The Prostate 75:679–692 (2015)

The Effect of Pomegranate Fruit Extract on Testosterone-Induced BPH in Rats Amr E. Ammar,1,2 Ahmed Esmat,1 Mohammed D.H. Hassona,2,3 Mariane G. Tadros,1 Ashraf B. Abdel-Naim,1 and Emma S. Tomlinson Guns,2* 1 2

Department of Pharmacology and Toxicology, Faculty of Pharmacy, Ain Shams University, Cairo, Egypt Vancouver Prostate Centre, Department of Urologic Sciences, University of British Columbia, Vancouver, British Columbia, Canada 3 Department of Pharmacology and Toxicology, Helwan University, Helwan, Egypt

BACKGROUND. Benign prostatic hyperplasia (BPH) affects many men after the age of 50 years. Inflammation and oxidative stress along with apoptotic changes are thought to play an important role in the pathology of BPH. Pomegranate contains a variety of polyphenolic compounds that have been studied in a medley of diseases for their anti-oxidant, anti-inflammatory and pro-apoptotic properties. Therefore, this study examined the effect of Pomegranate Fruit Extract (PFE) on the development of BPH using a testosterone-induced BPH model in rats. METHODS. A total of 48 rats were randomly divided into six groups of eight, one group served as the control, BPH was induced by testosterone 3 mg/kg S.C. daily in four groups, three of them received PFE by oral gavage daily at doses of 25, 50, and 100 mg/kg respectively, while one group received PFE at a dose of 50 mg/kg without induction of BPH. RESULTS. PFE at a dose of 100 mg/kg was the most effective in decreasing testosterone-induced increase in prostate weight, prostate weight/body weight ratio, and PAP levels by 30.8%, 55%, and 68% respectively and in preventing the accompanying histological changes. In the BPH model, testosterone significantly decreased GSH, SOD, and CAT to 0.45, 0.64, and 0.88 of the control group values respectively, and significantly increased MDA by >6-fold. In combination with testosterone, PFE dosed at 100 mg/kg significantly increased GSH, SOD, and CAT to 0.83, 0.92, and 0.93 of the control group values respectively, whereas MDA was significantly decreased by 72% compared with the testosterone treated group. In addition to this, at the range of doses studied, PFE lowered COX-II, iNOS, Ki-67 expression, and increased apoptotic index. CONCLUSION. The current findings elucidate the effectiveness of PFE in preventing testosterone-induced BPH in rats. This could be attributed, at least partly, to its anti-oxidant, anti-inflammatory, and pro-apoptotic properties. Prostate 75:679–692, 2015. # 2015 Wiley Periodicals, Inc.

KEY WORDS: Benign Prostatic Hyperplasia; Pomegranate extract; Oxidative stress; Inflammation; Testosterone-Induced BPH Rat Model

INTRODUCTION BPH has a significant incidence in men after the age of 50 years, the incidence of histological BPH increases from 8% in the fourth decade to 82% in the eighth decade [1]. BPH represents a benign enlargement of the prostatic tissue surrounding the urethra leading to constriction of the urethra and resulting in increased urgency and frequency of ß 2015 Wiley Periodicals, Inc.




Correspondence to: Emma S. Tomlinson Guns, Vancouver Prostate Centre, Department of Urologic Sciences, University of British Columbia, Vancouver, British Columbia, Canada. E-mail: [email protected] Received 12 July 2014; Accepted 4 December 2014 DOI 10.1002/pros.22951 Published online 25 January 2015 in Wiley Online Library (wileyonlinelibrary.com).

