# Immunomodulatory Effect of Red Onion (Allium cepa Linn) Scale Extract on Experimentally Induced Atypical Prostatic Hyperplasia in Wistar Rats

Authors: Ahmed A. Elberry; Shagufta Mufti; Jaudah Al-Maghrabi; Essam Abdel Sattar; Salah A. Ghareib; Hisham A. Mosli; Salah A. Gabr
Journal: Mediators of Inflammation
 (2014)

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## Abstract

Red onion scales (ROS) contain large amounts of flavonoids that are responsible for the reported antioxidant activity, immune enhancement, and anticancer property. Atypical prostatic hyperplasia (APH) was induced in adult castrated Wistar rats by both s.c. injection of testosterone (0.5 mg/rat/day) and by smearing citral on shaved skin once every 3 days for 30 days. Saw palmetto (100 mg/kg) as a positive control and ROS suspension at doses of 75, 150, and 300 mg/kg/day were given orally every day for 30 days. All medications were started 7 days after castration and along with testosterone and citral. The HPLC profile of ROS methanolic extract displayed two major peaks identified as quercetin and quercetin-4′-β-O-D-glucoside. Histopathological examination of APH-induced prostatic rats revealed evidence of hyperplasia and inflammation with cellular proliferation and reduced apoptosis Immunohistochemistry showed increased tissue expressions of IL-6, IL-8, TNF-α, IGF-1, and clusterin, while TGF-β1 was decreased, which correlates with the presence of inflammation. Both saw palmetto and RO scale treatment have ameliorated these changes. These ameliorative effects were more evident in RO scale groups and were dose dependent. In conclusion, methanolic extract of ROS showed a protective effect against APH induced rats that may be attributed to potential anti-inflammatory and immunomodulatory effects.


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## Body

## 1. Introduction

Atypical prostatic hyperplasia (APH) is a pseudoneoplastic lesion that can mimic prostate adenocarcinoma because of its architectural and cytological features. In this prostatic disease, there is an imbalance between prostate cell growth and apoptosis [1]. The occurrence rate of APH is not known and its associations with benign prostatic hypertrophy (BPH) and latent carcinoma of the prostate (LPC) have not been completely clarified [2]. The fact that APH arises always in prostates with concomitant BPH and exhibits several cancer-like features places APH as an intermediate lesion between BPH and the subset of well-differentiated cancers in a hypothetical pathway between BPH and LPC [3].The role of inflammation in prostate diseases is suggested by the presence of inflammatory cells within the BPH [4]. Inflammation is usually a self-limited event, with initial proinflammatory cytokines, growth factor release, and angiogenesis followed by an anti-inflammatory cytokine-mediated resolution [5]. Chronic inflammation produces free radicals as various reactive oxygen species [6].There is an interest in the potential health benefits of Allium vegetables, mainly onion (Allium cepa) and garlic (A. sativum). Intake of Allium vegetables may be inversely related to cancer at various sites, including prostate [7, 8]. Allium vegetables seem to have antiproliferative action on human cancers and to prevent diseases associated with aging [9]. Moreover, onion and garlic intake has been inversely associated with BPH [10]. Onions contain high levels of nonnutrient antioxidant compounds, flavonoids, and the alk(en)yl cysteine sulfoxides (ACSOs), which have protective effects against different degenerative pathologies such as cardiovascular disease, cancer, and other dysfunctions based on oxidative stress [11].The uses of bulb in food and manufacture introduce tons of its scales (red dried onion scales, ROS) as a waste product. Recently, these scales have attracted the attention of many researchers to maximize the benefits of this waste material. ROS contain large amounts of flavonoids, mainly ferulic, gallic, kaempferol, quercetin, quercetin dimer, quercetin trimer, and quercetin glycosides. These flavonoids are responsible for the reported antioxidant activity of ROS which is comparable to that ofα-tocopherol [12, 13]. They are also reported to have hepatoprotective effect, immune enhancement potential, anti-infectious, antistress and anticancer properties [14]. Moreover, quercetin was found to ameliorate significantly prostatitis symptoms by decreasing prostatic inflammation [15]. Most of the studies were done using an onion bulb per se, bulb extracts, or bulb juice without studying the protective effect of RO scales, the richest part of the onion with quercetin. The aim of this study was to investigate the protective effect of the ROS extract on enlarged rat prostate and the histopathological changes related to inflammation, proliferation, and/or apoptosis in APH induced experimentally in rats.


## 2.  Materials and Methods

### 2.1. Chemicals and Reagents

Solvents of HPLC grade used for HPLC analysis, in addition to analytical grade solvents for extraction and thin layer chromatography (TLC), were purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies against clusterin, phospho-Smad2, andβ-actin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA; anticlusterin for Western blot analysis); Upstate Biotechnology (Lake Placid, NY; anticlusterin for immunohistochemistry (IHC)); and Cell Signaling Technology Inc. (Danvers, MA). Antibodies against TGF-β1 ligands were purchased from Dakocytomation (Carpinteria, CA). Citral was obtained from Fluka Chemie AG, Buchs, Switzerland. Testosterone was obtained from Sigma-Aldrich and Dakocytomation (Carpinteria, CA), while saw palmetto was obtained from Futurebiotics (NY, USA).


### 2.2. Collection and Extraction of Dried Red Onion Scales

The red onion bulbs (Allium cepa L., Giza red cultivar) were collected from a single field cultivated in Sohag city, Sohag governorate, Egypt. It was cultivated in mid-November and collected in the beginning of May, 2010. The fully grown bulbs were peeled and the ROS were stored at 25°C in a dark place till extraction. The plant sample was identified by a staff member of the Horticulture Department, Faculty of Agriculture, Cairo University. ROS powder (1250 g) was extracted with 80% ethanol (3 × 5 liters) by using Ultra-Turrax T50 homogenizer (Janke and Kunkel, IKA-Labortechnik, Staufen, Germany) at a temperature not exceeding 25°C. The extract was concentrated by evaporation under reduced pressure, lyophilized to give 240 g of reddish semisolid residue, and protected from light at 4°C to use later.


### 2.3. Standardization of Red Onion Scale (ROS) Extract

Preliminary phytochemical tests to identify the main chemical constituents were carried out according to the methods of Trease and Evans [16]. Chromatographic TLC profile of the methanolic extract was performed using thin layer chromatography (solvent system : ethyl acetate-formic acid-glacial acetic acid-water, 100 : 11 : 11 : 26) as per fingerprinting using a chromatographic condition reported by Wagner and Bladt [17] against common reference flavonoids available in the Pharmacognosy Department, Faculty of Pharmacy, Cairo University. Total phenolics were determined as mg gallic acid equivalent per gram dry extract (mg GAE/g DE) employing the Folin-Ciocalteu method described by Singleton and Rossi [18]. Aluminum chloride colorimetric assay described by Hertog [19] was applied for determination of total flavonoids content as mg quercetin equivalent per gram of dry extract (mg QE/g DE) with a slight modification [20]. The free radical scavenging activity of the extracts, based on the scavenging activity of the stable 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical, was determined by the method described by Braca et al. [21]. The capability of the extracts to scavenge the DPPH radical was calculated using the following equation: %inhibition = (A
Control
-
A
Test) × 100/A
Control.


### 2.4. HPLC Analysis

Quercetin (Q) and quercetin-4′-β-O-D-glucoside (QG) were used as standard marker compounds. QG was separated from the ROS extract and identified using a procedure reported by Abou Zid and Elsherbeiny [22]. HPLC analysis was performed on Agilent HP1200 series HPLC system equipped with a G1322A quaternary pump and degasser, a G1314A variable wavelength UV detector, and 250 × 4.0 mm (particle size 5 μm) ODS column (Lichrospher 100, Merck, Darmstadt, Germany). Chromatographic separation was achieved by applying a linear gradient system using mobile phase A (5% formic acid in water v/v) and mobile phase B (methanol). A linear gradient elution was performed using 50% mobile phase B for 10 min, rising to 80% B over 10 min, then to 100% B over 11 min, and back to 50% B. The flow rate was maintained at 1.0 mL/min and UV was monitored at 360 nm.


