated by alcohol, cleared in xylene and embedded in paraffin at 56 °C in hot air oven for another 24 h. Sections of 3 μm thickness were stained with hematoxylin and eosin (H&E) and examined under the light microscope (Leica Microsystems, Germany). All histopathologic processing and assessment of specimens were performed by an experienced observer unaware of the identity of the sample being examined to avoid any bias. Immunohistochemical detection of COX-2 and iNOS. Paraffin embedded tissue sections of 3 μm thickness were rehydrated in xylene and then in graded ethanol solutions. The slides were heated in citrate buffer (pH 6) for 5 min and were blocked with 5% bovine serum albumin (BSA) in Tris buffered saline (TBS) for 2 h. The sections were then immunostained with primary polyclonal rabbit anti-COX-2 or anti-iNOS (Thermo Scientific, IL, USA) at a concentration of 1 μg/ml in 5% BSA in TBS and were incubated overnight at 4 °C. Following primary antibody incubation step, the slides were washed with TBS and were then incubated with goat anti-rabbit secondary antibody. Finally, the sections were washed with TBS and incubated for 5–10 min in a solution of 0.02% diaminobenzidine (DAB) containing 0.01% H2O2. Counter staining was performed using hematoxylin and the slides were visualized and digital images were generated using light microscope fitted with digital camera (Leica Microsystems, Germany). Estimation of serum testosterone, LH and FSH levels. Serum levels of testosterone were assessed using automated chemiluminescence immunoassay system (ADVIA Centaur; Bayer Vital, Fernwald, Germany) whereas serum LH and FSH were determined using the corresponding ELISA Kit (Shibayagi Co., Japan). Testicular MPO activity. The MPO activity, an index of neutrophil infiltration, was estimated according to the method of Krawisz et al. (1984). One unit of MPO activity is defined as the amount of enzyme converting 1 μmol of H2 O2 to water in 1 min at 25 °C. The testicular homogenates were subjected to 3 cycles of freezing/thawing, 30 s of sonication and centrifuged at 20,000 ×g for 20 min at 4 °C. Odianisidine hydrochloride (0.167%) and H2O2 (0.0005%) in potassium phosphate buffer (50 mmol/L, pH 6) were added to the supernatant and the absorbance rate was monitored at 460 nm for 4 min. Estimation of inflammatory cytokines (TNF-α and IL-10). The serum levels of TNF-α and IL-10 were measured using ELISA kits (R&D Systems Incorporation, USA). All the procedures were performed according to the manufacturer's instructions. The assay of these cytokines employs the quantitative sandwich enzyme immunoassay technique with specific antibodies pre-coated onto the microplate. The standards, control, and samples were pipetted into the wells and the rat cytokines were bound by the immobilized antibody. After washing away any unbound substances, an enzyme-linked secondary antibody specific for rat TNF-α or IL-10 was added to the wells. Following color development, the assay was stopped, and the absorbance was read at 450 nm. The intensity of the color was proportional to the amount of the corresponding cytokine bound in the initial step. Estimation of lipid peroxides. The determination of lipid peroxides, expressed as malondialdehyde (MDA), was carried out according to the thiobarbituric acid assay of Buege and Aust (1978). One part of the sample was mixed with double volume of a reagent consisting of 15% w/v trichloroacetic acid, 0.375% w/v thiobarbituric acid and

