-
Research ArticleImmunomodulatory Effect of Red Onion(Allium cepa
Linn) Scale Extract on Experimentally InducedAtypical Prostatic
Hyperplasia in Wistar Rats
Ahmed A. Elberry,1,2 Shagufta Mufti,3 Jaudah Al-Maghrabi,3 Essam
Abdel Sattar,4
Salah A. Ghareib,5 Hisham A. Mosli,6 and Salah A. Gabr7
1 Department of Clinical Pharmacy, Faculty of Pharmacy, King
Abdulaziz University, Jeddah 21589, Saudi Arabia2Department of
Pharmacology, Faculty of Medicine, Beni Suef University, Beni Suef,
Egypt3 Department of Pathology, Faculty of Medicine, King Abdulaziz
University, Jeddah 21589, Saudi Arabia4Department of Pharmacognosy,
Faculty of Pharmacy, Cairo University, Cairo, Egypt5 Department of
Pharmacology and Toxicology, Faculty of Pharmacy, King Abdulaziz
University, Jeddah 21589, Saudi Arabia6Department of Urology,
Faculty of Medicine, King Abdulaziz University, Jeddah 21589, Saudi
Arabia7Department of Isotopes Applications, Nuclear Research
Center, Atomic Energy Authority, Cairo, Egypt
Correspondence should be addressed to Ahmed A. Elberry; berry
[email protected]
Received 19 November 2013; Revised 21 February 2014; Accepted 26
February 2014; Published 13 April 2014
Academic Editor: Yona Keisari
Copyright © 2014 Ahmed A. Elberry et al. This is an open access
article distributed under the Creative Commons AttributionLicense,
which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properlycited.
Red onion scales (ROS) contain large amounts of flavonoids that
are responsible for the reported antioxidant activity,
immuneenhancement, and anticancer property. Atypical prostatic
hyperplasia (APH) was induced in adult castrated Wistar rats by
boths.c. injection of testosterone (0.5mg/rat/day) and by smearing
citral on shaved skin once every 3 days for 30 days. Saw
palmetto(100mg/kg) as a positive control and ROS suspension at
doses of 75, 150, and 300mg/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
methanolicextract displayed two major peaks identified as quercetin
and quercetin-4-𝛽-O-D-glucoside. Histopathological examination
ofAPH-induced prostatic rats revealed evidence of hyperplasia and
inflammation with cellular proliferation and reduced
apoptosisImmunohistochemistry showed increased tissue expressions
of IL-6, IL-8, TNF-𝛼, IGF-1, and clusterin, while TGF-𝛽1
wasdecreased, which correlates with the presence of inflammation.
Both saw palmetto and RO scale treatment have ameliorated
thesechanges. These ameliorative effects were more evident in RO
scale groups and were dose dependent. In conclusion,
methanolicextract of ROS showed a protective effect against APH
induced rats that may be attributed to potential anti-inflammatory
andimmunomodulatory effects.
1. Introduction
Atypical prostatic hyperplasia (APH) is a pseudoneoplasticlesion
that can mimic prostate adenocarcinoma because ofits architectural
and cytological features. In this prostaticdisease, there is an
imbalance between prostate cell growthand apoptosis [1]. The
occurrence rate of APH is notknown and its associationswith benign
prostatic hypertrophy(BPH) and latent carcinoma of the prostate
(LPC) havenot been completely clarified [2]. The fact that APH
arisesalways in prostates with concomitant BPH and exhibits
several cancer-like features places APH as an intermediatelesion
between BPH and the subset of well-differentiatedcancers in a
hypothetical pathway between BPH and LPC[3].
The role of inflammation in prostate diseases is suggestedby the
presence of inflammatory cells within the BPH [4].Inflammation is
usually a self-limited event, with initialproinflammatory
cytokines, growth factor release, and angio-genesis followed by an
anti-inflammatory cytokine-mediatedresolution [5]. Chronic
inflammation produces free radicalsas various reactive oxygen
species [6].
Hindawi Publishing CorporationMediators of InflammationVolume
2014, Article ID 640746, 13
pageshttp://dx.doi.org/10.1155/2014/640746
-
2 Mediators of Inflammation
There is an interest in the potential health benefits ofAllium
vegetables, mainly onion (Allium cepa) and garlic(A. sativum).
Intake of Allium vegetables may be inverselyrelated to cancer at
various sites, including prostate [7, 8].Allium vegetables seem to
have antiproliferative action onhuman cancers and to prevent
diseases associated with aging[9]. Moreover, onion and garlic
intake has been inverselyassociated with BPH [10]. Onions contain
high levels ofnonnutrient antioxidant compounds, flavonoids, and
thealk(en)yl cysteine sulfoxides (ACSOs), which have
protectiveeffects against different degenerative pathologies such
ascardiovascular disease, cancer, and other dysfunctions basedon
oxidative stress [11].
The uses of bulb in food and manufacture introducetons of its
scales (red dried onion scales, ROS) as a wasteproduct. Recently,
these scales have attracted the atten-tion of many researchers to
maximize the benefits of thiswaste material. ROS contain large
amounts of flavonoids,mainly ferulic, gallic, kaempferol,
quercetin, quercetin dimer,quercetin trimer, and quercetin
glycosides. These flavonoidsare responsible for the reported
antioxidant activity of ROSwhich is comparable to that of
𝛼-tocopherol [12, 13]. Theyare also reported to have
hepatoprotective effect, immuneenhancement potential,
anti-infectious, antistress and anti-cancer properties [14].
Moreover, quercetin was found toameliorate significantly
prostatitis symptoms by decreasingprostatic inflammation [15]. Most
of the studies were doneusing an onion bulb per se, bulb extracts,
or bulb juicewithout studying the protective effect of RO scales,
the richestpart of the onion with quercetin. The aim of this
studywas to investigate the protective effect of the ROS extracton
enlarged rat prostate and the histopathological changesrelated to
inflammation, proliferation, and/or apoptosis inAPH induced
experimentally in rats.
2. Materials and Methods
2.1. Chemicals and Reagents. Solvents of HPLC grade usedfor HPLC
analysis, in addition to analytical grade solvents forextraction
and thin layer chromatography (TLC), were pur-chased
fromSigma-Aldrich (St. Louis,MO,USA).Antibodiesagainst clusterin,
phospho-Smad2, and𝛽-actinwere obtainedfrom Santa Cruz Biotechnology
(Santa Cruz, CA; anticlus-terin forWestern blot analysis); Upstate
Biotechnology (LakePlacid, NY; anticlusterin for
immunohistochemistry (IHC));and Cell Signaling Technology Inc.
(Danvers, MA). Antibod-ies against TGF-𝛽1 ligands were purchased
from Dakocy-tomation (Carpinteria, CA). Citral was obtained from
FlukaChemie AG, Buchs, Switzerland. Testosterone was obtainedfrom
Sigma-Aldrich andDakocytomation (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) werecollected from
a single field cultivated in Sohag city, Sohaggovernorate, Egypt.
It was cultivated in mid-November andcollected in the beginning of
May, 2010. The fully grown
bulbs were peeled and the ROS were stored at 25∘C in a darkplace
till extraction.The plant sample was identified by a staffmember of
the Horticulture Department, Faculty of Agricul-ture, Cairo
University. ROS powder (1250 g) was extractedwith 80% ethanol (3 ×
5 liters) by using Ultra-Turrax T50homogenizer (Janke andKunkel,
IKA-Labortechnik, Staufen,Germany) at a temperature not exceeding
25∘C. The extractwas concentrated by evaporation under reduced
pressure,lyophilized to give 240 g of reddish semisolid residue,
andprotected from light at 4∘C to use later.