### 2.5. Experimental Animals

Adolescent male Wistar rats, aged 50–60 days, were obtained from the animal facility of King Fahd Research Center, King Abdulaziz University, Jeddah, Saudi Arabia. They were used in the study according to the guidelines of the Biochemical and Research Ethics Committee at King Abdulaziz University, in accordance with the NIH guidelines. Animals were housed in a well-ventilated, temperature-controlled room at 22± 3°C with a 12 h light-dark cycle. They were provided with standard rat chow pellets obtained from Grain Silos and Flour Mills organization F-1005, Jeddah, Saudi Arabia, and tap water ad libitum. All experimental procedures were performed between 8–10 a.m. and care was taken to avoid stressful conditions.Orchidectomy was performed aseptically, under ethyl ether anesthesia, by a midscrotal incision. Following ligation of the spermatic cord and vessels, testes and epididymis were removed. The remaining stump was pushed back through the inguinal canal into the abdominal cavity, and the scrotal sac was closed by sutures [23]. After castration, the rats were maintained under standard laboratory conditions for 7 days in order to allow a definite involution of the prostatic gland [24].APH was induced as previously described by Engelstein et al. [25] in castrated rats using citral and testosterone. Rats were subcutaneously injected with testosterone propionate in corn oil (0.5 mg/0.1 mL/rat) each day for 30 days. Citral, 1 M diluted in 70% ethanol, was smeared on a different shaved area of skin, each time, on the back at a final dose of 185 mg/kg every 4 days for 30 days. Control group rats were smeared with the solvent (ethanol) alone at the shaved skin. Considering the pungent lime fragrance of citral, the control groups were kept in a separate location from the citral-treated animals to avoid possible false results as citral has some influence via the olfactory tract.


### 2.6. Animal Treatment

Seven days after castration, the animals were randomly divided into six groups (n
=
7). Group I served as a control (shamed operation) group and received both corn oil (0.1 mL/rat) and 1% CMC-Na (0.3 mL/100 g body weight) daily during the period of the experiment. Groups from II–VI were castrated and had APH. Group II served as negative control and received CMC-Na 1% as previously mentioned. Group III served as positive control and received saw palmetto (100 mg/kg) suspended in 1% CMC-Na. Groups from IV–VI were treated with ROS at doses of 75, 150, or 300 mg/kg/day, respectively, by oral gavage. All the ROS extracts were suspended in 1% CMC-Na and were given to rats once daily by oral gavage along with testosterone injection and citral smearing for 30 days as described before [26]. After euthanization, ventral prostate of each rat in each group was removed from the body, 24 h after the last administration, and weighed. The half of the tissues were frozen in liquid nitrogen and stored at −75°C until use. The remaining tissues were fixed immediately in 0.1 M phosphate-buffered 10% formalin (pH 7.4) for 48 h and then embedded in paraffin and were used for histological studies. Serial 4 μm thick sections from each tissue specimen were prepared and mounted on poly-L-lysine coated glass slides. These were used for detection of IL-6, IL-8, TNF-α, IGF-1, clusterin, and TGF-β1 receptors in the prostatic tissue by immunohistochemistry.


### 2.7. Histopathology and Histoscore

A part of the ventral lobes was separated and fixed overnight in Stieve’s solution. Thereafter, the tissue was thoroughly rinsed with water and immersed overnight in ethanol 70%. Then, it was dehydrated, embedded in paraffin, and 5 mm thick sections were cut and stained by Harris’ hematoxylin eosin, according to the standard procedures of Lillie [27].A score-chart protocol (histoscore) developed by Scolnik et al. [26] was used to obtain an objective quantitative assessment. The examination, description, and scoring of the slides were performed in a blinded manner. The scoring system was presented in arbitrary units to make a better evaluation. In a second step, the cumulative score in each group was correlated to the final histological diagnosis in order to establish a score range for normal and hyperplasia. An additional histological inflammatory score described by de Nunzio et al. [28] was used to evaluate the inflammation. Score 0: no inflammation, score 1: scattered inflammatory cell infiltrate without nodules, score 2: no confluent lymphoid, and score 3: large inflammatory areas with confluence.


### 2.8. Immunohistochemistry and Immunhistoscoring

Hematoxylin and eosin staining was performed to observe histopathology. For analysis of IL-6 and TGF-β1 expression, sections from the paraffin embedded tissue blocks were mounted on charged glass slides and baked at 60°C for 1 h in the oven, then mounted on the Ventana staining machine, dewaxed by EZ Prep (Xylene substitute), and rehydrated. The tissue sections were heated in Ventana buffer CC1 (pH 6) to facilitate antigen retrieval and treated with H2O2 to eliminate endogenous peroxidase. This was followed by incubation for 60 min at room temperature with primary antibodies IL-6 and TGF-β1. The dilutions used were IL-6 (dilution 1 : 50) and TGF-β1 (dilution of 1 : 25). Subsequently, the sections were incubated with biotinylated secondary antibody using the avidin-biotin complex method. The immunoreaction was visualized using diaminobenzidine. All sections were lightly counterstained with hematoxylin as a background. The positive control used for IL-6 and TGF-β1 was from the colon. The negative controls comprised serial sections that were stained using equivalent concentrations of nonimmune mouse IgG in place of the primary antibodies. The level of staining was evaluated independently by three observers blinded to experimental conditions.Expression of TGF-β1 and IL-6 was evaluated according to a semiquantitative scale: 0, no detectable staining at all; 1, less than 10% of the cells stained positive; 2, 10−50% positive cells; and 3, more than 50% of cells positive [29]. Staining intensity was scored as 0 (no detectable stain), 1 (weak staining detected at intermediate to high power), 2 (moderate detected at low to intermediate power), to 3 (strong detected at low power) [30].


### 2.9. Reverse Transcription Polymerase Chain Reaction (RT-PCR)

Total RNA was extracted from the snap-frozen tissue samples using total RNA isolation kit (Macherey-Nagel) according to the manufacturer’s instructions. RT was performed in a 10μL reaction mixture. The RT reaction contained 1 μg RNA, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 2.5 mM dithiothreitol, 500 μmol/liter each of dATP, dCTP, dGTP, and dTTP (Bioline), 40 U RNasin (Bioline), 25 μg/mL oligo dT_pd(T)12–18 (Bioline), and 100UMoloney murine leukemia virus reverse transcriptase (Bioline). The reaction mixture was incubated at 42°C for 60 min and then heated to 80°C for 5 min. The resultant cDNA was used for PCR. For quantitative real-time RT-PCR, we prepared appropriate dilutions of each single-strand cDNA followed by normalizing of the cDNA content using β-actin as a quantitative control. Quantitative PCR amplification was performed with a 25 μL final volume consisting of 1 μL RT reaction mixture, 3 mM MgCl2, 10 pmol of each sense and antisense primer, and 12.5 μL (Roche Diagnostics). PCR conditions were as follows: initial denaturation at 95°C for 10 min and 35 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 1 min, and elongation at 72°C for 2 sec with a final elongation at 72°C for 10 min. Samples were migrated in 1% agarose gel using electrophoresis, UV visualized, and images were analyzed using total lab120 (Nonlinear Dynamic Ltd). Clusterin, TGF-β1, IGF-1, IL-6, IL-8, and TNF-α expression in the test samples were normalized to the corresponding β-actin level and were reported as the relative band intensity to the β-actin gene expression. Sequences of oligonucleotides used as primers for β-actin, IL-6, IL-8, TNFα, TGF-β1, IGF-1, and clusterin are summarized in Table 1.Table 1
Sequences of oligonucleotides used as primers.
Gene
Forward primer
Reverse primer
β-Actin
5′-GTCACCCACACTGTGCCCATCT-3′
5′-ACAGAGTACTTGCGCTCAGGAG-3′
IL-6
5′-GAACTCCTTCTCCACAAGCG-3′
5′-TTTTCTGCCAGTGCCTCTTT-3′
IL-8
5′-CTGCGCCAACACAGAAATTA-3′
5′-ATTGCATCTGGCAACCCTAC-3′
TNF-α
5′-CAGAGGGAAGAGTTCCCCAG-3′
5′-CCTTGGTCTGGTAGGAGACG-3′
TGF-β1
5′-GTTCTTCAATACGTCAGACATTCG-3′
5′-CATTATCTTTGCTGTCACAAGAGC-3′
IGF-1
5′-CACAGGCTATGGCTCCAGCAT-3′
5′-TCTCCAGCCTCCTCAGATCACA-3′
Clusterin
5′-CTGACCCAGCAGTACAACGA-3′
5′-TGACACGAGAGGGGACTTCT-3′