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0.25 N HCl. The mixture was heated at 90 °C in a water bath for 30 min. After cooling, the precipitate was removed by centrifugation at 1000 ×g for 10 min. The absorbance of the clear supernatant was recorded at 535 nm against a blank prepared by using bidistilled water instead of the sample. Nitric oxide (NO) estimation. Total NO was determined by measuring − its stable metabolites, particularly, nitrite (NO− 2 ) and nitrate (NO3 ) based on the method of Miranda et al. (2001) with the modification of replacing zinc sulfate instead of ethanol for the precipitation of proteins in the homogenate supernatant. Nitric oxide was extracted through centrifugation of 1 ml of 10% aqueous homogenate at 21,000 ×g for 15 min. To an aliquot of the resultant supernatant, vanadium trichloride (0.8% in 1 M HCl) was added for reduction of nitrate to nitrite, followed by rapid addition of Griess reagent consisting of N-(1-naphthyl) ethylenediamine dihydrochloride (0.1%) and sulfanilamide (2% in 5% HCl). The mixture was incubated for 30 min at 37 °C, allowed to cool and then the absorbance was measured at 540 nm. Estimation of caspase-3 activity. The activity of caspase-3 was colorimetrically assayed using R&D systems kit according to the manufacturer's instructions based on the method of Fernandes-Alnemri et al. (1994). Briefly, an aliquot of the homogenate supernatant was incubated with the labeled substrate DEVD-pNA (acetyl-Asp-Glu-ValAsp p-nitroanilide). The cleavage of the peptide by the caspase releases the chromophore pNA, which was read at 405 nm using a Biochrom Asys microplate reader (UK). Quantitative real-time RT-PCR Total RNA extraction. Total RNA was extracted from testicular tissues using RNeasy Mini kit (Qiagen, CA, USA). The isolated RNA was quantified using UV spectrophotometer (Beckman, USA) and the purity of RNA was verified by the 260/280 nm ratio ranging from 1.9 to 2.1. The integrity of RNA was confirmed by gel electrophoresis on a 1% agarose gel stained with ethidium bromide. cDNA synthesis and qPCR. Equal amounts of RNA (2 μg) were reverse transcribed into cDNA using Superscript Choice systems (Life Technologies, Breda, Netherlands) according to the manufacturer's instructions. To assess the mRNA expression of StAR and FasL, quantitative realtime PCR was performed using SYBR green PCR Master mix (Applied Biosystems, CA, USA) as described by the manufacturer. Briefly, in a 25 μl reaction volume, 5 μl of cDNA was added to 12.5 μl of 2 × SYBR green master mix and 200 ng of each primer. The sequences of primers are described in Table 1. The PCR reactions included 10 min at 95 °C for activation of AmpliTaq Gold DNA polymerase, followed by 40 cycles at 94 °C for 15 s (denaturing) and 60 °C for 1 min (annealing/extension). The expression level was calculated from the PCR cycle number (CT) where the increased fluorescence curve passes across a threshold value. The relative expression of target genes was obtained using comparative CT (ΔΔCT) method. The ΔCT was calculated by subtracting GAPDH CT from that of target gene whereas ΔΔCT was obtained by subtracting the ΔCT of calibrator sample (control gp) from that of test sample (AA, AA + CHR25, AA + CHR 50 or AA + CLX gp). The relative Table 1 Primer sequences used for real-time PCR. mRNA species

Accession number

Primer sequence

StAR

NM_031558.3

FasL

NM_012908.1

GAPDH

XR_147054.1

Forward 5′-ATGCCTGAGCAAAGCGGTGTC-3′ Reverse 5′-AAGTGGCTGGCGAACTCTATCTG-3′ Forward 5′-TCTGGTTGGAATGGGGTTAG-3′ Reverse 5′-TAAGGCTGTGGTTGGTGAAC-3′ Forward 5′-AGAAGGCTGGGGCTCATTTG-3′ Reverse 5′-AGGGGCCATCCACAGTCTTC-3′

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expression was calculated from the 2−ΔΔCT formula (Livak and Schmittgen, 2001). Statistical analysis. Data is expressed as mean ± SEM, and statistical comparisons were carried out using one-way analysis of variance (ANOVA). When differences were significant, Tukey–Kramer post hoc test was used for multiple comparisons between groups. The statistical analysis was carried out by SPSS program, version 17. The minimal level of significance was identified at p b 0.05. Results Chrysin and celecoxib suppress paw edema in adjuvant-induced arthritic rats We assessed the effects of chrysin in adjuvant-induced arthritis, an experimental immunopathy that shares many features with human RA. To investigate the severity of arthritis, the increase of paw edema was explored. Inoculation of FCA triggered a significant increase of paw volume starting from the 4th day till the end of the experiment on day 21 (Fig. 1). It is noteworthy that under our experimental conditions, all rats inoculated with FCA developed paw inflammation homogenously. Interestingly, chrysin dose-dependently suppressed paw edema with maximum efficacy on the 21st day by 20% and 62% inhibition for 25 mg/kg and 50 mg/kg doses, respectively, as compared to arthritic rats. The effects of 50 mg/kg chrysin on paw inflammation were even more pronounced than the standard celecoxib that exhibited a 32% inhibition on the final day of the experiment. Thus, these data suggest that chrysin and celecoxib attenuated the development of adjuvant-induced arthritis in rats. Chrysin and celecoxib attenuate testicular histopathological damage in rats with adjuvant arthritis We next assessed whether chrysin and celecoxib can protect against the histopathological damage in testes of rats with adjuvant