2.3. Standardization of Red Onion Scale (ROS) Extract.
Pre-liminary phytochemical tests to identify the main
chemicalconstituents were carried out according to the methodsof
Trease and Evans [16]. Chromatographic TLC profileof the methanolic
extract was performed using thin layerchromatography (solvent
system : ethyl acetate-formic acid-glacial acetic acid-water, 100 :
11 : 11 : 26) as per fingerprintingusing a chromatographic
condition reported by Wagner andBladt [17] against common reference
flavonoids available inthe Pharmacognosy Department, Faculty of
Pharmacy, CairoUniversity. Total phenolics were determined as mg
gallic acidequivalent per gram dry extract (mg GAE/g DE)
employingthe Folin-Ciocalteumethod described by Singleton and
Rossi[18]. Aluminum chloride colorimetric assay described byHertog
[19] was applied for determination of total flavonoidscontent as mg
quercetin equivalent per gram of dry extract(mgQE/gDE)with a
slightmodification [20].The free radicalscavenging activity of the
extracts, based on the scavengingactivity of the stable
1,1-diphenyl-2-picrylhydrazyl (DPPH)free radical, was determined by
the method described byBraca et al. [21]. The capability of the
extracts to scavenge theDPPH radical was calculated using the
following equation:%inhibition = (𝐴Control − 𝐴Test) ×
100/𝐴Control.
2.4. HPLCAnalysis. Quercetin (Q) and quercetin-4-𝛽-O-D-glucoside
(QG) were used as standard marker compounds.QGwas separated from
theROS extract and identified using aprocedure reported by Abou Zid
and Elsherbeiny [22]. HPLCanalysis was performed on Agilent HP1200
series HPLC sys-tem equipped with a G1322A quaternary pump and
degasser,a G1314A variable wavelength UV detector, and 250 ×4.0mm
(particle size 5 𝜇m) ODS column (Lichrospher 100,Merck, Darmstadt,
Germany). Chromatographic separationwas achieved by applying a
linear gradient system usingmobile phase A (5% formic acid in water
v/v) and mobilephase B (methanol). A linear gradient elution was
performedusing 50% mobile phase B for 10min, rising to 80% B
over10min, then to 100% B over 11min, and back to 50% B.The flow
rate was maintained at 1.0mL/min and UV wasmonitored at 360 nm.
2.5. Experimental Animals. Adolescent male Wistar rats,aged
50–60 days, were obtained from the animal facilityof King Fahd
Research Center, King Abdulaziz University,Jeddah, Saudi Arabia.
They were used in the study accordingto the guidelines of the
Biochemical and Research EthicsCommittee at King Abdulaziz
University, in accordance with
-
Mediators of Inflammation 3
the NIH guidelines. Animals were housed in a well-ventilated,
temperature-controlled room at 22 ± 3∘C with a12 h light-dark
cycle. They were provided with standard ratchow pellets obtained
from Grain Silos and Flour Mills orga-nization F-1005, Jeddah,
Saudi Arabia, and tap water ad libi-tum. All experimental
procedures were performed between8–10 a.m. and care was taken to
avoid stressful conditions.
Orchidectomy was performed aseptically, under ethylether
anesthesia, by a midscrotal incision. Following ligationof the
spermatic cord and vessels, testes and epididymis wereremoved. The
remaining stump was pushed back throughthe inguinal canal into the
abdominal cavity, and the scrotalsac was closed by sutures [23].
After castration, the rats weremaintained under standard laboratory
conditions for 7 daysin order to allow a definite involution of the
prostatic gland[24].
APH was induced as previously described by Engelsteinet al. [25]
in castrated rats using citral and testosterone. Ratswere
subcutaneously injected with testosterone propionatein corn oil
(0.5mg/0.1mL/rat) each day for 30 days. Citral,1M diluted in 70%
ethanol, was smeared on a differentshaved area of skin, each time,
on the back at a final doseof 185mg/kg every 4 days for 30 days.
Control group ratswere smeared with the solvent (ethanol) alone at
the shavedskin. Considering the pungent lime fragrance of citral,
thecontrol groups were kept in a separate location from
thecitral-treated animals to avoid possible false results as
citralhas some influence via the olfactory tract.
2.6. Animal Treatment. Seven days after castration, the ani-mals
were randomly divided into six groups (𝑛 = 7). Group Iserved as a
control (shamed operation) group and receivedboth corn oil
(0.1mL/rat) and 1% CMC-Na (0.3mL/100 gbody weight) daily during the
period of the experiment.Groups from II–VI were castrated and had
APH. GroupII served as negative control and received CMC-Na 1%
aspreviously mentioned. Group III served as positive controland
received saw palmetto (100mg/kg) suspended in 1%CMC-Na.Groups from
IV–VIwere treatedwithROS at dosesof 75, 150, or 300mg/kg/day,
respectively, by oral gavage.All the ROS extracts were suspended in
1% CMC-Na andwere given to rats once daily by oral gavage along
withtestosterone injection and citral smearing for 30 days
asdescribed before [26]. After euthanization, ventral prostateof
each rat in each group was removed from the body, 24 hafter the
last administration, and weighed. The half of thetissues were
frozen in liquid nitrogen and stored at −75∘Cuntil use. The
remaining tissues were fixed immediately in0.1M phosphate-buffered
10% formalin (pH 7.4) for 48 h andthen embedded in paraffin and
were used for histologicalstudies. Serial 4 𝜇m thick sections from
each tissue specimenwere prepared and mounted on poly-L-lysine
coated glassslides. These were used for detection of IL-6, IL-8,
TNF-𝛼,IGF-1, clusterin, and TGF-𝛽1 receptors in the prostatic
tissueby immunohistochemistry.
2.7. Histopathology and Histoscore. A part of the ventrallobes
was separated and fixed overnight in Stieve’s solution.
Thereafter, the tissue was thoroughly rinsed with water
andimmersed overnight in ethanol 70%.Then, it was
dehydrated,embedded in paraffin, and 5mm thick sections were cutand
stained by Harris’ hematoxylin eosin, according to thestandard
procedures of Lillie [27].
A score-chart protocol (histoscore) developed by Scolniket al.
[26] was used to obtain an objective quantitativeassessment. The
examination, description, and scoring ofthe slides were performed
in a blinded manner. The scoringsystem was presented in arbitrary
units to make a betterevaluation. In a second step, the cumulative
score in eachgroup was correlated to the final histological
diagnosis inorder to establish a score range for normal and
hyperplasia.An additional histological inflammatory score described
byde Nunzio et al. [28] was used to evaluate the inflammation.Score
0: no inflammation, score 1: scattered inflammatory cellinfiltrate
without nodules, score 2: no confluent lymphoid,and score 3: large
inflammatory areas with confluence.
