Toshiyuki Kamijo,1*
Shiegeru Sato2, and Tadaichi
Kitamura1
1. Department of Urology,
Faculty of Medicine, University of Tokyo, Japan
...
2. Ohme Research Laboratories,
Tobishi Pharmaceutical Co., Ltd., Tokyo, Japan
The treatment for chronic
nonbacterial prostatitis (NBP) has not been
established. Pollexia pollen extract (ref. CN-009) is
reported to have therapeutic effects for NBP. The effects and mechanisms of
CN-009 were investigated.
Pollexia consists of a water soluble fraction
(PolliAQ) and fat-soluble fraction (PolliLIP).
Ten-month-old rats were used with
administration of estradiol after castration, which
were similar to human NBP histologically. Since
CN-009 consists of PolliAq and PolliLip,
these extracts were administered, respectively. The prostate was evaluated histopathologically including glandular damage (epithelial
score), stromal ratio and immunohistochemical
assays for epithelial function (PAP), stromal
evaluation (Vimentin), cell proliferation (PCNA) and
apoptosis (deoxyuridine triphosphate
biotin nick end-labeling (TUNEL)).
Controls revealed severe acinar gland atrophy and stromal
proliferation. CN-009 showed diminished these damages. Epithelial score was
better (P<0.01) and PAP positive materials were more abundant in CN-009 and PolliLip than in Controls. The stromal
ratio was lower in CN-009 (P<0.01) and PolliAq
(P<0.05). There was no difference for PCNA positive cells in the epithelium
and stroma, and TUNEL positive cells in the
epithelium. While, the number of TUNEL positive cells in the stroma of CN-009 and PolliAQ
increased (P<0.01).
These findings suggest that CN-009
protects acinar epithelial cells mainly by POLLILIP
and also inhibits stromal proliferation in
association with enhanced apoptosis mainly by PolliAq.
Prostate 49: 122-131, 2001. © 2001 Wiley-Liss, Inc.
Three chronic prostatitis
syndromes have been recognized; chronic bacterial prostatitis
(CBP), chronic nonbacterial prostatitis (NBP) and prostatodynia. NBP is the most frequent disorder of 64% in
these three diseases [1]. The etiology of NBP is unknown. A number of organisms
or other factors have been reported to be the possible causes for NBP. They are
Trichomonas vaginalis,
Chlamydia trachomatis, genital mycoplasmas,
staphylococci, coryneforms, genital viruses [2], biofilms [3], stagnation of prostatic secretion, autoimmune
disease, allergy, disorder of sex hormone and psychological effects [4, 5]. For
the treatment of CBP or NBP, antibiotics of new-quinolone
or tetracycline have been administered. However, many cases resist these
treatments [6].
Cn-009 is a pollen extract, which contains 20:1 ratio of powdered aqueous and
organic extract. It is essentially a microbial digest of a standardized mixture
of eight plant species grown at the Scania area in
southern Sweden. The active ingredients consist of water-soluble (PolliAQ) and fat-soluble (PolliLIP)
fractions [7, 8]. It was reported that CN-009 showed urine discharge action
[9.10], anti-prostatic hypertrophic action [7] and anti-inflammatory effects to
the prostate [11] in a preliminary study. Since Ask-Upmark
[12] Reported CN-009 showed an efficacy to prostatitis,
it has been used for the treatment of chronic prostatitis
with high therapeutic effects. However, the mechanisms for these effects remain
unknown. To assess the mechanisms of the anti-prostatitis
effect by CN-009, the present study was performed using a nonbacterial prostatitis rat model [13, 14] induced by 17b -estradiol administrations and castration.
Sex Hormone-Induced
Nonbacterial Prostatitis Model
Ten-month-old Wistar aged male rats were purchased
from Japan Slc Co. (Tokyo, Japan). The rats were
housed in a climatised environment with a 12-hr
light/dark cycle, 40-70% humidity. Food and water were supplied ad libitum. The rats were castrated under ether anesthesia,
and then 17b-estradiol (Sigma, MI) 0.25 mg/ 2ml/kg diluted by sesame oil, as an
inducer for prostatitis, was subcutaneously injected
into the back of rats for 30 days from 1 day after castration [13,14].
Experimental Structure and Schedule
CN-009was suspended for 630 or 1,260 mg/5ml with 1% HCO-60 (Japan Surfactant, Tokyo,
Japan). PolliAQ and PolliLIP
were similarly prepared for 1,200 and 60-mg/5 ml, respectively. Testosterone
(TS) (Wako Chemicals, Tokyo, Japan), as a positive control, was diluted for
2.5-mg/2 ml with corn oil (Yuro Chemical, Tokyo,
Japan).