arthritis. Sections from the control group revealed an intact architecture of testicular tissues with functional seminiferous tubules and normal germ cell layers indicating complete spermatogenic series (Fig. 2A and Table 2). On the other hand, testes of arthritic rats suffered remarkable morphological alterations and tissue damage with massive degeneration of seminiferous tubules, reduction in germ cell layers and disruption of spermatogenesis. Interstitial edema, congestion of blood vessels and infiltration of immunoinflammatory cells were also observed. (Fig. 2B). Chrysin protected against these pathological changes and preserved the testicular architecture with restoration of spermatogenesis in the majority of seminiferous tubules in a dose-dependent manner (Fig. 2C, D). In the 25 mg/kg dose of chrysin, degeneration was observed in 25% of the seminiferous tubules with detection of spermatid giant cells, a degenerative lesion associated with various testicular injuries (Morton et al., 1986) (Fig. 2C and Table 2). Degeneration was also detected in 20% of the seminiferous tubules in the 50 mg/kg dose of chrysin (Fig. 2D and Table 2). Likewise, celecoxib mitigated the histopathological changes and protected the seminiferous tubules with degeneration detected in 25% of them as compared to the arthritic group (Fig. 2E and Table 2). These observations indicate that chrysin and celecoxib mitigated the testicular histopathological damage in adjuvant arthritic rats. Chrysin and celecoxib modulate serum levels of testosterone, LH and FSH and upregulate the testicular expression of StAR gene The testicular injury in rats with adjuvant arthritis was further verified via measuring the gonadosomatic index in addition to serum testosterone, LH and FSH hormones. Arthritic animals displayed lowered gonadosomatic index (54%) and testosterone levels (34%) (Fig. 3A, B) whereas serum LH and FSH displayed 3.1 and 4.8 fold increases (Fig. 4), respectively, as compared to the control group. Administration of chrysin at both doses reversed these alterations. The effects of 50 mg/kg of chrysin were comparable to celecoxib with respect to serum testosterone levels and were even more pronounced in case of gonadosomatic index, LH and FSH. Testicular steroidogenesis was assessed via detecting the mRNA expression of StAR gene using real-time RT-PCR. As depicted in Fig. 3C, arthritis provoked decreased testicular mRNA expression of StAR gene (37% of control value) whereas chrysin as well as celecoxib upregulated its expression. Together, these data indicate that chrysin and celecoxib can enhance steroidogenesis to mitigate the testicular suppression in rats with adjuvant arthritis. Chrysin and celecoxib modulate MPO, inflammatory cytokines and testicular COX-2 and iNOS protein expression

Fig. 1. Chrysin and celecoxib alleviate paw edema in arthritic rats. Animals were rendered arthritic by a single sc injection of Freund's complete adjuvant (FCA) into the subplantar surface of the right hind paw. Chrysin (25 & 50 mg/kg) and celecoxib (5 mg/kg) were orally administered once daily for 21 days starting from the day of arthritis induction (day 0). Values represent mean ± SEM (n = 6–8). *Significant difference from control gp at p b 0.05, #significant difference from arthritic gp at p b 0.05. AA; adjuvant-induced arthritis, CHR 25; chrysin (25 mg/kg), CHR 50; chrysin (50 mg/kg), CLX; celecoxib (5 mg/kg).

To gain an insight into the inflammatory milieu of animals with adjuvant arthritis, we investigated the testicular MPO, serum TNFα & IL-10 cytokines and the testicular protein expression of COX-2 and iNOS. Testes of rats with AA were infiltrated by immunoinflammatory cells as indicated by a 1.9-fold increase of MPO activity, a reliable index for neutrophil invasion (Eiserich et al., 1998), as compared to the control group (Fig. 5A). Chrysin as well as celecoxib administration afforded a significant decrease of MPO activity as compared to the arthritic group. In the same context, the inoculation of FCA resulted in severe inflammatory response in arthritic rats as indicated by elevation of serum TNF-α (9.4-fold) with concomitant reduction of serum IL-10 (0.5-fold) as compared to the control group (Fig. 5B, C). Administration of chrysin counteracted these changes with 50 mg/kg of chrysin exerting comparable effects to celecoxib. The immunohistochemical detection of testicular COX-2 and iNOS protein expression revealed an enhanced expression that was localized within the interstitial stroma adjacent to the basement membrane of degenerated tubules in testes of animals with adjuvant

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Fig. 2. Chrysin and celecoxib mitigate the histopathological damage in testes of rats with adjuvant arthritis. Representative photomicrographs of sections from testes harvested on the 21st day post FCA paw inoculation. (A) The control group displayed normal intact testicular tissues with mature seminiferous tubules and complete spermatogenic series (s). (B) Testes of AA group showing massive degeneration of seminiferous tubules (ds), reduction in germ cell layers and disruption of spermatogenesis. Interstitial edema and congestion of blood vessels (v) with infiltration of inflammatory cells (m) were also detected. (C & D) Testes of AA + CHR revealed attenuation of the pathological changes, preservation of testicular architecture with restoration of spermatogenesis. (C) Degeneration was detected in 25% of the seminiferous tubules in AA + CHR 25 gp with detection of spermatid giant cells (g) in the lumen of affected tubules. (D) In AA + CHR 50 gp, the degeneration of seminiferous tubules was detected in 20% of the seminiferous tubules. (E) Testes of AA + CLX gp were characterized by mitigation of the histopathological findings with degeneration of seminiferous tubules confined to 25% of them. Hematoxylin and eosin staining, original magnification: ×40. AA; adjuvant-induced arthritis, CHR 25; chrysin (25 mg/kg), CHR 50; chrysin (50 mg/kg), CLX; celecoxib (5 mg/kg).