### 2.10. Statistical Analysis

Data were expressed as mean± SE and were analyzed by analysis of variance (ANOVA) followed by Tukey-Kramer multiple comparisons test. Inflammation scores and their significance were calculated by Chi-square test with Yate’s corrections. Differences were considered significant with a P value less than 0.05. Statistical analyses were performed using the SPSS for Windows (v. 10.0).


## 2.1. Chemicals and Reagents

Solvents of HPLC grade used for HPLC analysis, in addition to analytical grade solvents for extraction and thin layer chromatography (TLC), were purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies against clusterin, phospho-Smad2, andβ-actin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA; anticlusterin for Western blot analysis); Upstate Biotechnology (Lake Placid, NY; anticlusterin for immunohistochemistry (IHC)); and Cell Signaling Technology Inc. (Danvers, MA). Antibodies against TGF-β1 ligands were purchased from Dakocytomation (Carpinteria, CA). Citral was obtained from Fluka Chemie AG, Buchs, Switzerland. Testosterone was obtained from Sigma-Aldrich and Dakocytomation (Carpinteria, CA), while saw palmetto was obtained from Futurebiotics (NY, USA).


## 2.2. Collection and Extraction of Dried Red Onion Scales

The red onion bulbs (Allium cepa L., Giza red cultivar) were collected from a single field cultivated in Sohag city, Sohag governorate, Egypt. It was cultivated in mid-November and collected in the beginning of May, 2010. The fully grown bulbs were peeled and the ROS were stored at 25°C in a dark place till extraction. The plant sample was identified by a staff member of the Horticulture Department, Faculty of Agriculture, Cairo University. ROS powder (1250 g) was extracted with 80% ethanol (3 × 5 liters) by using Ultra-Turrax T50 homogenizer (Janke and Kunkel, IKA-Labortechnik, Staufen, Germany) at a temperature not exceeding 25°C. The extract was concentrated by evaporation under reduced pressure, lyophilized to give 240 g of reddish semisolid residue, and protected from light at 4°C to use later.


## 2.3. Standardization of Red Onion Scale (ROS) Extract

Preliminary phytochemical tests to identify the main chemical constituents were carried out according to the methods of Trease and Evans [16]. Chromatographic TLC profile of the methanolic extract was performed using thin layer chromatography (solvent system : ethyl acetate-formic acid-glacial acetic acid-water, 100 : 11 : 11 : 26) as per fingerprinting using a chromatographic condition reported by Wagner and Bladt [17] against common reference flavonoids available in the Pharmacognosy Department, Faculty of Pharmacy, Cairo University. Total phenolics were determined as mg gallic acid equivalent per gram dry extract (mg GAE/g DE) employing the Folin-Ciocalteu method described by Singleton and Rossi [18]. Aluminum chloride colorimetric assay described by Hertog [19] was applied for determination of total flavonoids content as mg quercetin equivalent per gram of dry extract (mg QE/g DE) with a slight modification [20]. The free radical scavenging activity of the extracts, based on the scavenging activity of the stable 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical, was determined by the method described by Braca et al. [21]. The capability of the extracts to scavenge the DPPH radical was calculated using the following equation: %inhibition = (A
Control
-
A
Test) × 100/A
Control.


## 2.4. HPLC Analysis

Quercetin (Q) and quercetin-4′-β-O-D-glucoside (QG) were used as standard marker compounds. QG was separated from the ROS extract and identified using a procedure reported by Abou Zid and Elsherbeiny [22]. HPLC analysis was performed on Agilent HP1200 series HPLC system equipped with a G1322A quaternary pump and degasser, a G1314A variable wavelength UV detector, and 250 × 4.0 mm (particle size 5 μm) ODS column (Lichrospher 100, Merck, Darmstadt, Germany). Chromatographic separation was achieved by applying a linear gradient system using mobile phase A (5% formic acid in water v/v) and mobile phase B (methanol). A linear gradient elution was performed using 50% mobile phase B for 10 min, rising to 80% B over 10 min, then to 100% B over 11 min, and back to 50% B. The flow rate was maintained at 1.0 mL/min and UV was monitored at 360 nm.


## 2.5. Experimental Animals

Adolescent male Wistar rats, aged 50–60 days, were obtained from the animal facility of King Fahd Research Center, King Abdulaziz University, Jeddah, Saudi Arabia. They were used in the study according to the guidelines of the Biochemical and Research Ethics Committee at King Abdulaziz University, in accordance with the NIH guidelines. Animals were housed in a well-ventilated, temperature-controlled room at 22± 3°C with a 12 h light-dark cycle. They were provided with standard rat chow pellets obtained from Grain Silos and Flour Mills organization F-1005, Jeddah, Saudi Arabia, and tap water ad libitum. All experimental procedures were performed between 8–10 a.m. and care was taken to avoid stressful conditions.Orchidectomy was performed aseptically, under ethyl ether anesthesia, by a midscrotal incision. Following ligation of the spermatic cord and vessels, testes and epididymis were removed. The remaining stump was pushed back through the inguinal canal into the abdominal cavity, and the scrotal sac was closed by sutures [23]. After castration, the rats were maintained under standard laboratory conditions for 7 days in order to allow a definite involution of the prostatic gland [24].APH was induced as previously described by Engelstein et al. [25] in castrated rats using citral and testosterone. Rats were subcutaneously injected with testosterone propionate in corn oil (0.5 mg/0.1 mL/rat) each day for 30 days. Citral, 1 M diluted in 70% ethanol, was smeared on a different shaved area of skin, each time, on the back at a final dose of 185 mg/kg every 4 days for 30 days. Control group rats were smeared with the solvent (ethanol) alone at the shaved skin. Considering the pungent lime fragrance of citral, the control groups were kept in a separate location from the citral-treated animals to avoid possible false results as citral has some influence via the olfactory tract.


## 2.6. Animal Treatment

Seven days after castration, the animals were randomly divided into six groups (n
=
7). Group I served as a control (shamed operation) group and received both corn oil (0.1 mL/rat) and 1% CMC-Na (0.3 mL/100 g body weight) daily during the period of the experiment. Groups from II–VI were castrated and had APH. Group II served as negative control and received CMC-Na 1% as previously mentioned. Group III served as positive control and received saw palmetto (100 mg/kg) suspended in 1% CMC-Na. Groups from IV–VI were treated with ROS at doses of 75, 150, or 300 mg/kg/day, respectively, by oral gavage. All the ROS extracts were suspended in 1% CMC-Na and were given to rats once daily by oral gavage along with testosterone injection and citral smearing for 30 days as described before [26]. After euthanization, ventral prostate of each rat in each group was removed from the body, 24 h after the last administration, and weighed. The half of the tissues were frozen in liquid nitrogen and stored at −75°C until use. The remaining tissues were fixed immediately in 0.1 M phosphate-buffered 10% formalin (pH 7.4) for 48 h and then embedded in paraffin and were used for histological studies. Serial 4 μm thick sections from each tissue specimen were prepared and mounted on poly-L-lysine coated glass slides. These were used for detection of IL-6, IL-8, TNF-α, IGF-1, clusterin, and TGF-β1 receptors in the prostatic tissue by immunohistochemistry.