2.8. Immunohistochemistry and Immunhistoscoring. Hema-toxylin
and eosin staining was performed to observehistopathology. For
analysis of IL-6 and TGF-𝛽1 expression,sections from the paraffin
embedded tissue blocks weremounted on charged glass slides and
baked at 60∘C for 1 hin the oven, then mounted on the Ventana
staining machine,dewaxed by EZ Prep (Xylene substitute), and
rehydrated.Thetissue sections were heated in Ventana buffer CC1 (pH
6) tofacilitate antigen retrieval and treated with H
2O2to eliminate
endogenous peroxidase. This was followed by incubation for60min
at room temperature with primary antibodies IL-6and TGF-𝛽1. The
dilutions used were IL-6 (dilution 1 : 50)and TGF-𝛽1 (dilution of 1
: 25). Subsequently, the sectionswere incubated with biotinylated
secondary antibody usingthe avidin-biotin complex method. The
immunoreactionwas visualized using diaminobenzidine. All sections
werelightly counterstained with hematoxylin as a background.The
positive control used for IL-6 and TGF-𝛽1 was from thecolon. The
negative controls comprised serial sections thatwere stained using
equivalent concentrations of nonimmunemouse IgG in place of the
primary antibodies. The levelof staining was evaluated
independently by three observersblinded to experimental
conditions.
Expression of TGF-𝛽1 and IL-6 was evaluated accordingto a
semiquantitative scale: 0, no detectable staining at all; 1,less
than 10% of the cells stained positive; 2, 10−50% positivecells;
and 3, more than 50% of cells positive [29]. Stainingintensity was
scored as 0 (no detectable stain), 1 (weakstaining detected at
intermediate to high power), 2 (moderatedetected at low to
intermediate power), to 3 (strong detectedat low power) [30].
2.9. Reverse Transcription Polymerase Chain Reaction (RT-PCR).
Total RNA was extracted from the snap-frozen tissuesamples using
total RNA isolation kit (Macherey-Nagel)according to the
manufacturer’s instructions. RT was per-formed in a 10 𝜇L reaction
mixture. The RT reaction con-tained 1 𝜇g RNA, 10mM Tris-HCl (pH
8.3), 50mM KCl,1.5mM MgCl
2, 2.5mM dithiothreitol, 500𝜇mol/liter each
-
4 Mediators of Inflammation
Table 1: Sequences of oligonucleotides used as primers.
Gene Forward primer Reverse primer𝛽-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
of dATP, dCTP, dGTP, and dTTP (Bioline), 40U RNasin(Bioline), 25
𝜇g/mL oligo dT pd(T)12–18 (Bioline), and100UMoloney murine leukemia
virus reverse transcriptase(Bioline). The reaction mixture was
incubated at 42∘C for60min and then heated to 80∘C for 5min. The
resultantcDNA was used for PCR. For quantitative real-time RT-PCR,
we prepared appropriate dilutions of each single-strandcDNA
followed by normalizing of the cDNA content using𝛽-actin as a
quantitative control. Quantitative PCR amplifi-cation was performed
with a 25𝜇L final volume consistingof 1 𝜇L RT reaction mixture, 3mM
MgCl
2, 10 pmol of each
sense and antisense primer, and 12.5 𝜇L (Roche Diagnostics).PCR
conditions were as follows: initial denaturation at 95∘Cfor 10min
and 35 cycles of denaturation at 94∘C for 1min,annealing at 55∘C
for 1min, and elongation at 72∘C for2 sec with a final elongation
at 72∘C for 10min. Sampleswere migrated in 1% agarose gel using
electrophoresis, UVvisualized, and images were analyzed using total
lab120(NonlinearDynamic Ltd). Clusterin, TGF-𝛽1, IGF-1, IL-6, IL-8,
and TNF-𝛼 expression in the test samples were normalizedto the
corresponding 𝛽-actin level and were reported asthe relative band
intensity to the 𝛽-actin gene expression.Sequences of
oligonucleotides used as primers for𝛽-actin, IL-6, IL-8, TNF𝛼,
TGF-𝛽1, IGF-1, and clusterin are summarizedin Table 1.
2.10. Statistical Analysis. Data were expressed as mean ± SEand
were analyzed by analysis of variance (ANOVA) fol-lowed by
Tukey-Kramer multiple comparisons test. Inflam-mation scores and
their significance were calculated byChi-square test with Yate’s
corrections. Differences wereconsidered significant with a 𝑃 value
less than 0.05. Statis-tical analyses were performed using the SPSS
for Windows(v. 10.0).
3. Results
3.1. Characterization of ROS Extract. Screening of themethanolic
onion extract indicated mainly the presence ofsterols and/or
terpenoids, polyphenolic compounds suchas flavonoids, tannins, and
the absence of alkaloids andsaponins. Assay of total phenolic
content of methanolicextract was determined to be 12.9mg GAE/g DE.
Assay oftotal flavonoid content was determined to be 119mg QE/gDE.
The antioxidant is expressed as inhibition percentage
corresponding to a reduction of the absorbance of DPPHof 50%
(IC
50). Ascorbic acid, a commonly used reference
antioxidant, elicited 96.21% inhibition at 1mM/mL. Onionextract
was able to reduce the stable radical DPPH to theyellow colored
diphenylpicrylhydrazine. Thus, it exhibitedobservable scavenging
activity in a dose-related manner withIC50values of 368 𝜇g/mL.
Figures 1 and 2 showed HPLC and
TLC profile of ROS extract against Q and QG as markercompounds.
The HPLC profile of ROS methanolic extractdisplayed two major peaks
at tR 5.68 and 11.01 at 360 nmidentified as quercetin and
quercetin-4-𝛽-O-D-glucosidethrough spiking with standard quercetin
and the isolatedquercetin-4-𝛽-O-D-glucoside. The amount of
quercetin inROS extract was found to be 60.1mg/gm of dry extract
usingstandard quercetin (1.5mg/mL).
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 ofthe experiment
compared to the starting weight. Moreover,both APW and RPW were
increased significantly comparedto the normal control rats. All
treatments with saw palmettoor ROS failed to improve the BW at the
end of the experimentcompared to the starting weight. However, saw
palmettoinduced small but insignificant reduction in APW andRPW
compared to control rats. However, ROS also inducedsignificant and
dose-related reductions in both APW andRPW compared to control rats
with APH (Table 2).
3.3. Effect of ROS on Proinflammatory Cytokines, IL-6, IL-8,and
TNF-𝛼. Induction of APH in rats significantly increasedthe tissue
levels of IL-6, IL-8, and TNF-𝛼. Saw palmetto treat-ment
significantly decreased the IL-6 (20.3%), IL-8 (48.1%),and TNF-𝛼
(39.4%). ROS induced significant reductionsin IL-6, IL-8, and
TNF-𝛼, which were greater than thatproduced by saw palmetto (Figure
3). These reductions weredose 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%,
and91.3%).
3.4. Effect of ROS on Gene Expression of TGF-𝛽R1, IGF-1,and
Clusterin. Induction of APH is accompanied with asignificant
increase of the clusterin and IGF-1 expression inthe ventral lobe
of rat prostate, while the TGF-𝛽R1 expressionwas significantly
decreased. Saw palmetto treated rats with
-
Mediators of Inflammation 5
(min)
(mAU
)
400
300
200
100
0
0 5 10 15 20
2.129
2.990
11.011
16.116
18.209
19.131
VWD1 A, wavelength = 360nm (ESSAMROSD)
5.682
(a)
0 2 4 6 8 10 12 14
(min)
200
150
100
50
0
(mAU
)
VWD1 A, wavelength = 360nm (ESSAM Q4 GLCO)
5.115
(b)
Figure 1: (a) HPLC profile of red onion scales extract (ROS) and
(b) chromatogram of quercetin-4-glucoside standard (UVmax 366
nm).