arthritis (Figs. 6, 7 and Table 3). This is consistent with that fact that infiltrating immune cells are clustered in the interstitium but not inside seminiferous tubules (Jacobo et al., 2012). Administration of chrysin and celecoxib mitigated the expression of these proinflammatory proteins. These observations indicate that chrysin and celecoxib downregulation of these proinflammatory targets along with restoration of the anti-inflammatory IL-10 are implicated in the mitigation of testicular dysfunction in adjuvant arthritic rats.

Chrysin and celecoxib inhibit testicular oxidative stress in rats with adjuvant arthritis To explore the oxidative status, we measured the levels of lipid peroxides expressed as malondialdehyde (MDA) and nitric oxide (NO) in the testes of rats with adjuvant arthritis. As shown in Fig. 8, the levels of lipid peroxides and nitric oxide displayed 1.5 and 1.4 fold increases, respectively, as compared to the control group. Chrysin and celecoxib

Table 2 Histopathological alterations in testicular tissues of rats with adjuvant arthritis. Histopathological alteration

Control

AA

AA + CHR 25

AA + CHR 50

AA + CLX

Degeneration of seminiferous tubules Congestion in blood vessels Calcification of seminiferous tubules

− − −

++++ ++ −

++ − −

+ − −

++ − −

++++ very severe, +++ severe, ++ moderate, + mild, − Nil. AA; adjuvant-induced arthritis, CHR 25; chrysin (25 mg/kg), CHR 50; chrysin (50 mg/kg), CLX; celecoxib (5 mg/kg).

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significantly lowered the levels of these oxidative stress markers. These effects suggest that chrysin and celecoxib mitigation of oxidative perturbations plays a role in attenuation of testicular impairment in adjuvant arthritic rats.

Fig. 4. Chrysin and celecoxib restore serum luteinizing hormone (LH; A) and follicle stimulating hormone (FSH; B) levels in rats with adjuvant arthritis. Arthritis was induced by inoculation of FCA and chrysin and celecoxib were orally administered for 21 days starting from the day of arthritis induction. Values represent mean ± SEM (n = 6–8). *Significant difference from control gp at p b 0.05, #significant difference from arthritic gp at p b 0.05. AA; adjuvant-induced arthritis, CHR 25; chrysin (25 mg/kg), CHR 50; chrysin (50 mg/kg), CLX; celecoxib (5 mg/kg).

Chrysin and celecoxib downregulate the mRNA expression of FasL and inhibit caspase-3 activity We addressed the impact of chrysin on testicular apoptosis via assessing the activity of caspase-3, a reliable indicator of apoptosis (Nagata, 1999). Besides, we detected the mRNA expression of FasL which is involved in Fas–FasL system, the major pathway of germ cell apoptosis in various experimental models of testicular injury including autoimmune orchitis, toxicant- and hormone suppression-induced pathologies (Jacobo et al., 2012; Perez et al., 2013). Adjuvant arthritic animals suffered significant testicular apoptosis as indicated by a 1.8 fold Fig. 3. Chrysin and celecoxib enhance the gonadosomatic index (A), serum testosterone (B) and testicular StAR mRNA expression (C) in rats with adjuvant arthritis. Arthritis was induced by inoculation of FCA and chrysin and celecoxib were orally administered for 21 days starting from the day of arthritis induction. The mRNA expression of the steroidogenic acute regulatory (StAR) gene was detected by quantitative real-time RT-PCR. Values represent mean ± SEM (n = 6–8) for (A) and (B) while for (C), data were expressed as mean ± SD (n = 6). *Significant difference from control gp at p b 0.05, #significant difference from arthritic gp at p b 0.05. AA; adjuvant-induced arthritis, CHR 25; chrysin (25 mg/kg), CHR 50; chrysin (50 mg/kg), CLX; celecoxib (5 mg/kg).

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increase of caspase-3 activity along with a 5.4 fold elevation in the testicular FasL mRNA expression as compared to the control group (Fig. 9). Administration of chrysin and celecoxib significantly counteracted these changes with comparable efficacy of 50 mg/kg of chrysin to celecoxib, suggesting that chrysin and celecoxib attenuation of apoptosis is implicated in the protection against testicular injury in rats with adjuvant arthritis.