## 2.7. Histopathology and Histoscore

A part of the ventral lobes was separated and fixed overnight in Stieve’s solution. Thereafter, the tissue was thoroughly rinsed with water and immersed overnight in ethanol 70%. Then, it was dehydrated, embedded in paraffin, and 5 mm thick sections were cut and stained by Harris’ hematoxylin eosin, according to the standard procedures of Lillie [27].A score-chart protocol (histoscore) developed by Scolnik et al. [26] was used to obtain an objective quantitative assessment. The examination, description, and scoring of the slides were performed in a blinded manner. The scoring system was presented in arbitrary units to make a better evaluation. In a second step, the cumulative score in each group was correlated to the final histological diagnosis in order to establish a score range for normal and hyperplasia. An additional histological inflammatory score described by de Nunzio et al. [28] was used to evaluate the inflammation. Score 0: no inflammation, score 1: scattered inflammatory cell infiltrate without nodules, score 2: no confluent lymphoid, and score 3: large inflammatory areas with confluence.


## 2.8. Immunohistochemistry and Immunhistoscoring

Hematoxylin and eosin staining was performed to observe histopathology. For analysis of IL-6 and TGF-β1 expression, sections from the paraffin embedded tissue blocks were mounted on charged glass slides and baked at 60°C for 1 h in the oven, then mounted on the Ventana staining machine, dewaxed by EZ Prep (Xylene substitute), and rehydrated. The tissue sections were heated in Ventana buffer CC1 (pH 6) to facilitate antigen retrieval and treated with H2O2 to eliminate endogenous peroxidase. This was followed by incubation for 60 min at room temperature with primary antibodies IL-6 and TGF-β1. The dilutions used were IL-6 (dilution 1 : 50) and TGF-β1 (dilution of 1 : 25). Subsequently, the sections were incubated with biotinylated secondary antibody using the avidin-biotin complex method. The immunoreaction was visualized using diaminobenzidine. All sections were lightly counterstained with hematoxylin as a background. The positive control used for IL-6 and TGF-β1 was from the colon. The negative controls comprised serial sections that were stained using equivalent concentrations of nonimmune mouse IgG in place of the primary antibodies. The level of staining was evaluated independently by three observers blinded to experimental conditions.Expression of TGF-β1 and IL-6 was evaluated according to a semiquantitative scale: 0, no detectable staining at all; 1, less than 10% of the cells stained positive; 2, 10−50% positive cells; and 3, more than 50% of cells positive [29]. Staining intensity was scored as 0 (no detectable stain), 1 (weak staining detected at intermediate to high power), 2 (moderate detected at low to intermediate power), to 3 (strong detected at low power) [30].


## 2.9. Reverse Transcription Polymerase Chain Reaction (RT-PCR)

Total RNA was extracted from the snap-frozen tissue samples using total RNA isolation kit (Macherey-Nagel) according to the manufacturer’s instructions. RT was performed in a 10μL reaction mixture. The RT reaction contained 1 μg RNA, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 2.5 mM dithiothreitol, 500 μmol/liter each of dATP, dCTP, dGTP, and dTTP (Bioline), 40 U RNasin (Bioline), 25 μg/mL oligo dT_pd(T)12–18 (Bioline), and 100UMoloney murine leukemia virus reverse transcriptase (Bioline). The reaction mixture was incubated at 42°C for 60 min and then heated to 80°C for 5 min. The resultant cDNA was used for PCR. For quantitative real-time RT-PCR, we prepared appropriate dilutions of each single-strand cDNA followed by normalizing of the cDNA content using β-actin as a quantitative control. Quantitative PCR amplification was performed with a 25 μL final volume consisting of 1 μL RT reaction mixture, 3 mM MgCl2, 10 pmol of each sense and antisense primer, and 12.5 μL (Roche Diagnostics). PCR conditions were as follows: initial denaturation at 95°C for 10 min and 35 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 1 min, and elongation at 72°C for 2 sec with a final elongation at 72°C for 10 min. Samples were migrated in 1% agarose gel using electrophoresis, UV visualized, and images were analyzed using total lab120 (Nonlinear Dynamic Ltd). Clusterin, TGF-β1, IGF-1, IL-6, IL-8, and TNF-α expression in the test samples were normalized to the corresponding β-actin level and were reported as the relative band intensity to the β-actin gene expression. Sequences of oligonucleotides used as primers for β-actin, IL-6, IL-8, TNFα, TGF-β1, IGF-1, and clusterin are summarized in Table 1.Table 1
Sequences of oligonucleotides used as primers.
Gene
Forward primer
Reverse primer
β-Actin
5′-GTCACCCACACTGTGCCCATCT-3′
5′-ACAGAGTACTTGCGCTCAGGAG-3′
IL-6
5′-GAACTCCTTCTCCACAAGCG-3′
5′-TTTTCTGCCAGTGCCTCTTT-3′
IL-8
5′-CTGCGCCAACACAGAAATTA-3′
5′-ATTGCATCTGGCAACCCTAC-3′
TNF-α
5′-CAGAGGGAAGAGTTCCCCAG-3′
5′-CCTTGGTCTGGTAGGAGACG-3′
TGF-β1
5′-GTTCTTCAATACGTCAGACATTCG-3′
5′-CATTATCTTTGCTGTCACAAGAGC-3′
IGF-1
5′-CACAGGCTATGGCTCCAGCAT-3′
5′-TCTCCAGCCTCCTCAGATCACA-3′
Clusterin
5′-CTGACCCAGCAGTACAACGA-3′
5′-TGACACGAGAGGGGACTTCT-3′


## 2.10. Statistical Analysis

Data were expressed as mean± SE and were analyzed by analysis of variance (ANOVA) followed by Tukey-Kramer multiple comparisons test. Inflammation scores and their significance were calculated by Chi-square test with Yate’s corrections. Differences were considered significant with a P value less than 0.05. Statistical analyses were performed using the SPSS for Windows (v. 10.0).


## 3. Results

### 3.1. Characterization of ROS Extract

Screening of the methanolic onion extract indicated mainly the presence of sterols and/or terpenoids, polyphenolic compounds such as flavonoids, tannins, and the absence of alkaloids and saponins. Assay of total phenolic content of methanolic extract was determined to be 12.9 mg GAE/g DE. Assay of total flavonoid content was determined to be 119 mg QE/g DE. The antioxidant is expressed as inhibition percentage corresponding to a reduction of the absorbance of DPPH of 50% (IC50). Ascorbic acid, a commonly used reference antioxidant, elicited 96.21% inhibition at 1 mM/mL. Onion extract was able to reduce the stable radical DPPH to the yellow colored diphenylpicrylhydrazine. Thus, it exhibited observable scavenging activity in a dose-related manner with IC50 values of 368 μg/mL. Figures 1 and 2 showed HPLC and TLC profile of ROS extract against Q and QG as marker compounds. The HPLC profile of ROS methanolic extract displayed two major peaks att
R 5.68 and 11.01 at 360 nm identified as quercetin and quercetin-4′-β-O-D-glucoside through spiking with standard quercetin and the isolated quercetin-4′-β-O-D-glucoside. The amount of quercetin in ROS extract was found to be 60.1 mg/gm of dry extract using standard quercetin (1.5 mg/mL).(a) HPLC profile of red onion scales extract (ROS) and (b) chromatogram of quercetin-4′-glucoside standard (UVmax 366 nm).
(a)
(b)Figure 2
TLC profile of red onion scales, 1: methanol extract; 2: quercetin-4′-glucoside, 3: rutin; 4: quercetin; UV 366 nm.