Table 2: Effect of red onion scales (RO scales) extract on
absolute prostatic weight (APW) and relative prostatic weight (RPW)
of rats withAPH 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 ±
0.05Rats with APH (negative control) 187.6 ± 8.65 241.4b ± 12.55
0.756∗ ± 0.05 3.136∗ ± 0.19Rats with APH treated with
Saw palmetto (100mg/kg) 192.8 ± 4.59 258.3b ± 10.22 0.698∗ ±
0.03 2.705∗ ± 0.21RO scales (75mg/kg) 227.5 ± 7.44 271.1b ± 7.69
0.681∗ ± 0.02 2.512∗a ± 0.12RO scales (150mg/kg) 225.5 ± 7.15
271.6b ± 9.20 0.593∗a ± 0.023 2.183∗a ± 0.14RO scales (300mg/kg)
196.2 ± 7.39 262.7b ± 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.
Figure 2: TLC profile of red onion scales, 1: methanol extract;
2:quercetin-4-glucoside, 3: rutin; 4: quercetin; UV 366 nm.
APH showed significant increase in clusterin and TGF-𝛽R1
expression, an effect which was accompanied with thedecrease in
IGF-1 expression. All the treatment groups withROS 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 expressionwas
not significantly changed compared to the APH group(Figure 4).
3.5. Histological Changes. Ventral prostates from the
normalgroup showed normal histology with average
histoscorecorresponding to 24.3 ± 1.2 (Figure 5(a)). Ventral
prostatesfrom the APH-induced rats showed increase in the num-ber
of acini (hyperplasia) with irregular distribution. Theacini were
arranged back to back with intraluminal andstromal papillary
projections. All prostatic acini were linedby tall columnar cells
with epithelial pilling and nuclearstratification seen among six of
seven prostatic sections inthe group. Prostatic sections from two
rats in this groupshowed 1-2 mitotic figures. The interstitial
stroma was scantand showed edema with congested blood vessels and
mixedacute and chronic perivascular inflammatory cells.
Polymor-phonuclear leukocytes with eosinophilic granules were
con-sidered as acute inflammatory cells. Chronic inflammatorycells
seen were lymphocytes which were distinguished bytheir darkly
stained ink-dot nucleus and thin rim basophiliccytoplasm. These
findings were associated with a significant
-
6 Mediators of Inflammation
0.0
0.1
0.2
0.3
0.4
*
0.00
0.02
0.04
0.06
0.08
0.10IL
-6/𝛽
-act
in (R
.B.I)
IL-8
/𝛽-a
ctin
(R.B
.I)
∗
#
##
#
#
# # #
∗
0.0
0.1
0.2
0.3
0.4
NormalAPHSaw palmetto
RO scales 75mg/kgRO scales 150mg/kgRO scales 300mg/kg
TNF-𝛼
/𝛽-a
ctin
(R.B
.I)
∗
##
#
#
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 differentdose levels. All values are expressed as mean ± S.E. of
the relative band intensity (R.B.I.) using 𝛽-actin as a reference.
∗Significantly differentfrom normal shame control rats at 𝑃 <
0.001. #Significantly different from APH control rats received CMC
at 𝑃 < 0.001.
increase in the histoscore corresponding to 41.1 ± 1.8.(Figures
5(b)–5(d)).
The ventral prostate in the saw palmetto group showeddiminution
regarding the scale of pathological changes andhistoscore
corresponding to 32.2 ± 2.1 (Figure 5(e)). Theprostatic acini were
less crowded. However, hyperplasticchanges such as acini lined by
tall columnar cells withepithelial pilling and nuclear
stratification were seen amongall rats in the group. The nuclei
were round to oval, regular,and basally placed. There was no loss
of polarity notedin the epithelium. Prostatic sections from two
rats in thisgroup also showed 1-2 mitotic figures. The interstitial
stromawas considerable, fibromuscular to edematous, and
showedvascular congestion. Prostatic sections from one rat
showedsmall foci of mixed inflammatory cells.
Histological changes in the ventral lobe of APH-inducedrats
treated with ROS extract (75mg/kg) showed irregularand enormous
dilatation of prostatic acini with intraluminalsecretions among
five of six rats in the group. These changeswere more pronounced in
the central regions of the prostatictissue. Dilated acini showed
epithelial changes in the form offlattening or low cuboidal
transformation (Figure 6(a)). Fewacini showed loss of lining
epithelium although the basementmembrane appeared still intact.
Acini seen at the peripheral
part of the section were still hyperplastic being lined by
tallcolumnar cells showing nuclear stratification (Figure
6(b)).Mitotic figures were absent and the surrounding stromashowed
edema and congested blood vessels. Mixed acute andchronic
inflammatory cells were seen among four of six ratsin the group
alongwith clustering of stromalmast cells. Kary-orrhectic debris
was seen within the gland lumina amongfour of six rats in the
group. These findings corresponded toa histoscore of 35.3 ±
1.2.
Sections examined from ventral prostates of APH-induced rats
treated with ROS extract (150mg/kg) showedprostatic acini lined by
tall columnar epithelium exhibitingnuclear stratification in all
five rats. The acinar distensionwas slightly diminished. Few acini
showed scalloped luminalsecretions among three of five rats.
Mitotic figures wereabsent and surrounding stroma showed edema and
congestedblood vessels. Small foci of mixed acute and chronic
inflam-matory cells were seen among three of five rats in the
group.Intraluminal sloughing of karyorrhectic debris was also
seenamong three of five rats (Figures 6(c) and 6(d)). Clusteringof
stromal mast cells was seen among three of five rats. Thesefindings
corresponded to a histoscore of 34.0 ± 0.5.
Histological changes in ventral prostates of rats, withAPH
treated with ROS extract (300mg/kg) showed prostatic
-
Mediators of Inflammation 7
0.0
0.2
0.4
0.6
0.8
0.0
0.5
1.0
1.5
∗
#∗
##
#
#
TGF-𝛽
R1/𝛽
-act
in (R
.B.I)
IGF-1
/𝛽-a
ctin
(R.B
.I)
0.0
0.5
1.0
1.5
∗
#
#
##
Clus
terin
/𝛽-a
ctin
(R.B
.I)
NormalAPHSaw palmetto
RO scales 75mg/kgRO scales 150mg/kgRO scales 300mg/kg
Figure 4: The expression of TGF-𝛽R1, IGF-1, and clusterin target
genes in ventral prostate of Wistar rats after treatment with RO
scales atdifferent dose levels. All values are expressed as mean ±
S.E. of the relative band intensity (R.B.I.) using 𝛽-actin as a
reference. ∗Significantlydifferent from normal shame control rats
at 𝑃 < 0.001. #Significantly different from APH control rats
receiving CMC at 𝑃 < 0.001.
acini lined by tall columnar epithelium exhibiting
nuclearstratification in all six rats. Acinar distension was
slightlydiminished. Scatteredmixed acute and chronic
inflammatorycells in the stroma along with intraluminal sloughing
of kary-orrhectic debris were seen in three of six rats (Figure
6(e)).Mitotic figures were absent and surrounding stroma
showededema and congested blood vessels. Clustering of stromalmast
cells was seen among two rats. These findings corre-sponded to a
histoscore of 32.2 ± 0.8.