Discussion

Fig. 5. Chrysin and celecoxib suppress MPO and TNF-α with enhancement of IL-10 in rats with adjuvant arthritis. (A) Testicular myeloperoxidase (MPO) activity. (B) Serum tumor necrosis factor-α (TNF-α). (C) Serum interleukin-10 (IL-10). Arthritis was induced by inoculation of FCA and chrysin and celecoxib were orally administered for 21 days starting from the day of arthritis induction. Values represent mean ± SEM (n = 6–8). *Significant difference from control gp at p b 0.05, #significant difference from arthritic gp at p b 0.05. AA; adjuvant-induced arthritis, CHR 25; chrysin (25 mg/kg), CHR 50; chrysin (50 mg/kg), CLX; celecoxib (5 mg/kg).

Testicular dysfunction and impaired fertility are serious complications that threaten men with RA (Bove, 2013; Cutolo et al., 2003). Thus, it seems imperative to search for agents that can protect against the progression of RA along with the associated testicular dysfunction in those patients. Interestingly, chrysin suppressed paw edema in adjuvant arthritic rats, an experimental model of RA, with comparable efficacy to celecoxib. More important, the current study highlights, for the first time, the beneficial protective actions of chrysin and celecoxib against the testicular suppression in adjuvant arthritic rats. These favorable effects were linked with upregulation of testicular StAR gene expression, enhanced steroidogenesis and preservation of spermatogenesis. Modulation of the inflammatory cytokines and testicular COX-2 and iNOS expression along with inhibition of oxidative stress also mediated the beneficial actions of the two agents. In addition, attenuation of FasL expression and caspase-3 activity protected against testicular apoptosis and associated injury. Adjuvant-induced arthritis has been regarded as a convenient model to investigate the mechanisms of immune modulation of the reproductive system (Bruot and Clemens, 1992; Clemens and Bruot, 1989). Our data revealed marked testicular dysfunction in rats with adjuvant arthritis as indicated by diminished serum testosterone with elevation of serum LH and FSH. Besides, the histopathological analysis revealed degeneration of seminiferous tubules with disruption of spermatogenesis. These findings are in concert with previous experimental studies (Bruot and Clemens, 1992; Clemens and Bruot, 1989). In addition, Gordon et al. (1986) have described similar hormonal disturbances in men with RA. The deficiency of testosterone during the clinically active phase of AA has been attributed to testicular macrophages which secreted a proinflammatory factor that abrogated testosterone production (Bruot and Clemens, 1992). Interestingly, chrysin as well as celecoxib upregulated StAR gene expression with consequent boosting of serum testosterone and restoration of LH and FSH hormones. They also preserved the testicular architecture with conservation of spermatogenesis. These findings are in line with the reported beneficial effects of chrysin in normal and aging male rat gonads (Ciftci et al., 2012; Dhawan et al., 2002). Virtually, testosterone is generated from testicular Leydig cells under the influence of StAR protein which is crucial for spermatogenesis and male virility (Wang et al., 2009). In this context, Jana et al. (2008) reported an in vitro upregulation of StAR gene expression and steroidogenesis by chrysin via increasing the sensitivity of Leydig cells to cAMP stimulation. Other studies also reported in vivo testosterone boosting actions of chrysin via inhibition of peripheral aromatase, an enzyme that blocks the conversion of androgens to estrogens (Dhawan et al., 2002; Wang et al., 2009). In aging male rats, chrysin has been demonstrated to enhance serum testosterone and associated libido (Dhawan et al., 2002). Besides, chrysin has been reported to increase sperm count and motility in normal adult rats (Ciftci et al., 2012). However, Gambelunghe et al. (2003) have reported no change in urinary testosterone following a 3-week chrysin administration in human subjects. This discrepancy could be related to the low dose of chrysin utilized in that study. Regarding celecoxib, it has previously displayed protective effects against LPS-induced testicular injury with elevation of serum testosterone (Winnall et al., 2009), an effect that was also observed in normal rats (Selmanoglu et al., 2006).

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Fig. 6. Chrysin and celecoxib downregulate testicular cyclo-oxygenase-2 (COX-2) protein expression in rats with adjuvant arthritis. Representative images for the immunohistochemical detection of COX-2 expression from testicular samples harvested on the 21st day post FCA paw inoculation (magnification: ×200). (A) Control gp: minimal expression; (B) AA gp: extensive expression (brown color) confined to the interstitial stroma (m) but absent in the seminiferous tubules (s); (C–E) AA + CHR 25, AA + CHR 50 and AA + CLX gps: attenuated expression. AA; adjuvant-induced arthritis, CHR 25; chrysin (25 mg/kg), CHR 50; chrysin (50 mg/kg), CLX; celecoxib (5 mg/kg).