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urination, hesitancy as well as compromised urine flow [2]. Acute and chronic inflammation contribute to the development of BPH by stimulating cellular growth through various pathways particularly oxidative stress [3]. Although controlled production of reactive oxygen species (ROS) is necessary for cellular signaling, proliferation, apoptosis, and protection against microorganisms, higher concentrations are associated with a variety of diseases such as cancer, ischemia, along with immune, and hormonal abnormalities [4]. Prostate tissue is normally protected from oxidative damage by free radical scavengers and enzymatic antioxidants such as glutathione, catalase, and superoxide dismutase [5]. BPH also involves increased growth of both smooth muscle and epithelial cells mainly in the transition zone of the prostate [6]. Interestingly, Di Silverio et al. [7] showed that COX-II inhibition in human prostatic tissue can produce a significant increase in apoptosis in prostatic tissue. Additionally, in a study by Minutoli et al. [8], a combination of Serenoa repens, selenium, and lycopene was effective in decreasing prostate weight and growth in testosterone-induced BPH in rats by promoting apoptosis through induction of Caspase-3 expression. PFE contains a medley of polyphenolic compounds including hydrolysable tannins which possess anti-inflammatory [9], anti-proliferative, pro-apoptotic [10,11], and anticancer effects [12] and therefore, offers a potential benefit for BPH patients. Pomegranate anti-inflammatory effects involve inhibition of both COX and LOX enzymes [13] while also inhibiting prostaglandin release from cells [14]. Furthermore, polyphenolic compounds in pomegranate juice (PJ) possess antioxidant properties [15], this was also shown in treating rats with liver fibrosis with 50 mg/kg pomegranate peel extract for four weeks which lowered malondialdehyde (MDA) levels and myeloperoxidase activity along with TNF-a and IL-1b levels [16]. Moreover, PJ have demonstrated antiproliferative effects in clinical and pre-clinical trials [17]. Similarly, other studies have revealed that PJ has both anti-proliferative and pro-apoptotic effects in LNCaP, PC-3, and DU-145 [10,11] mainly through its effects on cell cycle and apoptosis, these effects were also detected in human colon cancer cells [18], breast cancer cells [19] and PC3 prostate cancer cell lines [20]. Therefore, the aim of current study was to investigate the role of PFE in the development of BPH using a testosterone-induced BPH model in rats.

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MATERIALS AND METHODS Drugs Pomegranate Fruit Extract (PFE): was provided by Verdure Sciences Inc. Noblesville, IN. Testosterone: (Steroid S.P.A, Cologno Monzese (MI), Italy) was kindly supplied by (Chemical Development Industries Co (CID), Cairo, Egypt). PFE was dissolved in a mixture of 68.75% propylene glycol, 15.625% ethanol, and 15.625% water. Thiobarbituric acid (TBA) and 1,1,3,3 tetramethoxypropane were purchased from Sigma Aldrich Chemical Co. (St. Louis, MO). All other chemicals were of the highest available analytical grade. Compositional Analysis of Pomegranate Fruit Extract (PFE) Our previously published work described the compositional characteristics of the PFE used in this report [21]. Several chemical constituents of the water soluble extract, including gallic acid, ellagic acid, caffeic acid, luteolin, hexahydroxydiphenic acid, cyanidin, and gallagyldilactone were identified using preparative thin layer chromatography (TLC), HPLC (PDA), and LC/MS methods including MS scan and MRM, and compared with the authentic standards [21]. Animals The protocol for animal handling and treatment was approved by Bioethical and Research Committee of Ain Shams University, Cairo, Egypt. Forty-eight male Sprague–Dawley rats (200–250 g) aged 10 weeks, were purchased from Nile Co. for Pharmaceutical and Chemical industries, Cairo, Egypt. Rats were housed in an air-conditioned atmosphere, at a temperature of 25C with alternate 12-hr light and dark cycles. Animals were acclimated for 1 week before experimentation. They were kept on a standard diet and water ad libitum. Experimental Design Animals were randomly divided into six groups, eight animals each. A group of rats served as the control and was administered 1.2 ml olive oil subcutaneously (S.C.) and propylene glycol orally. A second group was administered 3 mg/kg testosterone propionate (S.C.), dissolved in olive oil, daily for four weeks to induce BPH comparable to that occurring in patients as described by Shin et al. [22]. Groups 3, 4, and 5 were given PFE by oral gavage at three different doses (25 mg/kg, 50 mg/kg, and 100 mg/kg), respectively

The Effect of Pomegranate Fruit Extract on PBH along with testosterone. Group 6 rats were given PFE 50 mg/kg orally and olive oil S.C. Rats were sacrificed 72 hr after the last testosterone injection and the prostates were immediately removed and weighed, then the ratio of the prostate weight to body weight was calculated. Sections of the ventral prostate lobe were fixed in 10% neutral buffered formalin and embedded in paraffin for both histological and immunohistochemical examinations. Histopathological Examination Ventral prostate tissues were embedded in 10% formalin and processed for paraffin sections of 4 mm thickness. After de-waxing and rehydration, sections were stained with haematoxylin and eosin (H&E) for routine histopathological examination using light microscopy (Image J, 1.46a, NIH, USA).