3.6. Immunohistochemistry. Normal control rats showed
noexpression of both immunohistochemical markers with pos-itive
internal controls (Figures 7(a) and 7(b)). TGFBR-1expression
corresponded to score 3 with strong intensityin the secretory
epithelial cells of acini with foci of strongexpression among basal
cells also in the prostatic sectionsfrom APH-induced rats. IL-6
expression corresponded toscore 1 and showed weak intensity in the
secretory epithelialcells only. No expression was observed in basal
or stromalcells (Figures 7(c) and 7(d)).
Prostatic sections from saw palmetto treated APH-induced rats
revealed TGFBR-1 expression corresponding toscore 3 exhibiting
strong intensity in the secretory epithelialcells of acini with
foci of strong expression also in the basalcells. IL-6 was variable
between score 2-3 with a staining
intensity between moderate to strong. No expression wasobserved
in the basal or stromal cells (Figures 8(a) and 8(b)).
Prostatic sections fromRO scale revealed IL-6
expressioncorresponding to score 1 with weak intensity.
TGFBR-1expression was score 0. No expression was observed in
thebasal or stromal cells. The immunohistochemical profileexpressed
by the prostatic tissue showed no notable dozerelated variation
(Figures 8(c) and 8(d)).
4. Discussion
It was reported that APH usually arises with concomitantBPH and
exhibits several cancer-like features, which makesAPH an
intermediate lesion between BPH and the subset
ofwell-differentiated prostate cancers [31]. In the present
study,APH-induced rats revealed prostatic enlargement which
wasevidenced by an increase in the APW and RPW of theventral
prostate lobe as well as acinar hyperplasia that maybe attributed
to the influence of androgen on prostate growth[32]. The
development of hyperplasia was associated withenhanced
proliferation and suppressed apoptosis of prostaticcells [25, 33].
Citral, used in the current study, was reported tohave an
estrogenic-like effect, as it binds to estrogen receptorsthat are
located in prostatic epithelial cells of both human[34] and rat
[35]. Type 2 estrogen-binding sites in rat ventral
-
8 Mediators of Inflammation
(a) (b)
(c) (d)
(e)
Figure 5: (a) Prostate of normal rats with round acini and
intact basement membranes. Arrow points to acini lined by two
layers of lowcuboidal epithelium. (b) Prostate of testosterone and
citral treated castrated rats with atypical prostatic hyperplasia
(APH) reveals increasein the number of prostatic acini
(hyperplasia) which are placed back to back with no intervening
stroma. Thin arrow points to acini linedby tall columnar cells.
Thick arrow points to nuclear stratification and stromal
projections. (c) Interstitial stroma shows congested bloodvessels
and inflammatory cells. (d) Thick vertical down arrow points to
polymorphonuclear acute inflammatory cells and the thin
slantingarrows point to chronic inlammatory cells (lymphocytes).
Very thin arrow points to luminal papillary projections, thick long
arrow pointsto 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 nuclearstratification. Thin arrow points to
a small focus of polymorphonuclear acute inflammatory cells in
stroma. Acinus at the base of the figureshows intraluminal
projection (at 60X).
prostate could be responsible for this proliferative effect
[35].Moreover, the present study revealed an increase in the
IGF-1and a decrease in the TGF-𝛽1 which may cause the
prostateenlargement in APH group. TGF-𝛽1 is known to suppresstissue
proliferation and induce cell apoptosis [36].
Circulatingtestosterone acts locally in the prostate via the
productionof growth factors such as IGF and TGF families that act
in
a manner which influences prostate cell growth, survival,
orapoptosis [37].
Fruits and vegetables have health benefits and are goodsources
of antioxidants; therefore a lot of recent literaturehas focused on
nutritional and herbal medicine for prostatichyperplasia [38–40].
In the present study, administration ofROS extract induced
significant and dose-related reduction
-
Mediators of Inflammation 9
(a) (b)
(c) (d)
(e)
Figure 6: (a)-(b) Prostate of rats treatedwith RO scale extract
in a dose of 75mg/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 cellsshowing nuclear stratification.
Arrow points to polymorphonuclear acute inflammatory cell
infiltrate in stroma near the acini. (c)-(d) Prostateof rats
treated with RO scale extract in a dose of 150mg/kg showing. (c)
Arrow pointing to reduced acinar distension which are lined by
tallcolumnar cells exhibiting nuclear stratification. (d) Arrow
pointing to luminal karyorrhectic debris. (e) Prostate of rats
treated with RO scalesin a dose of 300mg/kg showing. Thin arrow
points to luminal karyorrhectic debris and thick arrow points to
hyperplasia as seen by tallcolumnar cells lining the acini with
nuclear stratification (at 60X).
in both the absolute and relative weight of prostate inthe
APH-induced rats, an effect that was greater than thatinduced by
saw palmetto. These effects may be due to thefact that ROS are rich
in flavonols which represent 60% ofthe total active constituents.
These mainly are kaempferol,quercetin, quercetin dimer, quercetin
trimer, and otherquercetin glycoside. Flavonols were found to be
responsiblefor the antioxidant, hepatoprotective, anticancer,
antimicro-bial, antistress, and other biological activities [12,
13]. Amongthe major constituents of the ROS extract, 16% are
quercetin,
which was found to be mainly responsible for the
protectiveeffects against different degenerative pathological
diseases[11, 14]. It was reported that administration of quercetin
alongwith finasteride resulted in reduction in prostate weight
inrats, through a cell cycle-related pathway that may
functionindependently of androgens [41].
Moreover, the ROS extract has ameliorated the his-topathological
changes, significantly modulated the expres-sion of the
proinflammatory cytokines, and decreased the in-flammatory scores
in a dosedependentmanner. In the current
-
10 Mediators of Inflammation
(a) (b)
(c) (d)
Figure 7: Immunohistochemical expression of TGF𝛽R-1 and IL-6 in
prostates of normal and APH rats. (a) Normal rats TGF𝛽R-1
expressionscore 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 60X).
(a) (b)
(c) (d)
Figure 8: (a)-(b) Immunohistochemical expression of TGF𝛽R-1 and
IL-6 in saw palmetto 100 g treated rats. (a) TGF𝛽R-1 score 3. (b)
IL-6expression score 2-3. (c)-(d) Immunohistochemical expression of
TGFBR-1 and IL-6 in RO scales 75mg 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).
-
Mediators of Inflammation 11
study, ROS significantly decreased IL-6, IL-8, and TNF-𝛼 inthe
ventral lobe of prostatic tissues of rats with APH. Thesefindings
are in accordance with results that are reported byJung et al.
[42].
In the context of chronic inflammation and expressionof
proinflammatory cytokines, IL-6 is one of the majorphysiologic
mediators of acute phase reaction that influenceimmune responses
and inflammatory reactions [43]. IL-6 isalso secreted by both
normal and prostatic epithelial cellsand acts as a growth factor
for normal prostatic epithelialcells [44]. TNF-𝛼, a proinflammatory
cytokine, may induceinflammation by induction of COX-2, superoxide
radical,and hydrogen peroxide in both human and rat mesangialcells
[45]. TGF-𝛽, an inflammatory cytokine, has been shownto regulate
stromal proliferation and differentiation in BPH,and it is a key
factor in the androgen control of prostaticgrowth. Recently,
Descazeaud et al. [46] investigated theTGF-𝛽 receptor II protein
(TGFBRII) expression in BPHpatients. They observed a significant
association betweenTGFBRII stromal staining and prostatic
volume.