A unique observation in our study is the alleviation of joint inflammation along with attenuation of testicular suppression which signifies the protective actions of chrysin and celecoxib against RA progression and associated testicular injury. Generally, the elevation of serum testosterone, with its proven anti-inflammatory and immune response suppressor actions, has been associated with improvement of clinical and chemical markers of disease activity in RA (Cutolo et al., 2002; Page et al., 2006). The anti-inflammatory impact of testosterone on immune system is likely mediated via mechanisms that involve halting of proinflammatory cytokine release by monocytes and macrophages, boosting of the anti-inflammatory IL10 by T cells and suppression of NF-κB provoked activation of IL-6 promoter in human fibroblasts (Bove, 2013). The benefits of testosterone therapy have been described in the clinical setting in men with RA (Cutolo et al., 2002). One clinical trial using oral testosterone undecanoate revealed elevation of serum testosterone and CD8 + T cells with decline in CD4 + T helper cells along with attenuation of IgM rheumatoid factor and RA disease activity (Cutolo et al., 1991). Nevertheless, another trial using monthly testosterone enanthate injection displayed enhanced testosterone levels in RA patients without affecting the disease activity (Hall et al., 1996). Interestingly, synergy has been described between androgens and immunosuppressive agents including cyclosporine A and methotrexate and anti-IL-6 receptor antibody. Thus, these studies may provide a

rationale for using androgen therapy as an adjunct to the disease modifying drugs (Bove, 2013). Our data further described an upregulated inflammatory response in rats with AA as evidenced by enhanced MPO activity, TNF-α levels and COX-2 and iNOS expression with concomitant lowering of IL-10. The elevated MPO levels indicate the testicular invasion by polymorphonuclear leukocytes (Eiserich et al., 1998). Upon tissue infiltration, neutrophils and macrophages release proinflammatory cytokines such as TNF-α and IL-1β which block gonadal androgen biosynthesis at the level of steroid 17 alpha-hydroxylase/17,20 lyase (cytochrome P450c17) enzyme (Ernestam et al., 2007). In the same context, the observed upregulation of testicular COX-2 also contributes to suppressed steroidogenesis via inhibition of StAR gene expression in Leydig cells (Wang et al., 2005). Within RA synovium, proinflammatory cytokines stimulate peripheral aromatase with conversion of androgens, natural suppressors of immune response, to estrogens, enhancers of immune response, with consequent triggering of mitogenic and proliferative signals to synovial cells (Cutolo et al., 2003). The observed decline of IL-10 likely reflects the severe inflammation in arthritic rats since IL-10 possesses anti-inflammatory features which halt the release of proinflammatory cytokines from activated macrophages, neutrophils and CD4+ T lymphocytes (Ozturk et al., 2013). Interestingly, chrysin and celecoxib reversed the alterations of the inflammatory markers signifying their beneficial anti-inflammatory

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Fig. 7. Chrysin and celecoxib downregulate testicular inducible nitric oxide synthase (iNOS) protein expression in rats with adjuvant arthritis. Representative images for the immunohistochemical detection of iNOS expression from testicular samples harvested on the 21st day post FCA paw inoculation (magnification: ×200). (A) Control gp: minimal expression; (B) AA gp: enhanced expression (brown color) confined to the interstitium (m); (C–E) AA + CHR 25, AA + CHR 50 and AA + CLX gps: attenuated expression. AA; adjuvant-induced arthritis, CHR 25; chrysin (25 mg/kg), CHR 50; chrysin (50 mg/kg), CLX; celecoxib (5 mg/kg).

effects in attenuating injury in testes of rats with AA. Previously, the marked anti-inflammatory effects of chrysin mediated its protection against renal injury (Rehman et al., 2013). Chrysin has also been reported to suppress COX-2 expression via inhibition of nuclear factor for IL-6 (NF-IL6) DNA-binding activity in LPS-stimulated macrophages (Woo et al., 2005). The above data do not agree with Winnall et al. (2009) who reported that celecoxib, despite its alleviation of LPS-induced testicular injury and testosterone suppression, didn't affect the inflammatory mediators. The differences could be ascribed to the entirely different experimental design, type of inflammagen (CFA vs LPS) and celecoxib dose (5 mg/kg vs 78–100 mg/kg). In fact, the marked antiinflammatory features of celecoxib have been described in several experimental models including adjuvant arthritis (Khayyal et al., 2005) and ischemia–reperfusion injury (Malek and Saleh, 2009) via downregulation of COX-2 and proinflammatory cytokines.

In rats with adjuvant arthritis, elevation of testicular lipid peroxides, NO and MPO were observed indicating enhanced oxidative stress which has been intimately linked to the impairment of sperm function and infertility (Vaithinathan et al., 2010). Virtually, testes are highly vulnerable to the detrimental actions of reactive oxygen species (ROS) and lipid peroxidation owing to their high content of polyunsaturated lipids in cellular membranes (Vaithinathan et al., 2010). Furthermore, increased synthesis of NO, generated by inducible nitric oxide synthase (iNOS), has been implicated in mediating the injury in several testicular pathologies (Taneli et al., 2005). Via its reaction with superoxide anion, NO generates peroxynitrite radical, a potent oxidizing agent, which inflicts cellular injury via DNA fragmentation and lipid peroxidation (Valko et al., 2007). MPO enzyme also contributes to oxidative stress via overshooting of hypochlorous acid, a potent cytotoxic oxidant that provokes tissue injury (Eiserich et al., 1998).