Assessment of Oxidative Stress Markers Prostatic tissues were homogenized in ice cooled phosphate-buffered saline (50 mM potassium phosphate, pH 7.5). Reduced glutathione (GSH) was assessed in the tissue homogenates using the commercially available kit (Biodiagnostic, Cairo, Egypt). Total glutathione content [GSH þ GSSG] was determined by glutathione reductase enzyme recycling assay [23]. Additionally, catalase (CAT) and superoxide dismutase (SOD) activities were determined using the commercially available kit (Biodiagnostic, Cairo, Egypt), according to the manufacturing instructions. Lipid peroxidation was estimated spectrophotometrically using the thiobarbituric acid reactive substance (TBARS) method by measuring malondialdehyde (MDA) level, as described by Mihara and Uchiyama [24]. Protein content was determined according to Biuret method using the commercially available kit (Biodiagnostic, Cairo, Egypt).

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Immunohistochemical Detection of COX-II, iNOS, Ki-67 and Apoptotic Index Paraffin embedded tissue sections of 3 mm thickness, taken from three rats per group, were deparaffinized first with xylene then hydrated using an ethanol series and heated in citrate buffer (pH 6.0) for 5 min. After that, the sections were blocked with 5% bovine serum albumin (BSA) in tris buffered saline (TBS) for 2 hr. The sections were then incubated overnight at 4 °C with one of the following primary antibodies; COX-II rabbit polyclonal antibody (cat#PA5–23638, Thermo Fischer Scientific), iNOS rabbit polyclonal antibody (cat#RB-9242-P, Thermo Fisher Scientific), Ki-67 rabbit monoclonal antibody (cat#RM-9106 Thermo Fisher Scientific), AR rabbit polyconal antibody (cat#SC-816, Santa Cruz), NF-kB rabbit polyclonal antibody (cat#SC-372, Santa Cruz) , ER-a (cat#NCL-L-ER-6F11, Novacastra) and phospho AKT rabbit monoclonal antibodies (cat#4060, Cell Signaling). Tunnel assay was performed using the following enzymes, terminal transferase (cat# 03 333 566 001), digoxigenin-11-dUTP, alkali-stable (cat# 11 093 088 910) from Roche and dATP sodium salt solution (cat# D6920, Sigma Aldrich Chemical Co). Antibodies were purchased at a concentration of 1 mg/ml containing 5% BSA in TBS. After washing the slides with TBS; the sections were incubated with the corresponding secondary antibody. Sections were then washed with TBS and incubated for 10 min in a solution of 0.02% diaminobenzidine containing 0.01% H2O2. Counter staining was performed using hematoxylin and the slides were visualized under a light microscope [25]. The immunohistochemical quantitation was performed by using image analysis software (Image J, 1.46a, NIH, USA). It was represented as the optical density of stained (positive) cells across ten different fields for each rat section. Immunohistochemical Evaluation of Proliferation index and Apoptotic index: This study was done on

TABLE I. Effect of Three Different Doses of PFE on Prostate Weight, Prostate Weight /body Weight Ratio and Prostatic Acid Phosphatase in Testosterone-Induced BPH in Rats: Data Are Expressed as Mean  S.D. Statistical Analysis Was Performed Using One-Way ANOVA. Groups Control Testosterone 3 mg/kg Testosterone þ PFE (25 mg/kg) Testosterone þ PFE (50 mg/kg) Testosterone þ PFE (100 mg/kg) PFE (50 mg/kg)

Prostate weight

Prostate weight/body weight

b

100% 253.98%a 182.69%b 164.69%b 175.62%b 112.50%b

b

100% 256.96%a 155.37%b 140%b 115%b 102.45%b

PAP 100%b 409.18%a 275.77%a,b 193.90%b 131.86%b 95.44%b

Statistically significant from the control group at P < 0.05. Statistically significant from the corresponding testosterone-treated group at P < 0.05.