The ROS extract contains large amounts of antioxidantflavonoids,
mainly, ferulic, gallic, kaempferol, and quercetinor their
glycosides [12, 13]. Jung et al. [42] found thathepatic expressions
of TNF-𝛼 and IL-6 in diabetic rats weresuppressed by quercetin.
These results are in agreementwith previous reports, in which
quercetin was found tohave antioxidative and anti-inflammatory
activities [47].Quercetin significantly decreases prostatitis
symptoms bydecreasing prostatic inflammation [15]. Moreover,
quercetincan affect the prostate cancer biology by inhibiting
arachi-donic acid metabolism through the blocking of phospho-lipase
A2 and 5 as well as 12-lipooxygenase enzymes [48]and inhibiting
androgen receptor mutations [49]. Quercetinhas also demonstrated
the ability to interrupt the spreadof prostate cancer (metastases)
and to promote cell death.It was able to decrease the activity of
specific enzymesknown to be involved in tumor invasion and
metastases [50].Furthermore, quercetin was found to have
antiproliferativeactivity in vitro against several cancer cells
[51] and arrestedcell cycle progression either at the G1/S phase
[50] or at theG2/M transitional boundary [52]. Quercetin is also a
possibleoption to relieve symptoms for men who have
prostateproblems, and it has been identified as being beneficial
incases of prostatitis [53].
The ROS extract significantly reduced the absolute andrelative
weight of ventral lobe prostate of rats with APH com-pared with the
negative control rats. This effect may be par-tially due to that
ROS extract may have antiproliferative andapoptotic action on the
ventral lobe of prostatic tissues. TheROS extract significantly
decreased the expression of IGF-1and increased clusterin
expression.The prostate enlargementand the increased net weight of
the ventral lobe in case ofAPHmay be partly due to the increased
IGF-1 and decreasedTGF-𝛽1 expression. This modulation in expression
of thesecytokines may play a crucial role in prostate cell
proliferationand apoptosis. These findings are in agreement with
that ofWu et al. [54]. IGF-1, a mitogenic factor, interacts with
IGF-1 receptor stimulating cell proliferation [55] and inducing
proliferative prostatic diseases [56]. Moreover, Senthilkumaret
al. [57] suggested that quercetin can decrease the survivalof
androgen-independent prostate cancer cells by changingthe
expression of IGF-1 signaling and inducing apoptosis incancer
patients.
The antiproliferative effect of quercetin is probably medi-ated
by interactionwith the type II estrogen binding sites
[58].Quercetin also inhibits cell invasion and induces
apoptosisthrough a pathway involving heat shock proteins [59].
Theability of RO extract to decrease the prostate weight in
ratswith APH may also be due to that it has some active
con-stituents with a specific ability to inhibit
phosphodiesterasesubtype 5A (PDE-5A). These findings are in
agreement withthat which was reported by Lines and Ono [60] who
foundthat the improvement in sexual function might be due
toquercetin 1. Quercetin 1 was reported to have specific
PDE-5Ainhibitory activity [55]. Chronic administration of NO
donordrugs and PDE5-inhibitors may also induce
antiproliferativeand/or apoptotic effects in the prostate [61].
In conclusion, the results of the present study showed thatthe
methanolic extract of ROS have the ability to decreasethe prostate
weight in APH-induced rats. This effect may bedue to the
anti-inflammatory and immunomodulatory effectsof the extract. All
of these effects are dose-dependent andmay not only be related to
the antioxidant activities of theflavonoids or their glycosides but
also due to its phenoliccontent in ROS.
Conflict of Interests
The authors declare that they have no conflict of interests.
Acknowledgments
This work was supported by Sheikh Ahmed H. Fetaihi,Chair for
Research on Prostatic Diseases, King AbdulazizUniversity, Jeddah,
Saudi Arabia.The authors would also liketo thankMr. IslamFarouk,
Department of Pharmacology andToxicology, Faculty of Pharmacy, King
Abdulaziz University,Jeddah, Saudi Arabia, for his great effort and
help in theexperimental study.
References
[1] A. Sciarra, G. Mariotti, S. Salciccia et al., “Prostate
growth andinflammation,” Journal of Steroid Biochemistry and
MolecularBiology, vol. 108, no. 3–5, pp. 254–260, 2008.
[2] K. Stamatiou, A. Alevizos,M. Natzar et al., “Associations
amongbenign prostate hypertrophy, atypical adenomatous
hyperplasiaand latent carcinoma of the prostate,” Asian Journal of
Androl-ogy, vol. 9, no. 2, pp. 229–233, 2007.
[3] D. G. Bostwick, J. Srigley, D. Grignon et al., “Atypical
ade-nomatous hyperplasia of the prostate: morphologic criteriafor
its distinction from well-differentiated carcinoma,”
HumanPathology, vol. 24, no. 8, pp. 818–832, 1993.
[4] G. Novara, A. Galfano, R. B. Berto, V. Ficarra, R. V.
Navarrete,and W. Artibani, “Inflammation, apoptosis, and BPH: what
isthe evidence?” European Urology, vol. 5, no. 4, pp.
401–409,2006.
-
12 Mediators of Inflammation
[5] J. S. Sandhu, “Prostate cancer and chronic prostatitis,”
CurrentUrology Reports, vol. 9, no. 4, pp. 328–332, 2008.
[6] B. Rigas and Y. Sun, “Induction of oxidative stress as
amechanismof action of chemopreventive agents against
cancer,”British Journal of Cancer, vol. 98, no. 7, pp. 1157–1160,
2008.
[7] K. Rahman andG.M. Lowe, “Garlic and cardiovascular disease:a
critical review,” Journal of Nutrition, vol. 136, 3, pp.
736S–740S,2006.
[8] C. Galeone, C. Pelucchi, F. Levi et al., “Onion and garlic
use andhuman cancer,” American Journal of Clinical Nutrition, vol.
84,no. 5, pp. 1027–1032, 2006.
[9] A. de Martino, G. Filomeni, K. Aquilano, M. R. Ciriolo, and
G.Rotilio, “Effects of water garlic extracts on cell cycle and
viabilityof HepG2 hepatoma cells,” Journal of Nutritional
Biochemistry,vol. 17, no. 11, pp. 742–749, 2006.
[10] F. Bravi, C. Bosetti, L. dal Maso et al., “Food groups and
risk ofbenign prostatic hyperplasia,” Urology, vol. 67, no. 1, pp.
73–79,2006.
[11] G. Griffiths, L. Trueman, T. Crowther, B.Thomas, and B.
Smith,“Onions—aglobal benefit to health,”PhytotherapyResearch,
vol.16, no. 7, pp. 603–615, 2002.
[12] N. L. Tram, C. Hazama, M. Shimoyamada, H. Ando, K. Kato,and
R. Yamauchi, “Antioxidative compounds from the outerscales of
onion,” Journal of Agricultural and Food Chemistry, vol.53, no. 21,
pp. 8183–8189, 2005.
[13] A. Gülşen, D. P. Makris, and P. Kefalas, “Biomimetic
oxidationof quercetin: isolation of a naturally occurring quercetin
het-erodimer and evaluation of its in vitro antioxidant
properties,”Food Research International, vol. 40, no. 1, pp. 7–14,
2007.