Table 3 Immunohistochemical detection of COX-2 and iNOS protein expression in testes of rats with adjuvant arthritis.

COX-2 expression (in the interstitial stroma) iNOS expression (in the interstitial stroma)

Control

AA

AA + CHR 25

AA + CHR 50

AA + CLX

− +

++++ +++

++ ++

++ +

+ ++

++++ very extensive, +++ extensive, ++ moderate, + mild, − Nil. AA; adjuvant-induced arthritis, CHR 25; chrysin (25 mg/kg), CHR 50; chrysin (50 mg/kg), CLX; celecoxib (5 mg/kg).

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Fig. 8. Chrysin and celecoxib suppress testicular oxidative stress in rats with adjuvant arthritis. (A) Levels of lipid peroxides expressed as malondialdehyde (MDA). (B) Levels of nitric oxide (NO). Arthritis was induced by inoculation of FCA and chrysin and celecoxib were orally administered for 21 days starting from the day of arthritis induction. Values represent mean ± SEM (n = 6–8). *Significant difference from control gp at p b 0.05, #significant difference from arthritic gp at p b 0.05. AA; adjuvant-induced arthritis, CHR 25; chrysin (25 mg/kg), CHR 50; chrysin (50 mg/kg), CLX; celecoxib (5 mg/kg).

The observed alleviating actions of chrysin and celecoxib on testicular oxidative stress markers highlight the implication of their antioxidant properties in attenuation of testicular injury. These findings coincide with previous reports that highlighted the involvement of chrysin antioxidant features for protection against tissue injury in several experimental models (Khan et al., 2012; Pushpavalli et al., 2010; Rehman et al., 2013). Likewise, celecoxib displayed antioxidant features that partly mediated its protection against adjuvant arthritis (Khayyal et al., 2005) and ischemia–reperfusion injury (Malek and Saleh, 2009). Besides the antioxidant properties, inhibition of neutrophil recruitment by chrysin and celecoxib could explain halting of oxidative stress. Our data also revealed enhanced testicular apoptosis as evidenced by increased expression of FasL mRNA and activity of caspase-3, indicating a central role of Fas death-receptor pathway in germ cell apoptosis and sloughing in testes of rats with AA. In fact, Fas–FasL system has been recognized as the major pathway of germ cell apoptosis in several models of testicular damage including autoimmune orchitis (Jacobo et al., 2012; Perez et al., 2013). ROS, proinflammatory cytokines and NO, secreted by infiltrating immune cells, have been implicated as signals for driving the death-receptor apoptotic pathway (Chandra et al., 2000; Perez et al., 2013). Fas is a type I transmembrane receptor protein

Fig. 9. Chrysin and celecoxib downregulate testicular FasL mRNA expression (A) and caspase-3 activity (B) in rats with adjuvant arthritis. Arthritis was induced by inoculation of FCA and chrysin and celecoxib were orally administered for 21 days starting from the day of arthritis induction. The mRNA expression of FasL was detected by quantitative real-time RT-PCR. Values represent mean ± SD (n = 6) for (A) whereas the data are expressed as mean ± SEM (n = 6–8) for (B). *Significant difference from control gp at p b 0.05, #significant difference from arthritic gp at p b 0.05. AA; adjuvant-induced arthritis, CHR 25; chrysin (25 mg/kg), CHR 50; chrysin (50 mg/kg), CLX; celecoxib (5 mg/kg).

while FasL is a type II transmembrane protein. Following their crosslinking, activation of caspase-8 is ensued which ultimately activates caspase-3 that induces DNA fragmentation via activation of specific endonucleases (Nagata, 1999). In RA, synovial overexpression of FasL was detected in patients with severe RA than in those with mild symptoms (Matsuno et al., 2001). Besides, in autoimmune orchitis, soluble FasL, generated by CD4 + and CD8 + T lymphocytes, has been reported to penetrate into the adluminal compartments of seminiferous tubules inducing apoptosis of Fas-bearing germ cells (Jacobo et al., 2012). In addition to its classical role in apoptosis, FasL has also been implicated in triggering the inflammatory process via the interplay with nuclear factor kappa B (NF-κB) pathway (Ahn et al., 2001). Interestingly, administration of chrysin and celecoxib attenuated germ cell apoptosis as evidenced by suppression of FasL overexpression and caspase-3 activity. These findings signify that the anti-apoptotic effects of chrysin and celecoxib partly mediated the alleviation of testicular impairment in rats with AA. These findings are in agreement with previous reports that demonstrated the anti-apoptotic actions of chrysin in cisplatin-induced colon and jejunum toxicities (Khan et al.,