a

b

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Fig. 1. Histological examination. A: section taken from the control group showing normal histological structure and columnar lining epithelium. B: section taken from the testosterone treated group showing cystic hypertrophic wide lumen with papillary projections and increased epithelial thickness. C: section taken from the group co-treated with PFE 25 mg/kg and testosterone showing hypertrophy with cystic dilatation in some acini and mild reduction of epithelial thickness. D: section taken from the group co-treated with PFE 50 mg/kg and testosterone showing mild hypertrophy with cystic dilatation in some acini and some reduction of epithelial thickness. E: section from the group co-treated with PFE and testosterone 100 mg/kg showing normal histological structure indicating marked reduction in hypertrophy and hyperplasia. F: section taken from the prostate treated with PFE 50 mg/kg showed normal histological structure.

the 5 mm sections from six Rat formalin fixed paraffin embedded prostate specimens. Immunohistochemical staining of Ki-67 and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining were conducted by Ventana autostainer model Discover XT (Ventana Medical System, Tuscan, Arizona) with enzyme labeled biotin streptavidin system and solvent resistant DAB Map kit by using 1/500 of Ki67 rabbit monoclonal antibody from Thermo scientific, Waltham, MA and DAB-Map TUNEL Assay from Ventana using 1/10 concentrations of TdT enzyme. The Prostate

All IHC slides were scanned by 40x objective of Leica scanner 400 and the images were selected of X10 of the image hub software. Statistical Analysis Data are presented as mean  SD. Multiple comparisons were performed using one-way ANOVA followed by Tukey–Kramer as a post hoc test. The 0.05 level of probability was used as the criterion for significance. All statistical analyses were performed

The Effect of Pomegranate Fruit Extract on PBH using GraphPad Instat software version 3. Graphs were sketched using GraphPad Prism software version 4 (GraphPad Software, Inc., La Jolla, CA, USA). RESULTS Prostate Weight, Prostate Weight/Body Weight Ratio

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ALD was significantly decreased compared to testosterone group to 195.79 mm (Fig. 1E). Treatment with PFE 50 mg/kg alone showed intact normal histological structure similar to that of the control, ALD was 191.66 mm, significantly lower than the testosterone treated group (Fig. 1F). No remarkable inflammatory response was observed at the molecular level of the testosterone or PFE treated groups.

Animals treated with testosterone showed a significant increase in prostate weight, prostate weight/ body weight ratio to 253%, 256% compared to the control group. PFE dosed to rats at 25 mg/kg, 50 mg/ kg, and 100 mg/kg, significantly decreased prostate weight by 28.4%, 35.1%, and 30.8% respectively compared to the untreated BPH group, similarly, the increase in prostate weight/body weight ratio was decreased by 39.4%, 42.9%, and 55% respectively compared to the testosterone-induced BPH group. Whereas the group treated with PFE 50 mg/kg only, did not show significant difference in the above parameters from the control group (Table I).

Animals treated with testosterone showed a significant increase in prostate PAP level to 409.177% compared to the control. Co-treatment with PFE (25, 50 and 100 mg/kg) reduced the increase in PAP levels significantly by 32.7%, 52.8%, and 68% respectively. Co-treatment with PFE 100 mg/kg was effective in decreasing PAP to a level which is significantly different from the induced-BPH group but not from the control group. The group treated with PFE 50 mg/kg only did not show significant difference in PAP level from the control group (Fig. 2, Table I).

Histopathological Examination

GSH

Sections from the ventral prostates of the control group stained with hematoxylin-eosin showed no histological alteration in the acini or the lining epithelium. Epithelial cells were cuboidal in shape and of regular size; Average Lumen Diameter (ALD) is 239.125 mm (Fig. 1A). Testosterone administration induced disrupted morphology in the prostate epithelia indicated by marked thickening and hypertrophy as well as hyperplasia with papillary projections in the lining epithelium of the acini, widening of the lumen diameter was also observed for this group, no remarkable expansion was observed in the stroma, ALD significantly increased compared with the control to 374.5 mm for this group (Fig. 1B). Treatment with 25 mg/kg and 50 mg/kg PFE showed mild hypertrophy with cystic dilatation in some acini. The decrease in the epithelial thickness was insignificant compared to the testosterone-treated group and ALD was decreased to 255.5 mm and 229 mm respectively (Fig. 1C and 1D). On the other hand, PFE in the 100 mg/kg dose was effective in reducing the hypertrophy and hyperplasia seen in the testosterone-treated group maintaining the normal histological structure indicated by decreasing epithelial thickness and acini diameter to values similar to that of the control ones where the size of epithelial cells was decreased and the long secretory luminal cells changed to small flat cuboidal ones, also, papillary projections were absent. In this group,