### 3.2. Changes in Body Weight (BW), Absolute Prostatic Weight (APW), and Relative Prostatic Weight (RPW)

In APH-induced rats, BW has increased significantly at the end of the experiment compared to the starting weight. Moreover, both APW and RPW were increased significantly compared to the normal control rats. All treatments with saw palmetto or ROS failed to improve the BW at the end of the experiment compared to the starting weight. However, saw palmetto induced small but insignificant reduction in APW and RPW compared to control rats. However, ROS also induced significant and dose-related reductions in both APW and RPW compared to control rats with APH (Table2).Table 2
Effect of red onion scales (RO scales) extract on absolute prostatic weight (APW) and relative prostatic weight (RPW) of rats with APH treated for 30 days at different doses.
Treatment
Body weight (g)
APW (g)
RPW (mg/g)
Start
End
Normal rats
199.14
±
5.16
229
±
8.81
0.106
±
0.01
0.452
±
0.05
Rats with APH (negative control)
187.6
±
8.65
241.4
b
±
12.55
0.756
*
±
0.05
3.136
*
±
0.19
Rats with APH treated with
Saw palmetto (100 mg/kg)
192.8
±
4.59
258.3
b
±
10.22
0.698
*
±
0.03
2.705
*
±
0.21
RO scales  (75 mg/kg)
227.5
±
7.44
271.1
b
±
7.69
0.681
*
±
0.02
2.512
*
a
±
0.12
RO scales  (150 mg/kg)
225.5
±
7.15
271.6
b
±
9.20
0.593
*
a
±
0.023
2.183
*
a
±
0.14
RO scales  (300 mg/kg)
196.2
±
7.39
262.7
b
±
16.22
0.547
*
a
±
0.075
2.082
*
a
±
0.21
*Significantly different from normal rats at P
<
0.05.
aSignificantly different from rats with APH at P
<
0.05.
bSignificantly different from the value before starting the experiment at P
<
0.05.


### 3.3. Effect of ROS on Proinflammatory Cytokines, IL-6, IL-8, and TNF1α

Induction of APH in rats significantly increased the tissue levels of IL-6, IL-8, and TNF-α. Saw palmetto treatment significantly decreased the IL-6 (20.3%), IL-8 (48.1%), and TNF-α (39.4%). ROS induced significant reductions in IL-6, IL-8, and TNF-α, which were greater than that produced by saw palmetto (Figure 3). These reductions were dose dependant regarding IL-6 (54.7%, 59%, and 65.6%), IL-8 (93%, 95.3%, and 97.9%), and TNF-α (50.8%, 65.1%, and 91.3%).Figure 3
The expression of IL-6, IL-8, and TNF-α target genes in ventral prostate of Wistar rats after treatment with RO scales at different dose levels. All values are expressed as mean ± S.E. of the relative band intensity (R.B.I.) using β-actin as a reference. *Significantly different from normal shame control rats at P
<
0.001. #Significantly different from APH control rats received CMC at P
<
0.001.


### 3.4. Effect of ROS on Gene Expression of TGF-βR1, IGF-1, and Clusterin

Induction of APH is accompanied with a significant increase of the clusterin and IGF-1 expression in the ventral lobe of rat prostate, while the TGF-βR1 expression was significantly decreased. Saw palmetto treated rats with APH showed significant increase in clusterin and TGF-βR1 expression, an effect which was accompanied with the decrease in IGF-1 expression. All the treatment groups with ROS extract showed a significant increase in clusterin, while the expression of IGF-1 was significantly decreased in a dose-dependent fashion. On the other hand, TGF-βR1 expression was not significantly changed compared to the APH group (Figure 4).Figure 4
The expression of TGF-βR1, IGF-1, and clusterin target genes in ventral prostate of Wistar rats after treatment with RO scales at different dose levels. All values are expressed as mean ± S.E. of the relative band intensity (R.B.I.) using β-actin as a reference. *Significantly different from normal shame control rats at P
<
0.001. #Significantly different from APH control rats receiving CMC at P
<
0.001.


### 3.5. Histological Changes

Ventral prostates from the normal group showed normal histology with average histoscore corresponding to24.3
±
1.2 (Figure 5(a)). Ventral prostates from the APH-induced rats showed increase in the number of acini (hyperplasia) with irregular distribution. The acini were arranged back to back with intraluminal and stromal papillary projections. All prostatic acini were lined by tall columnar cells with epithelial pilling and nuclear stratification seen among six of seven prostatic sections in the group. Prostatic sections from two rats in this group showed 1-2 mitotic figures. The interstitial stroma was scant and showed edema with congested blood vessels and mixed acute and chronic perivascular inflammatory cells. Polymorphonuclear leukocytes with eosinophilic granules were considered as acute inflammatory cells. Chronic inflammatory cells seen were lymphocytes which were distinguished by their darkly stained ink-dot nucleus and thin rim basophilic cytoplasm. These findings were associated with a significant increase in the histoscore corresponding to 41.1
±
1.8. (Figures 5(b)–5(d)).(a) Prostate of normal rats with round acini and intact basement membranes. Arrow points to acini lined by two layers of low cuboidal epithelium. (b) Prostate of testosterone and citral treated castrated rats with atypical prostatic hyperplasia (APH) reveals increase in the number of prostatic acini (hyperplasia) which are placed back to back with no intervening stroma. Thin arrow points to acini lined by tall columnar cells. Thick arrow points to nuclear stratification and stromal projections. (c) Interstitial stroma shows congested blood vessels and inflammatory cells. (d) Thick vertical down arrow points to polymorphonuclear acute inflammatory cells and the thin slanting arrows point to chronic inlammatory cells (lymphocytes). Very thin arrow points to luminal papillary projections, thick long arrow points to nuclear stratification in the acini, and small arrow points to polymorphonuclear acute inflammatory cells in between prostatic acini. (e) Prostate of saw palmeto treated rats showing hyperplastic changes. Thick arrow points to acini lined by tall columnar cells showing nuclear stratification. Thin arrow points to a small focus of polymorphonuclear acute inflammatory cells in stroma. Acinus at the base of the figure shows intraluminal projection (at 60X).
(a)
(b)
(c)
(d)
(e)The ventral prostate in the saw palmetto group showed diminution regarding the scale of pathological changes and histoscore corresponding to32.2
±
2.1 (Figure 5(e)). The prostatic acini were less crowded. However, hyperplastic changes such as acini lined by tall columnar cells with epithelial pilling and nuclear stratification were seen among all rats in the group. The nuclei were round to oval, regular, and basally placed. There was no loss of polarity noted in the epithelium. Prostatic sections from two rats in this group also showed 1-2 mitotic figures. The interstitial stroma was considerable, fibromuscular to edematous, and showed vascular congestion. Prostatic sections from one rat showed small foci of mixed inflammatory cells.Histological changes in the ventral lobe of APH-induced rats treated with ROS extract (75 mg/kg) showed irregular and enormous dilatation of prostatic acini with intraluminal secretions among five of six rats in the group. These changes were more pronounced in the central regions of the prostatic tissue. Dilated acini showed epithelial changes in the form of flattening or low cuboidal transformation (Figure6(a)). Few acini showed loss of lining epithelium although the basement membrane appeared still intact. Acini seen at the peripheral part of the section were still hyperplastic being lined by tall columnar cells showing nuclear stratification (Figure 6(b)). Mitotic figures were absent and the surrounding stroma showed edema and congested blood vessels. Mixed acute and chronic inflammatory cells were seen among four of six rats in the group along with clustering of stromal mast cells. Karyorrhectic debris was seen within the gland lumina among four of six rats in the group. These findings corresponded to a histoscore of 35.3
±
1.2.(a)-(b) Prostate of rats treated with RO scale extract in a dose of 75 mg/kg showing. (a) Irregularly dilated acini filled with secretions. Arrow points to thinning and flattening of lining epithelium. (b) Acinus at the periphery of the field is lined bytall columnar epithelial cells showing nuclear stratification. Arrow points to polymorphonuclear acute inflammatory cell infiltrate in stroma near the acini. (c)-(d) Prostate of rats treated with RO scale extract in a dose of 150 mg/kg showing. (c) Arrow pointing to reduced acinar distension which are lined by tall columnar cells exhibiting nuclear stratification. (d) Arrow pointing to luminal karyorrhectic debris. (e) Prostate of rats treated with RO scales in a dose of 300 mg/kg showing. Thin arrow points to luminal karyorrhectic debris and thick arrow points to hyperplasia as seen by tall columnar cells lining the acini with nuclear stratification (at 60X).
(a)
(b)
(c)
(d)
(e)Sections examined from ventral prostates of APH-induced rats treated with ROS extract (150 mg/kg) showed prostatic acini lined by tall columnar epithelium exhibiting nuclear stratification in all five rats. The acinar distension was slightly diminished. Few acini showed scalloped luminal secretions among three of five rats. Mitotic figures were absent and surrounding stroma showed edema and congested blood vessels. Small foci of mixed acute and chronic inflammatory cells were seen among three of five rats in the group. Intraluminal sloughing of karyorrhectic debris was also seen among three of five rats (Figures6(c) and 6(d)). Clustering of stromal mast cells was seen among three of five rats. These findings corresponded to a histoscore of 34.0
±
0.5.Histological changes in ventral prostates of rats, with APH treated with ROS extract (300 mg/kg) showed prostatic acini lined by tall columnar epithelium exhibiting nuclear stratification in all six rats. Acinar distension was slightly diminished. Scattered mixed acute and chronic inflammatory cells in the stroma along with intraluminal sloughing of karyorrhectic debris were seen in three of six rats (Figure6(e)). Mitotic figures were absent and surrounding stroma showed edema and congested blood vessels. Clustering of stromal mast cells was seen among two rats. These findings corresponded to a histoscore of 32.2
±
0.8.