[14] B. N. Singh, B. R. Singh, R. L. Singh et al.,
“Polyphenolicsfrom various extracts/fractions of red onion (Allium
cepa) peelwith potent antioxidant and antimutagenic activities,”
Food andChemical Toxicology, vol. 47, no. 6, pp. 1161–1167,
2009.
[15] D. A. Shoskes, S. I. Zeitlin, A. Shahed, and J. Rajfer,
“Quercetinin men with category III chronic prostatitis: a
preliminaryprospective, double-blind, placebo-controlled trial,”
Urology,vol. 54, no. 6, pp. 960–963, 1999.
[16] G. E. Trease and W. C. Evans, Pharmacognosy, Bailliere
TindallPress, London, UK, 1983.
[17] H. Wagner and S. Bladt, Plant Drug Analysis: A Thin
LayerChromatography Atlas, Springer, London, UK, 1996.
[18] V. L. Singleton and J. A. Rossi, “Colorimetry of total
phenolicswith phosphomolybdic-phosphotungstic acid
reagents,”Ameri-can Journal of Enology and Viticulture, vol. 16,
pp. 144–158, 1965.
[19] M. G. L. Hertog, “Content of potentially
anticarcinogenicflavonoids of 28 vegetables and 9 fruits commonly
consumedin the Netherlands,” Journal of Agricultural and Food
Chemistry,vol. 40, no. 12, pp. 2379–2383, 1992.
[20] E. A. Abdel-Sattar, S. M. Mouneir, G. F. Asaad, and H.M.
Abdallah, “Protective effect of Calligonum comosum
onhaloperidol-induced oxidative stress in rat,” Toxicology
andIndustrial Health, vol. 30, no. 2, pp. 147–153, 2014.
[21] A. Braca, D. Nunziatina, D. B. Lorenzo, P. Cosimo, P.Mateo,
andM. Ivano, “Antioxidant principles from Bauhinia
terapotensis,”Journal of Natural Products, vol. 64, pp. 892–895,
2001.
[22] S. F. Abou Zid and G. M. Elsherbeiny, “Increase in
flavonoidscontent in red onion peel bymechanical shredding,”
Journal ofMedicinal Plants Research, vol. 2, no. 9, pp. 258–260,
2008.
[23] E. Golomb, A. Kruglikova, D. Dvir, and A.
Abramovici,“Induction of atypical prostatic hyperplasia in rats by
sympath-omimetic stimulation,”The Prostate, vol. 34, pp. 214–221,
1998.
[24] L. Sandford, J. W. Searle, and J. F. Kerr, “Successive
wavesof apoptosis in the rat prostate after repeated withdrawal
oftestosterone stimulation,” Pathology, vol. 16, no. 4, pp.
406–410,1984.
[25] D. Engelstein, J. Shmueli, S. Bruhis, C. Servadio, and
A.Abramovici, “Citral and testosterone interactions in
inducingbenign and atypical prostatic hyperplasia in rats,”
ComparativeBiochemistry and Physiology, vol. 115, no. 2, pp.
169–177, 1996.
[26] M. D. Scolnik, C. Servadio, and A. Abramovici,
“Comparativestudy of experimentally induced benign and atypical
hyperpla-sia in the ventral prostate of different rat strains,”
Journal ofAndrology, vol. 15, no. 4, pp. 287–297, 1994.
[27] R. D. Lillie,Histopathologic Techniques and Practical
Histochem-istry, McGraw-Hill, New York, NY, USA, 1965.
[28] C. de Nunzio, G. Kramer, M. Marberger et al., “The
contro-versial relationship between benign prostatic hyperplasia
andprostate cancer: the role of inflammation,” European
Urology,vol. 60, no. 1, pp. 106–117, 2011.
[29] A.Hobisch,H. Rogatsch, andA.Hittmair, “Immunohistochem-ical
localization of interleukin-6 and its receptor in
benign,premalignant and malignant prostate tissue,” The Journal
ofPathology, vol. 191, pp. 239–244, 2000.
[30] S. K. Gounder, V. T. Chang, D. Hoover et al., “Prostate
cancerimmunohistochemical (IHC) stains and survival in stage
D3patients,” J Clin Oncol, vol. 26, 22187, no. 15, 2008.
[31] D. G. Bostwick, W. H. Cooner, L. Denis, G. W. Jones, P.T.
Scardino, and G. P. Murphy, “The association of benignprostatic
hyperplasia and cancer of the prostate,” Cancer, vol.70, no. 1, pp.
291–301, 1992.
[32] A. Abramovici, C. Servadio, J. Shmuely, and U.
Sandbank,“Experimental induction of atypical hyperplasia in rat
ventralprostate,” Progress in clinical and biological research,
vol. 243, pp.559–568, 1987.
[33] A.A.Geldof, C. Engel, andB. R. Rao, “Estrogenic action of
com-monly used fragrant agent citral induces prostatic
hyperplasia,”Urological Research, vol. 20, no. 2, pp. 139–144,
1992.
[34] J. Brolin, L. Skoog, and P. Ekman, “Immunohistochemistryand
biochemistry in detection of androgen, progesterone, andestrogen
receptors in benign and malignant human prostatictissue,” Prostate,
vol. 20, no. 4, pp. 281–295, 1992.
[35] S. M. Ho, I. Leav, F. B. Merk, M. Yu, P. W. L. Kwan, and
J.Ziar, “Induction of atypical hyperplasia, apoptosis, and type
IIestrogen- binding sites in the ventral prostates of noble
ratstreated with testosterone and pharmacologic doses of
estradiol-17𝛽,” Laboratory Investigation, vol. 73, no. 3, pp.
356–365, 1995.
[36] N. Soulitzis, I. Karyotis, D. Delakas, and D. A.
Spandidos,“Expression analysis of peptide growth factors VEGF,
FGF2,TGFB1, EGF and IGF1 in prostate cancer and benign
prostatichyperplasia,” International Journal of Oncology, vol. 29,
no. 2, pp.305–314, 2006.
[37] D.Marinese, R. Patel, and P. D.Walden, “Mechanistic
investiga-tion of the adrenergic induction of ventral prostate
hyperplasiain mice,” Prostate, vol. 54, no. 3, pp. 230–237,
2003.
[38] I. S. Shin, M. Y. Lee, H. K. Ha, C. S. Seo, and H. K.
Shin,“Inhibitory effect of Yukmijihwang-tang, a traditional
herbalformula against testosterone-induced benign prostatic
hyper-plasia in rats,” BMC Complementary and Alternative
Medicine,vol. 20, pp. 12–48, 2012.
[39] C. C. Peng, J. H. Liu, C. H. Chang et al., “Action
mechanismof Ginkgo biloba leaf extract intervened by exercise
therapyin treatment of benign prostate hyperplasia,”
Evidence-Based
-
Mediators of Inflammation 13
Complementary and Alternative Medicine, vol. 2013, Article
ID408734, 12 pages, 2013.
[40] C. H. Ma, W. L. Lin, S. L. Lui et al., “Efficacy and safety
ofChinese herbal medicine for benign prostatic hyperplasia:
sys-tematic review of randomized controlled trials,” Asian
Journalof Andrology, vol. 15, pp. 471–482, 2013.
[41] Z. Ma, T. H. Nguyen, T. H. Huynh, P. T. Do, and H.