Mean GSH level in the control group was 0.12 mmol/mg protein. Animals treated with testosterone showed a significant decrease in prostate GSH level to 0.45 fold compared to the control. Treatment with PFE (25 and 50 mg/kg) resulted in an increase of GSH level to 0.56 and 0.63 fold of the control values respectively which are statistically significant from both the testosterone and control group. However, treatment with PFE 100 mg/kg resulted in significant

PAP Activity

Fig. 2. The effect of PFE on PAP level in prostate homogenate in testosterone-induced prostatic hyperplasia. a: Statistically significant from the control group at P < 0.05, b: Statistically significant from the corresponding testosterone treated group at P < 0.05 The Prostate

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increase of GSH to 0.83 fold that of the control which is statistically significant from the testosterone group but not from the control group. Whereas the group treated with PFE 50 mg/kg showed no significant variation in the GSH levels as compared to the control group (Fig. 3A, Table II). Total Glutathione Total glutathione levels in animals treated with PFE 25, 50, and 100 mg/kg did not show significant difference either from the control group, testosterone

3 mg/kg or the PFE 50 mg/kg only treated group (Fig. 3B, Table II). SOD SOD level in the control group was 38.11 U/mg protein. Animals treated with testosterone showed a significant decrease in prostate SOD level to 0.64 the value of the control. Treatment with PFE dosed at 25 and 50 mg/kg resulted in an increase of SOD levels to 0.72 and 0.81 the value of the control values respectively which is still statistically significant from the

Fig. 3. The effect of PFE on oxidative stress parameters. A: GSH B: Total glutathione. C: Superoxide dismutase level. D: Malondialdehyde level. E: Catalase level. a: Statistically significant from the control group at P < 0.05 b: Statistically significant from the corresponding testosterone treated group at P < 0.05 The Prostate

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TABLE II. Effect of PFE 25, 50 and 100 mg/kg on GSH, Total GSH and MDA Levels in Prostate Homogenates of Testosterone-Induced BPH in Rats. Data Are Expressed as Mean  S.D. Statistical Analysis Was Performed Using One-Way ANOVA. Group

GSH (mmol/mg protein)

Total glutathione (mmol/mg protein)

MDA (nmol/ mg protein)

0.125  0.010b 0.056  0.007a a,b 0.071  0.001 a,b 0.08  0.001 0.105  0.012b 0.126  0.006b

0.158  0.021 0.155  0.024 0.15  0.021 0.16  0.027 0.159  0.029 0.158  0.020

0.224  0.050b 1.371  0.140a a,b 1.09  0.068 a,b 0.873  0.121 0.363  0.034b 0.244  0.047b

Control Testosterone Testo þ PFE 25mg/kg Testo þ PFE 50 mg/kg TestoþPFE 100 mg/kg PFE 50 mg/kg

Statistically significant from the control group at P < 0.05. Statistically significant from the corresponding testosterone-treated group at P < 0.05.

a

b

control group and not from the testosterone group. However, treatment with PFE 100 mg/kg resulted in significant increase of SOD to 0.91 the value of the control which is statistically significant from the testosterone group and not from the control group. Whereas the group treated with PFE 50 mg/kg only showed no significant variation in the SOD levels compared to the control group (Fig. 3C). MDA Testosterone-treated group exhibited a significant increase in MDA level by 612% compared to the control group. Treatment with PFE 25 and 50 mg/kg alleviated this increase by 20% and 34.96% respectively yielding MDA values that are significantly different both from the control and the testosterone treated group. However treatment with 100 mg/kg PFE lead to a significant decrease in MDA level by 72% compared to the induced-BPH group yielding an MDA level statistically different from the induced-BPH group and not from the control. Whereas the group treated with PFE 50 mg/kg only showed no significant variation in the MDA levels compared to the control group (Fig.3D, Table II). CAT Testosterone-treated group exhibited a significant decrease in CAT level to 88.44% compared to the control group. Treatment with PFE 25 and 50 mg had no significant effect on CAT level having values of 88.41% and 89.78% compared to the control. However, treatment with 100 mg/kg PFE lead to an increase in CAT level to 93.81% albeit not significantly different from the testosterone treated group or the control. Whereas the group treated with PFE 50 mg/kg only showed no significant variation in the CAT levels compared to the control group (Fig.3E).