### 3.6. Immunohistochemistry

Normal control rats showed no expression of both immunohistochemical markers with positive internal controls (Figures7(a) and 7(b)). TGFBR-1 expression corresponded to score 3 with strong intensity in the secretory epithelial cells of acini with foci of strong expression among basal cells also in the prostatic sections from APH-induced rats. IL-6 expression corresponded to score 1 and showed weak intensity in the secretory epithelial cells only. No expression was observed in basal or stromal cells (Figures 7(c) and 7(d)).Immunohistochemical expression of TGFβR-1 and IL-6 in prostates of normal and APH rats. (a) Normal rats TGFβR-1 expression score 0. (b) Normal rats IL-6 expression score 0. (c) APH rats TGFBR-I expression score 3. (d) APH rats IL-6 expression score 1 (at 60 X).
(a)
(b)
(c)
(d)Prostatic sections from saw palmetto treated APH-induced rats revealed TGFBR-1 expression corresponding to score 3 exhibiting strong intensity in the secretory epithelial cells of acini with foci of strong expression also in the basal cells. IL-6 was variable between score 2-3 with a staining intensity between moderate to strong. No expression was observed in the basal or stromal cells (Figures8(a) and 8(b)).(a) (b) Immunohistochemical expression of TGFβR-1 and IL-6 in saw palmetto 100 g treated rats. (a) TGFβR-1 score 3. (b) IL-6 expression score 2-3. (c) (d) Immunohistochemical expression of TGFBR-1 and IL-6 in RO scales 75 mg treated rats. (c) TGFβR-1 score 0. Note negativity with positive internal control within the stroma in the upper right of the field, (d) IL-6 expression score 2 (at 60X).
(a)
(b)
(c)
(d)Prostatic sections from RO scale revealed IL-6 expression corresponding to score 1 with weak intensity. TGFBR-1 expression was score 0. No expression was observed in the basal or stromal cells. The immunohistochemical profile expressed by the prostatic tissue showed no notable doze related variation (Figures8(c) and 8(d)).


## 3.1. Characterization of ROS Extract

Screening of the methanolic onion extract indicated mainly the presence of sterols and/or terpenoids, polyphenolic compounds such as flavonoids, tannins, and the absence of alkaloids and saponins. Assay of total phenolic content of methanolic extract was determined to be 12.9 mg GAE/g DE. Assay of total flavonoid content was determined to be 119 mg QE/g DE. The antioxidant is expressed as inhibition percentage corresponding to a reduction of the absorbance of DPPH of 50% (IC50). Ascorbic acid, a commonly used reference antioxidant, elicited 96.21% inhibition at 1 mM/mL. Onion extract was able to reduce the stable radical DPPH to the yellow colored diphenylpicrylhydrazine. Thus, it exhibited observable scavenging activity in a dose-related manner with IC50 values of 368 μg/mL. Figures 1 and 2 showed HPLC and TLC profile of ROS extract against Q and QG as marker compounds. The HPLC profile of ROS methanolic extract displayed two major peaks att
R 5.68 and 11.01 at 360 nm identified as quercetin and quercetin-4′-β-O-D-glucoside through spiking with standard quercetin and the isolated quercetin-4′-β-O-D-glucoside. The amount of quercetin in ROS extract was found to be 60.1 mg/gm of dry extract using standard quercetin (1.5 mg/mL).(a) HPLC profile of red onion scales extract (ROS) and (b) chromatogram of quercetin-4′-glucoside standard (UVmax 366 nm).
(a)
(b)Figure 2
TLC profile of red onion scales, 1: methanol extract; 2: quercetin-4′-glucoside, 3: rutin; 4: quercetin; UV 366 nm.


## 3.2. Changes in Body Weight (BW), Absolute Prostatic Weight (APW), and Relative Prostatic Weight (RPW)

In APH-induced rats, BW has increased significantly at the end of the experiment compared to the starting weight. Moreover, both APW and RPW were increased significantly compared to the normal control rats. All treatments with saw palmetto or ROS failed to improve the BW at the end of the experiment compared to the starting weight. However, saw palmetto induced small but insignificant reduction in APW and RPW compared to control rats. However, ROS also induced significant and dose-related reductions in both APW and RPW compared to control rats with APH (Table2).Table 2
Effect of red onion scales (RO scales) extract on absolute prostatic weight (APW) and relative prostatic weight (RPW) of rats with APH treated for 30 days at different doses.
Treatment
Body weight (g)
APW (g)
RPW (mg/g)
Start
End
Normal rats
199.14
±
5.16
229
±
8.81
0.106
±
0.01
0.452
±
0.05
Rats with APH (negative control)
187.6
±
8.65
241.4
b
±
12.55
0.756
*
±
0.05
3.136
*
±
0.19
Rats with APH treated with
Saw palmetto (100 mg/kg)
192.8
±
4.59
258.3
b
±
10.22
0.698
*
±
0.03
2.705
*
±
0.21
RO scales  (75 mg/kg)
227.5
±
7.44
271.1
b
±
7.69
0.681
*
±
0.02
2.512
*
a
±
0.12
RO scales  (150 mg/kg)
225.5
±
7.15
271.6
b
±
9.20
0.593
*
a
±
0.023
2.183
*
a
±
0.14
RO scales  (300 mg/kg)
196.2
±
7.39
262.7
b
±
16.22
0.547
*
a
±
0.075
2.082
*
a
±
0.21
*Significantly different from normal rats at P
<
0.05.
aSignificantly different from rats with APH at P
<
0.05.
bSignificantly different from the value before starting the experiment at P
<
0.05.


## 3.3. Effect of ROS on Proinflammatory Cytokines, IL-6, IL-8, and TNF1α

Induction of APH in rats significantly increased the tissue levels of IL-6, IL-8, and TNF-α. Saw palmetto treatment significantly decreased the IL-6 (20.3%), IL-8 (48.1%), and TNF-α (39.4%). ROS induced significant reductions in IL-6, IL-8, and TNF-α, which were greater than that produced by saw palmetto (Figure 3). These reductions were dose dependant regarding IL-6 (54.7%, 59%, and 65.6%), IL-8 (93%, 95.3%, and 97.9%), and TNF-α (50.8%, 65.1%, and 91.3%).Figure 3
The expression of IL-6, IL-8, and TNF-α target genes in ventral prostate of Wistar rats after treatment with RO scales at different dose levels. All values are expressed as mean ± S.E. of the relative band intensity (R.B.I.) using β-actin as a reference. *Significantly different from normal shame control rats at P
<
0.001. #Significantly different from APH control rats received CMC at P
<
0.001.