Huynh,“Reduction of rat prostate weight by combined
quercetin-finasteride treatment is associated with cell cycle
deregulation,”Journal of Endocrinology, vol. 181, no. 3, pp.
493–507, 2004.
[42] J. Y. Jung, Y. Lim, M. S. Moon, J. Y. Kim, and O. Kwon,
“Onionpeel extracts ameliorate hyperglycemia and insulin
resistancein high fat diet/streptozotocin-induced diabetic rats,”
Nutritionand Metabolism, vol. 8, article 18, 2011.
[43] B. Djavan, E. Eckersberger, G. Espinosa et al.,
“ComplexMechanisms in Prostatic Inflammatory Response,”
EuropeanUrology, vol. 8, no. 13, pp. 872–878, 2009.
[44] D.Giri,M.Ozen, andM. Ittmann, “Interleukin-6 is an
autocrinegrowth factor in human prostate cancer,”The American
Journalof Pathology, vol. 159, no. 6, pp. 2159–2165, 2001.
[45] H. H. Radeke, B. Meier, N. Topley, J. Floge, G. G.
Habermehl,and K. Resch, “Interleukin 1-𝛼 and tumor necrosis
factor-𝛼induce oxygen radical production in mesangial cells,”
KidneyInternational, vol. 37, no. 2, pp. 767–775, 1990.
[46] A. Descazeaud, N. Weinbreck, G. Robert et al.,
“Transform-ing growth factor 𝛽-receptor II protein expression in
benignprostatic hyperplasia is associated with prostate volume
andinflammation,” BJU International, vol. 108, no. 2, pp.
E23–E28,2011.
[47] O. Coskun, M. Kanter, A. Korkmaz, and S. Oter, “Quercetin,a
flavonoid antioxidant, prevents and protects streptozotocin-induced
oxidative stress and 𝛽-cell damage in rat pancreas,”Pharmacological
Research, vol. 51, no. 2, pp. 117–123, 2005.
[48] J. S. Bland, Clinical Nutrition: A Functional Approach,
TheInstitute for Functional Medicine, Gig Harbor, Wash,
USA,1999.
[49] N. Xing, Y. Chen, S. H. Mitchell, and C. Y. F. Young,
“Quercetininhibits the expression and function of the androgen
receptorin LNCaP prostate cancer cells,” Carcinogenesis, vol. 22,
no. 3,pp. 409–414, 2001.
[50] M. R. Vijayababu, A. Arunkumar, P. Kanagaraj, P.
Venkatara-man,G.Krishnamoorthy, and J. Arunakaran, “Quercetin
down-regulates matrix metalloproteinases 2 and 9 proteins
expressionin prostate cancer cells (PC-3),” Molecular and Cellular
Bio-chemistry, vol. 287, no. 1-2, pp. 109–116, 2006.
[51] N. Bhatia, C. Agarwal, and R. Agarwal, “Differential
responsesof skin cancer-chemopreventive agents silibinin,
quercetin,and epigallocatechin 3-gallate on mitogenic signaling and
cellcycle regulators in human epidermoid carcinoma A431
cells,”Nutrition and Cancer, vol. 39, no. 2, pp. 292–299, 2001.
[52] J. A. Choi, J. Y. Kim, J. Y. Lee et al., “Induction of cell
cyclearrest and apoptosis in human breast cancer cells by
quercetin,”International journal of oncology, vol. 19, no. 4, pp.
837–844,2001.
[53] D. A. Shoskes, J. C. Nickel, and M. W. Kattan,
“Phenotypicallydirected multimodal therapy for chronic
prostatitis/chronicpelvic pain syndromepp. A prospective study
using UPOINT,”Urology, vol. 75, no. 6, pp. 1249–1253, 2010.
[54] S. Wu, H. Sun, X. Qi, and Z. Tu, “Effect of epristeride on
theexpression of IGF-1 and TGF-𝛽 receptors in
androgen-inducedcastrated rat prostate,” Experimental Biology and
Medicine, vol.226, no. 10, pp. 954–960, 2001.
[55] R. W. Furlanetto, J. N. DiCarlo, and C. Wisehart, “The type
IIinsulin-like growth factor receptor does not mediate
deoxyri-bonucleic acid synthesis in human fibroblasts,” Journal
ofClinical Endocrinology and Metabolism, vol. 64, no. 6, pp.
1142–1149, 1987.
[56] Z. Culig, A. Hobisch, M. V. Cronauer et al., “Regulation
ofprostatic growth and function by peptide growth
factors,”Prostate, vol. 28, pp. 392–405, 1996.
[57] K. Senthilkumar, P. Elumalai, R. Arunkumar et al.,
“Quercetinregulates insulin like growth factor signaling and
inducesintrinsic and extrinsic pathway mediated apoptosis in
andro-gen independent prostate cancer cells (PC-3),” Molecular
andCellular Biochemistry, vol. 344, no. 1-2, pp. 173–184, 2010.
[58] F.O. Ranelletti, R. Ricci, L.M. Larocca et al.,
“Growth-inhibitoryeffect of quercetin and presence of type-II
estrogen-bindingsites in human colon-cancer cell lines and primary
colorectaltumors,” International Journal of Cancer, vol. 50, no. 3,
pp. 486–492, 1992.
[59] Y. Q.Wei, X. Zhao, Y. Kariya, H. Fukata, K. Teshigawara,
and A.Uchida, “Induction of apoptosis by quercetin: involvement
ofheat shock protein,” Cancer Research, vol. 54, no. 18, pp.
4952–4957, 1994.
[60] T. C. Lines and M. Ono, “FRS 1000, an extract of red
onionpeel, strongly inhibits phosphodiesterase 5A (PDE 5A),”
Phy-tomedicine, vol. 13, no. 4, pp. 236–239, 2006.
[61] C. H. Deng, H. R. Chen, S. P. Qiu, J. Z. Liu, K. L.
Zheng,and H. Mei, “Effect of nitric oxide donor and
alpha1-receptorantagonist on proliferation/apoptosis of
hyperplastic prostaticstromal cells in vitro,” Zhonghua Wai Ke Za
Zhi, vol. 42, no. 4,pp. 201–204, 2004.
-
Submit your manuscripts athttp://www.hindawi.com
Stem CellsInternational
Hindawi Publishing Corporationhttp://www.hindawi.com Volume
2014
Hindawi Publishing Corporationhttp://www.hindawi.com Volume
2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttp://www.hindawi.com Volume
2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttp://www.hindawi.com Volume
2014
Hindawi Publishing Corporationhttp://www.hindawi.com Volume
2014
Disease Markers
Hindawi Publishing Corporationhttp://www.hindawi.com Volume
2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttp://www.hindawi.com Volume
2014
Hindawi Publishing Corporationhttp://www.hindawi.com Volume
2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttp://www.hindawi.com Volume
2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation
http://www.hindawi.com Volume 2014
Immunology ResearchHindawi Publishing
Corporationhttp://www.hindawi.com Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttp://www.hindawi.com Volume
2014
Hindawi Publishing Corporationhttp://www.hindawi.com Volume
2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttp://www.hindawi.com Volume
2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttp://www.hindawi.com Volume
2014
Hindawi Publishing Corporationhttp://www.hindawi.com Volume
2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttp://www.hindawi.com Volume
2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttp://www.hindawi.com Volume
2014
Parkinson’s Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing
Corporationhttp://www.hindawi.com