iNOS Expression The expression of iNOS in the acinar epithelia was assessed immunohistochemically. The control group showed limited iNOS expression (Fig.4A). Significant elevation in the expression of iNOS in the epithelial cells was seen in the testosterone treated group compared to the control group (Fig.4B). Treatment with PFE 25, 50, and 100 mg/kg significantly decreased iNOS expression compared to testosterone-treated group (Fig. 4C–4E). PFE 50 mg/kg only treated group did not show significant variation in iNOS level compared to the control (Fig.4F). Statistical analyses for these data sets are provided as a Supplementary file. COX-II Expression The expression of COX-II in the acinar epithelia was assessed immunohistochemically. The control group showed limited expression of COX-II (Fig.5A). Significant elevation in the expression of COX-II in the epithelial cells was seen in the testosterone-treated group compared to the control group (Fig.5B). Treatment with PFE 25, 50, and 100 mg/kg significantly decreased COX-II expression compared to testosterone-treated group (Fig.5C–5E). PFE 50 mg/kg only treated group did not show significant variation in COX-II expression compared to the control (Fig.5F). Statistical analyses for these data sets are provided as a Supplementary file. Ki-67 Expression The expression of Ki-67 protein in the epithelial cells was assessed immunohistochemically. The control group showed limited expression (Fig.6A). Significant elevation in the expression of Ki-67 was seen in the testosterone treated group as evidenced by extensive degree of staining (Fig.6B) indicating The Prostate

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Fig. 4. Immunohistochemical localization of iNOS in the prostate tissues. A: Expression of iNOS in the prostate epithelial cells of the control group showing light staining. B: Expression of iNOS in the testosterone only treated group showing an extensive degree of staining. C: Expression of iNOS in the prostate epithelial cells of the group co-treated with PFE 25 mg/kg and testosterone shows a minimal degree of expression. D: iNOS expression in the group co-treated with PFE 50 mg/kg and testosterone shows a minimal degree of expression. E: iNOS expression in the group co-treated with PFE 100 mg/kg and testosterone shows a minimal degree of expression. F: iNOS expression in the group treated with PFE 50 mg/kg showing a minimal degree of expression. Magnification is 10X using Image Hub software.

increased cellular proliferation. Treatment with PFE 25 mg/kg, 50, and 100 mg/kg significantly decreased Ki-67 as manifested by light staining compared to the testosterone treated group (Fig.6C–6E). The group receiving PFE 50 mg/kg only did not show significant variation in expression of Ki-67 compared to the control group (Fig.6F). Statistical analyses for these data sets are provided as a Supplementary file. The Prostate

TUNEL Assay TUNEL assay is used to identify cells undergoing apoptosis by detecting DNA terminal nicks expressing deoxynucleotidyl transferase enzyme. Control group showed high expression as manifested by extensive degree of staining indicating increased apoptosis (Fig.7A). In the testosterone treated group, a significant

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Fig. 5. Immunohistochemical staining of COX-II enzyme in the prostate tissues. A: Expression of COX-II in the prostate epithelial cells of the control group showing a light degree of staining. B: Expression of COX-II in prostate epithelial cells of testosterone only treated group showing an extensive degree of staining. C: Expression of COX-II in the group treated with PFE 25 mg/kg showing a light degree of staining. D: Expression of COX-II the group treated with PFE 50 mg/kg showing a light degree of staining. E: Expression of COX-II expression the group treated with 100 mg/kg showing a light degree of staining. F: Expression of COX-II in the group treated with 50 m/kg PFE. Magnification is 10X using Image Hub software

decrease of optical density, indicating decreased apoptotic activity and increased cellular proliferation was observed (Fig.7B). Treatment with PFE 25 mg/kg, 50, and 100 mg/kg significantly increased the number of cells undergoing apoptosis as indicated by heavy staining (Fig.7C–7E). The group receiving PFE 50 mg/kg only did not show significant variation from the control (Fig.7F). Statistical analyses for these data sets are provided as a Supplementary file.