## 3.4. Effect of ROS on Gene Expression of TGF-βR1, IGF-1, and Clusterin

Induction of APH is accompanied with a significant increase of the clusterin and IGF-1 expression in the ventral lobe of rat prostate, while the TGF-βR1 expression was significantly decreased. Saw palmetto treated rats with APH showed significant increase in clusterin and TGF-βR1 expression, an effect which was accompanied with the decrease in IGF-1 expression. All the treatment groups with ROS extract showed a significant increase in clusterin, while the expression of IGF-1 was significantly decreased in a dose-dependent fashion. On the other hand, TGF-βR1 expression was not significantly changed compared to the APH group (Figure 4).Figure 4
The expression of TGF-βR1, IGF-1, and clusterin target genes in ventral prostate of Wistar rats after treatment with RO scales at different dose levels. All values are expressed as mean ± S.E. of the relative band intensity (R.B.I.) using β-actin as a reference. *Significantly different from normal shame control rats at P
<
0.001. #Significantly different from APH control rats receiving CMC at P
<
0.001.


## 3.5. Histological Changes

Ventral prostates from the normal group showed normal histology with average histoscore corresponding to24.3
±
1.2 (Figure 5(a)). Ventral prostates from the APH-induced rats showed increase in the number of acini (hyperplasia) with irregular distribution. The acini were arranged back to back with intraluminal and stromal papillary projections. All prostatic acini were lined by tall columnar cells with epithelial pilling and nuclear stratification seen among six of seven prostatic sections in the group. Prostatic sections from two rats in this group showed 1-2 mitotic figures. The interstitial stroma was scant and showed edema with congested blood vessels and mixed acute and chronic perivascular inflammatory cells. Polymorphonuclear leukocytes with eosinophilic granules were considered as acute inflammatory cells. Chronic inflammatory cells seen were lymphocytes which were distinguished by their darkly stained ink-dot nucleus and thin rim basophilic cytoplasm. These findings were associated with a significant increase in the histoscore corresponding to 41.1
±
1.8. (Figures 5(b)–5(d)).(a) Prostate of normal rats with round acini and intact basement membranes. Arrow points to acini lined by two layers of low cuboidal epithelium. (b) Prostate of testosterone and citral treated castrated rats with atypical prostatic hyperplasia (APH) reveals increase in the number of prostatic acini (hyperplasia) which are placed back to back with no intervening stroma. Thin arrow points to acini lined by tall columnar cells. Thick arrow points to nuclear stratification and stromal projections. (c) Interstitial stroma shows congested blood vessels and inflammatory cells. (d) Thick vertical down arrow points to polymorphonuclear acute inflammatory cells and the thin slanting arrows point to chronic inlammatory cells (lymphocytes). Very thin arrow points to luminal papillary projections, thick long arrow points to nuclear stratification in the acini, and small arrow points to polymorphonuclear acute inflammatory cells in between prostatic acini. (e) Prostate of saw palmeto treated rats showing hyperplastic changes. Thick arrow points to acini lined by tall columnar cells showing nuclear stratification. Thin arrow points to a small focus of polymorphonuclear acute inflammatory cells in stroma. Acinus at the base of the figure shows intraluminal projection (at 60X).
(a)
(b)
(c)
(d)
(e)The ventral prostate in the saw palmetto group showed diminution regarding the scale of pathological changes and histoscore corresponding to32.2
±
2.1 (Figure 5(e)). The prostatic acini were less crowded. However, hyperplastic changes such as acini lined by tall columnar cells with epithelial pilling and nuclear stratification were seen among all rats in the group. The nuclei were round to oval, regular, and basally placed. There was no loss of polarity noted in the epithelium. Prostatic sections from two rats in this group also showed 1-2 mitotic figures. The interstitial stroma was considerable, fibromuscular to edematous, and showed vascular congestion. Prostatic sections from one rat showed small foci of mixed inflammatory cells.Histological changes in the ventral lobe of APH-induced rats treated with ROS extract (75 mg/kg) showed irregular and enormous dilatation of prostatic acini with intraluminal secretions among five of six rats in the group. These changes were more pronounced in the central regions of the prostatic tissue. Dilated acini showed epithelial changes in the form of flattening or low cuboidal transformation (Figure6(a)). Few acini showed loss of lining epithelium although the basement membrane appeared still intact. Acini seen at the peripheral part of the section were still hyperplastic being lined by tall columnar cells showing nuclear stratification (Figure 6(b)). Mitotic figures were absent and the surrounding stroma showed edema and congested blood vessels. Mixed acute and chronic inflammatory cells were seen among four of six rats in the group along with clustering of stromal mast cells. Karyorrhectic debris was seen within the gland lumina among four of six rats in the group. These findings corresponded to a histoscore of 35.3
±
1.2.(a)-(b) Prostate of rats treated with RO scale extract in a dose of 75 mg/kg showing. (a) Irregularly dilated acini filled with secretions. Arrow points to thinning and flattening of lining epithelium. (b) Acinus at the periphery of the field is lined bytall columnar epithelial cells showing nuclear stratification. Arrow points to polymorphonuclear acute inflammatory cell infiltrate in stroma near the acini. (c)-(d) Prostate of rats treated with RO scale extract in a dose of 150 mg/kg showing. (c) Arrow pointing to reduced acinar distension which are lined by tall columnar cells exhibiting nuclear stratification. (d) Arrow pointing to luminal karyorrhectic debris. (e) Prostate of rats treated with RO scales in a dose of 300 mg/kg showing. Thin arrow points to luminal karyorrhectic debris and thick arrow points to hyperplasia as seen by tall columnar cells lining the acini with nuclear stratification (at 60X).
(a)
(b)
(c)
(d)
(e)Sections examined from ventral prostates of APH-induced rats treated with ROS extract (150 mg/kg) showed prostatic acini lined by tall columnar epithelium exhibiting nuclear stratification in all five rats. The acinar distension was slightly diminished. Few acini showed scalloped luminal secretions among three of five rats. Mitotic figures were absent and surrounding stroma showed edema and congested blood vessels. Small foci of mixed acute and chronic inflammatory cells were seen among three of five rats in the group. Intraluminal sloughing of karyorrhectic debris was also seen among three of five rats (Figures6(c) and 6(d)). Clustering of stromal mast cells was seen among three of five rats. These findings corresponded to a histoscore of 34.0
±
0.5.Histological changes in ventral prostates of rats, with APH treated with ROS extract (300 mg/kg) showed prostatic acini lined by tall columnar epithelium exhibiting nuclear stratification in all six rats. Acinar distension was slightly diminished. Scattered mixed acute and chronic inflammatory cells in the stroma along with intraluminal sloughing of karyorrhectic debris were seen in three of six rats (Figure6(e)). Mitotic figures were absent and surrounding stroma showed edema and congested blood vessels. Clustering of stromal mast cells was seen among two rats. These findings corresponded to a histoscore of 32.2
±
0.8.