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2012; Rehman et al., 2013) and in methotrexate-induced hepatic injury (Ali et al., 2014) via downregulation of p53 expression and caspase-3 activity. Since ROS, proinflammatory cytokines and NO are the main signals for germ cell apoptosis (Chandra et al., 2000; Perez et al., 2013), the inhibition of these mediators by chrysin and celecoxib is probably associated with the observed attenuation of apoptosis. In conclusion, the current study highlights the dual protective effects of chrysin and celecoxib against paw edema and the testicular suppression in a rat model of RA. In particular, the alleviating actions against testicular impairment were mediated via boosting testicular StAR gene expression and associated testosterone production. In addition, the two agents modulated neutrophil infiltration, inflammatory cytokines, and COX-2 and iNOS expression besides the oxidative stress. They also inhibited FasL mRNA expression and caspase-3 activity. Thus, our data suggest the administration of celecoxib, a widely used NSAID for the management of RA (Steinbach et al., 2000), for halting the progression of testicular suppression in men with RA. Our findings also advocate chrysin as a safe complementary approach for the management of testicular dysfunction in those patients. Comparing the favorable actions of chrysin and celecoxib with the standard testosterone therapy currently used in the clinical practice would determine their relative potency for alleviating the testicular suppression associated with RA. Thus, future investigation addressing this comparison is warranted. In the same context, additional detailed investigations exploring the localization of inflammatory mediators such as TNF-α, iNOS, chemotactic factors and MPO in testicular injury associated with RA are also warranted to delineate the exact molecular events of chrysin and celecoxib actions in RA testicular injury. In addition, the efficacy of chrysin and celecoxib in the clinical setting needs to be established. Conflict of interest statement The authors declare that there are no conflicts of interest. Acknowledgment The authors are grateful to Prof. Kholoussy A.B. (Department of Pathology, Faculty of Veterinary Medicine, Cairo University, Egypt) for his kind help in histopathology and immunohistochemistry. References Agha, A.M., Gad, M.Z., 1995. Lipid peroxidation and lysosomal integrity in different inflammatory models in rats: the effects of indomethacin and naftazone. Pharmacol. Res. 32, 279–285. Ahn, J.H., Park, S.M., Cho, H.S., Lee, M.S., Yoon, J.B., Vilcek, J., Lee, T.H., 2001. Non-apoptotic signaling pathways activated by soluble Fas ligand in serum-starved human fibroblasts. Mitogen-activated protein kinases and NF-kappaB-dependent gene expression. J. Biol. Chem. 276, 47100–47106. Ali, N., Rashid, S., Nafees, S., Hasan, S.K., Sultana, S., 2014. Beneficial effects of chrysin against methotrexate-induced hepatotoxicity via attenuation of oxidative stress and apoptosis. Mol. Cell. Biochem. 385, 215–223. Arab, H.H., El-Sawalhi, M.M., 2013. Carvedilol alleviates adjuvant-induced arthritis and subcutaneous air pouch edema: modulation of oxidative stress and inflammatory mediators. Toxicol. Appl. Pharmacol. 268, 241–248. Bain, J., 2007. The many faces of testosterone. Clin. Interv. Aging 2, 567–576. Bove, R., 2013. Autoimmune diseases and reproductive aging. Clin. Immunol. 149, 251–264. Breikaa, R.M., Mosli, H.A., Nagy, A.A., Abdel-Naim, A.B., 2014. Adverse testicular effects of Botox in mature rats. Toxicol. Appl. Pharmacol. 275, 182–188. Bruot, B.C., Clemens, J.W., 1992. Regulation of testosterone production in the adjuvantinduced arthritic rat. J. Androl. 13, 87–92. Buege, J.A., Aust, S.D., 1978. Microsomal lipid peroxidation. Methods Enzymol. 52, 302–310. Buvat, J., Maggi, M., Guay, A., Torres, L.O., 2013. Testosterone deficiency in men: systematic review and standard operating procedures for diagnosis and treatment. J. Sex. Med. 10, 245–284. Chandra, J., Samali, A., Orrenius, S., 2000. Triggering and modulation of apoptosis by oxidative stress. Free Radic. Biol. Med. 29, 323–333. Ciftci, O., Ozdemir, I., Aydin, M., Beytur, A., 2012. Beneficial effects of chrysin on the reproductive system of adult male rats. Andrologia 44, 181–186. Clemens, J.W., Bruot, B.C., 1989. Testicular dysfunction in the adjuvant-induced arthritic rat. J. Androl. 10, 419–424.

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