Other Parameters Immunohistochemical staining of androgen receptor (AR) and NF-kB were positive with no significant difference between the different groups (data not shown). On the other hand, estrogen receptor a (ER-a) and phospho AKT (protein kinase B) were negative with no significant difference among the different groups (data not shown). The Prostate

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Fig. 6. Immunohistochemical staining of Ki-67 protein in the prostate tissues. A: Expression of Ki-67 in the prostate epithelial cells of the control group showing light staining. B: Expression of Ki-67 in the testosterone only treated group showing an extensive degree of staining. C: Expression of Ki-67 in the group co-treated with PFE 25 mg/kg and testosterone showing a light degree of staining. D: Expression of Ki-67 in the group co-treated with PFE 50 mg/kg and testosterone showing a light degree of staining. E: Expression of Ki-67 in the group co-treated with PFE 100 mg/kg and testosterone showing a light degree of staining. F: Expression of Ki-67 protein in prostate epithelial cells treated with PFE 50 mg/kg. Magnification is 10X using Image Hub software

DISCUSSION Benign prostatic hyperplasia (BPH) is an enlargement of the transition zone of the prostate and is present in most aging men, resulting in symptoms which require clinical intervention in approximately one third of men over the age of 60 [26], incidence of histological enlargement of the prostate is over 70% at age 60 years and The Prostate

over 90% at age 70 years [27]. It is usually associated with a variety of urological symptoms that range from bothersome to significantly impacting patient’s quality of life [28]. The aim of current study was to investigate the role of PFE in the development of BPH using a testosterone-induced BPH model in rats. In the current study; treatment of rats with 25, 50, and 100 mg/kg PFE orally for 28 days significantly

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Fig. 7. TUNEL assay of the prostatic tissues. A: Section from control group showing heavy staining. B: Section from testosterone only treated group showing a light degree of staining. C: section from the group co-treated with PFE 25 mg/kg and testosterone showing heavy staining. D: section from the group co-treated with PFE 50 mg/kg and testosterone showing heavy staining. E: section from group co-treated with PFE 100 mg/kg showing heavy staining. F: section from the group treated with PFE 50 mg/kg showing heavy staining. Magnification is 10X using Image Hub software

inhibited testosterone mediated increase in the prostate weight, prostate weight/body weight ratio and PAP levels compared to the testosterone treated group. This inhibition of BPH associated markers was found to be directly proportional to the dose of PFE. Additionally, histological examination confirmed the previous results where PFE 25 mg/kg, and 50 mg/kg treated group showed a trend decrease in ALD diameter and epithelial thickness compared to the

testosterone treated group which was not statistically significant, whereas PFE 100 mg/kg doses was effective in preserving the normal values of the ALD along with significant reduction of the intracini spaces and absence of papillary projections in the epithelial wall. Figure 8 summarizes the scoring data from the images shown in Figure 1 thus summarizing the ALD data graphically. Statistical analyses for these data sets are provided as a Supplementary file. The Prostate

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Fig. 8. The effect of PFE treatment on Average Lumen Diameter (ALD) in rat prostate tissues compared with control. a: Statistically significant from the control group at P < 0.05 b: Statistically significant from the corresponding testosterone treated group at P < 0.05. Lumen diameter was calculated as the average of 5 luminal diameters measured in the widest area at 10X magnification using the Image hub Software

Oxidative stress is defined as an abnormal ratio of free radicals and reactive metabolites known as ROS due to their overproduction, to the ratio of cellular antioxidants responsible for their destruction. This imbalance results in injury to the cells, tissues, and possibly to all body organs through damaging important biomolecules and cells [29]. Antioxidants are compounds or enzymes which are able to compete with oxidizable substrates, or instead destroy ROS at low concentrations, thus, preserving the cellular structure [30]. For example, glutathione (GSH) is the antioxidant with highest concentrations found in cells; it plays an important role in counteracting oxidative stress [31]. In the elderly, GSH levels are depleted as a result of its conversion to the oxidized forms, glutathione disulfide (GSSG) and glutathione protein mixed disulfides (GSSP) [32,33]. This depletion is linked to aging and its accompanying disorders in which the antioxidant capacity of the cells is reduced. In the current study 3 free radical scavengers were assessed, GSH, CAT, and SOD, all of them were significantly decreased in the testosterone treated group compared to the control. On the other hand, treatment with three different doses of PFE 25, 50, and 100 mg/kg increased levels of GSH; however, SOD was only significantly increased with PFE dosed at 100 mg/kg, while CAT showed an increased trend which is still not significant from either the control or the testosterone treated group. Total glutathione and lipid peroxides measured by MDA levels were assessed as an indicator of the oxidative status inside the cell. Total glutathione did not show any significant difference between the treatment, testosterone, and control groups, which The Prostate