## 3.6. Immunohistochemistry

Normal control rats showed no expression of both immunohistochemical markers with positive internal controls (Figures7(a) and 7(b)). TGFBR-1 expression corresponded to score 3 with strong intensity in the secretory epithelial cells of acini with foci of strong expression among basal cells also in the prostatic sections from APH-induced rats. IL-6 expression corresponded to score 1 and showed weak intensity in the secretory epithelial cells only. No expression was observed in basal or stromal cells (Figures 7(c) and 7(d)).Immunohistochemical expression of TGFβR-1 and IL-6 in prostates of normal and APH rats. (a) Normal rats TGFβR-1 expression score 0. (b) Normal rats IL-6 expression score 0. (c) APH rats TGFBR-I expression score 3. (d) APH rats IL-6 expression score 1 (at 60 X).
(a)
(b)
(c)
(d)Prostatic sections from saw palmetto treated APH-induced rats revealed TGFBR-1 expression corresponding to score 3 exhibiting strong intensity in the secretory epithelial cells of acini with foci of strong expression also in the basal cells. IL-6 was variable between score 2-3 with a staining intensity between moderate to strong. No expression was observed in the basal or stromal cells (Figures8(a) and 8(b)).(a) (b) Immunohistochemical expression of TGFβR-1 and IL-6 in saw palmetto 100 g treated rats. (a) TGFβR-1 score 3. (b) IL-6 expression score 2-3. (c) (d) Immunohistochemical expression of TGFBR-1 and IL-6 in RO scales 75 mg treated rats. (c) TGFβR-1 score 0. Note negativity with positive internal control within the stroma in the upper right of the field, (d) IL-6 expression score 2 (at 60X).
(a)
(b)
(c)
(d)Prostatic sections from RO scale revealed IL-6 expression corresponding to score 1 with weak intensity. TGFBR-1 expression was score 0. No expression was observed in the basal or stromal cells. The immunohistochemical profile expressed by the prostatic tissue showed no notable doze related variation (Figures8(c) and 8(d)).


## 4. Discussion

It was reported that APH usually arises with concomitant BPH and exhibits several cancer-like features, which makes APH an intermediate lesion between BPH and the subset of well-differentiated prostate cancers [31]. In the present study, APH-induced rats revealed prostatic enlargement which was evidenced by an increase in the APW and RPW of the ventral prostate lobe as well as acinar hyperplasia that may be attributed to the influence of androgen on prostate growth [32]. The development of hyperplasia was associated with enhanced proliferation and suppressed apoptosis of prostatic cells [25, 33]. Citral, used in the current study, was reported to have an estrogenic-like effect, as it binds to estrogen receptors that are located in prostatic epithelial cells of both human [34] and rat [35]. Type 2 estrogen-binding sites in rat ventral prostate could be responsible for this proliferative effect [35]. Moreover, the present study revealed an increase in the IGF-1 and a decrease in the TGF-β1 which may cause the prostate enlargement in APH group. TGF-β1 is known to suppress tissue proliferation and induce cell apoptosis [36]. Circulating testosterone acts locally in the prostate via the production of growth factors such as IGF and TGF families that act in a manner which influences prostate cell growth, survival, or apoptosis [37].Fruits and vegetables have health benefits and are good sources of antioxidants; therefore a lot of recent literature has focused on nutritional and herbal medicine for prostatic hyperplasia [38–40]. In the present study, administration of ROS extract induced significant and dose-related reduction in both the absolute and relative weight of prostate in the APH-induced rats, an effect that was greater than that induced by saw palmetto. These effects may be due to the fact that ROS are rich in flavonols which represent 60% of the total active constituents. These mainly are kaempferol, quercetin, quercetin dimer, quercetin trimer, and other quercetin glycoside. Flavonols were found to be responsible for the antioxidant, hepatoprotective, anticancer, antimicrobial, antistress, and other biological activities [12, 13]. Among the major constituents of the ROS extract, 16% are quercetin, which was found to be mainly responsible for the protective effects against different degenerative pathological diseases [11, 14]. It was reported that administration of quercetin along with finasteride resulted in reduction in prostate weight in rats, through a cell cycle-related pathway that may function independently of androgens [41].Moreover, the ROS extract has ameliorated the histopathological changes, significantly modulated the expression of the proinflammatory cytokines, and decreased the inflammatory scores in a dose-dependent manner. In the current study, ROS significantly decreased IL-6, IL-8, and TNF-α in the ventral lobe of prostatic tissues of rats with APH. These findings are in accordance with results that are reported by Jung et al. [42].In the context of chronic inflammation and expression of proinflammatory cytokines, IL-6 is one of the major physiologic mediators of acute phase reaction that influence immune responses and inflammatory reactions [43]. IL-6 is also secreted by both normal and prostatic epithelial cells and acts as a growth factor for normal prostatic epithelial cells [44]. TNF-α, a proinflammatory cytokine, may induce inflammation by induction of COX-2, superoxide radical, and hydrogen peroxide in both human and rat mesangial cells [45]. TGF-β, an inflammatory cytokine, has been shown to regulate stromal proliferation and differentiation in BPH, and it is a key factor in the androgen control of prostatic growth. Recently, Descazeaud et al. [46] investigated the TGF-β receptor II protein (TGFBRII) expression in BPH patients. They observed a significant association between TGFBRII stromal staining and prostatic volume.The ROS extract contains large amounts of antioxidant flavonoids, mainly, ferulic, gallic, kaempferol, and quercetin or their glycosides [12, 13]. Jung et al. [42] found that hepatic expressions of TNF-α and IL-6 in diabetic rats were suppressed by quercetin. These results are in agreement with previous reports, in which quercetin was found to have antioxidative and anti-inflammatory activities [47]. Quercetin significantly decreases prostatitis symptoms by decreasing prostatic inflammation [15]. Moreover, quercetin can affect the prostate cancer biology by inhibiting arachidonic acid metabolism through the blocking of phospholipase A2 and 5 as well as 12-lipooxygenase enzymes [48] and inhibiting androgen receptor mutations [49]. Quercetin has also demonstrated the ability to interrupt the spread of prostate cancer (metastases) and to promote cell death. It was able to decrease the activity of specific enzymes known to be involved in tumor invasion and metastases [50]. Furthermore, quercetin was found to have antiproliferative activity in vitro against several cancer cells [51] and arrested cell cycle progression either at the G1/S phase [50] or at the G2/M transitional boundary [52]. Quercetin is also a possible option to relieve symptoms for men who have prostate problems, and it has been identified as being beneficial in cases of prostatitis [53].The ROS extract significantly reduced the absolute and relative weight of ventral lobe prostate of rats with APH compared with the negative control rats. This effect may be partially due to that ROS extract may have antiproliferative and apoptotic action on the ventral lobe of prostatic tissues. The ROS extract significantly decreased the expression of IGF-1 and increased clusterin expression. The prostate enlargement and the increased net weight of the ventral lobe in case of APH may be partly due to the increased IGF-1 and decreased TGF-β1 expression. This modulation in expression of these cytokines may play a crucial role in prostate cell proliferation and apoptosis. These findings are in agreement with that of Wu et al. [54]. IGF-1, a mitogenic factor, interacts with IGF-1 receptor stimulating cell proliferation [55] and inducing proliferative prostatic diseases [56]. Moreover, Senthilkumar et al. [57] suggested that quercetin can decrease the survival of androgen-independent prostate cancer cells by changing the expression of IGF-1 signaling and inducing apoptosis in cancer patients.The antiproliferative effect of quercetin is probably mediated by interaction with the type II estrogen binding sites [58]. Quercetin also inhibits cell invasion and induces apoptosis through a pathway involving heat shock proteins [59]. The ability of RO extract to decrease the prostate weight in rats with APH may also be due to that it has some active constituents with a specific ability to inhibit phosphodiesterase subtype 5A (PDE-5A). These findings are in agreement with that which was reported by Lines and Ono [60] who found that the improvement in sexual function might be due to quercetin 1. Quercetin 1 was reported to have specific PDE-5A inhibitory activity [55]. Chronic administration of NO donor drugs and PDE5-inhibitors may also induce antiproliferative and/or apoptotic effects in the prostate [61].In conclusion, the results of the present study showed that the methanolic extract of ROS have the ability to decrease the prostate weight in APH-induced rats. This effect may be due to the anti-inflammatory and immunomodulatory effects of the extract. All of these effects are dose-dependent and may not only be related to the antioxidant activities of the flavonoids or their glycosides but also due to its phenolic content in ROS.



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