indicates that although the total glutathione levels were kept constant potent oxidative stress converted most of GSH into GSSG and GSSP. Treatment with PFE 25, 50, and 100 mg/kg significantly precluded the increase in MDA found in the induced-BPH group; however, only PFE 100 mg/kg decreased MDA to levels significantly different from the induced-BPH group and not from the control group. Patients with pronounced prostatic inflammation have larger prostate volumes, which is accompanied by higher incidence of acute urinary retention and poor prognosis [34,35]. Inflammation induces oxidative stress reactions resulting in the generation of arachidonic acid from membranes, which can be converted by COX enzymes to prostaglandins promoting inflammation and cellular growth [4]. Inflammation is also linked to the development of hyperplasia, as indicated in a mouse model of chronic prostatitis where hyperplastic tissues were found adjacent to inflammatory tissues [36]. Prostatic inflammation results in high expression of inflammatory enzymes including COX-II [37,38]. In this study COX-II enzyme expression in epithelial cells was assessed using immunohistochemistry. Overexpression of COX-II enzyme was demonstrated in the epithelial cells of induced-BPH cells. Treatment with PFE 25, 50, and 100 mg/kg precluded this increase in COX-II levels as indicated by light staining similar to the control group. This is in harmony with a number of studies that have demonstrated the efficacy of anti-inflammatory strategies in patients with BPH [39]. In one study, loxoprofen given at a dose of 60 mg in patients with lower urinary tract symptoms (LUTS) not responding to standard treatment, showed an improvement of the nocturia in 74% of the cases with better results in more severe cases [40]. Moreover, using COX-II inhibitor with a 5a- reductase induced apoptosis in BPH cells [41]. Inflammatory cells that are present in the prostate are the principal factor activating reactive nitrogen that can damage cells. Interestingly, iNOS expression in human prostate tissue has been characterized by an increased expression in the epithelial cells of patients with BPH when compared to normal tissues [42]. NO also is thought to potentiate COX-II activity generating pro-inflammatory prostaglandins. In this study iNOS enzyme expression in epithelial cells was assessed using immunohistochemistry. Significantly high levels of iNOS were demonstrated in the epithelial cells of the induced-BPH group. Treatment with PFE 25, 50, and 100 mg/kg precluded this increase in iNOS levels as indicated by light staining similar to that of the control group. Apoptosis is programmed cell death which allows the clearance of abnormal and damaged cells while

The Effect of Pomegranate Fruit Extract on PBH safeguarding other cells from the effect of harmful substances [43,44]. Wang et al. [42] found that COX-II enzyme expression in prostate epithelium is associated with increased cellular proliferation and decreased apoptotic activity through induction of Bcl-2. Antiproliferative and proapoptotic properties of pomegranate fruit extract (PFE) against human prostate cancer cells were demonstrated both in cell cultures and in a mouse xenograft model [45,46]. In another study carried out by our lab (Ming et al. [21]), PFE inhibited cell growth at a concentration of 20 mg/ml in prostate cancer cell lines LNCaP and 22RV1. Also, PC3 cells treated with PFE showed a dose-dependent decrease in cell growth with an increase in apoptosis [45]. In harmony with these results, we see an overexpression of Ki-67 in the testosterone-induced BPH group. Ki-67 is a nuclear protein which is associated with cellular proliferation. Treatment with PFE 25, 50, and 100 mg/kg resulted in a significant reduction in the expression of Ki-67 indicated by comparatively light staining. This supports the ability of PFE to alleviate BPH through its proapoptotic and anti-proliferative properties. In the same manner, the apoptotic index measured by TUNEL assay demonstrated low levels of cells undergoing apoptosis after induction of BPH by testosterone. On the other hand, treatment with PFE 25, 50, and 100 mg/kg was able to restore normal apoptotic activity indicated by an extensive degree of staining confirming the proapoptotic properties of PFE. In conclusion, the current findings elucidate the effectiveness of PFE in preventing testosterone-induced BPH in rats. These results are very striking and could be attributed, at least in part, to its anti-oxidant, anti-inflammatory and pro-apoptotic properties.

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Additional supporting information may be found in the online version of this article at the publisher’s web-site.

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