The experimental structure is shown in Table I and the experimental schedule is
illustrated in Figure 1. The rats were divided into seven groups consisting of
Sham-operation (Sham-ope), Control, CN-009 630,
CN-009 1260, PolliAQ, PolliLIP
and TS with five or six animals in each group.In the
Sham-ope group, the rats were treated with only
Sham-castration and without any drugs. In the Control group, the rats were
injected subcutaneously environment with a 12-hr light/dark cycle, 40-70%
humidity. Food and water were supplied ad libitum.
The rats were castrated under ether anesthesia, and then 17b-estradiol (Sigma,
MI) 0.25 mg/ 2ml/kg diluted by sesame oil, as an inducer for prostatitis, was subcutaneously injected into the back of
rats for 30 days from 1 day after castration [13,14].
Experimental Structure and Schedule
CN-009 was suspended for 630 or 1,260 mg/5ml with 1% HCO-60 (Japan Surfactant,
Tokyo, Japan). PolliAQ and PolliLIP
were similarly prepared for 1,200 and 60-mg/5 ml.,
|
Table 1. The |
|
|
|
|
Group |
No. of animals |
Inflammatory agent |
Drug treatment |
|
Sham-ope. |
5 |
No-treatment |
No-treatment |
|
Each parenthesis represents the route of administration. s.c, subcutaneous injection; p.o.,
oral administration |
|||
... respectively.
Testosterone (TS) (Wako Chemicals, Tokyo, Japan), as a positive control, was
diluted for 2.5-mg/2 ml with corn oil (Yuro
Chemical, Tokyo, Japan)
The experimental structure is shown in Table I and the experimental schedule is
illustrated in Figure 1. The rats were divided into seven groups consisting of
Sham-operation (Sham-ope), Control, CN-009 630,
CN-009 1260, PolliAQ, PolliLIP
and TS with five or six animals in each group.
In the Sham-ope group, the rats were treated with
only Sham-castration and without any drugs. In the Control group, the rats were
injected subcutaneously
With 17b-estradiol for 30 days from the day following castration and
administered orally with only 1% HCO- 60 5ml/kg for 14 days from Day 17. In the
CN-009 630, CN-009 1260, PolliAQ 1200 and PolliLIP groups, similar protocols were performed with oral
administration of CN-009 630, CN-009 1260, PolliAQ
1200 and PolliLIP 60 mg/kg, respectively. Also in the
TS group, the rats were subconsciously with 17b-estradiol for 30 days from the
next day of castration. After 14 days, TS 2.5 mg/kg was injected subconsciously
for 14days. All studies were conducted in accordance with institutional
guidelines of animal care in accordance with Japanese Governmental Animal
Protection and Management Law.
Prostate Weights and Histopathological Evaluation
The rats were sacrificed on the day following the final administration. The prostate
was extirpated and weighed. Relative prostatic weight was calculated from body
weight and absolute weight.
After fixation in 10% neutral buffered formalin, each prostate was cut into
coronal blocks. The tissue samples were dehydrated and embedded in paraffin.
Sections (3-4 mm thickness) were stained with Hemotoxyline-Eosin
(HE), Periodic acid Shiff (PAS) and Masson's
tri-chrome. The specimens were evaluated histopathologically.
Immunohistochemistry
Immunohistochemical studies were performed with anti-prostatic
cid phosphatase (PAP), and vimentin.
PAP staining was performed for the evalutation of
glandular epithelial function. In PAP stained specimens, anti-PAP polyclonal
antibody (Chemicon International, New York, NY) was
diluted by PBS including 0.1% BSA of a 1:100 ratio, and incubated for 2 hr at
37¡C. Biotinylated anti-rabbit IgG
and the avidin-biotin peroxidase
complex (ABC) method were performed. Unitect rabbit Immunohistochemistry detection systems (Oncogene
Science, New York, NY) were reacted by those methods. Vimentin
staining was performed for the evaluation of stromal
proliferation. An ImmunoCruz staining system (Santa
Cruz BioTech, Santa Cruz, CA) for Vimentin
Staining was used according tot eh manufacture's instructions.
Cell Proliferation and
Apoptosis
Cell proliferation and apoptosis were investigated with proliferating cell
nuclear antigen (PCNA) and terminal deoxynucleotidyl transferase mediated deoxyuridine
tri phosphate biotin nick end-labeling (TNEL), respectively. PCNA staining was
performed with PCNA staining kit (ZYMED Laboratories, South San Francisco, CA).
TUNEL method was performed with ApoTag Peroxidase In Situ Apoptosis Detection kit (Intervene, New
York, NY). In PCNA and TUNEL specimen, 5,000 cells were counted under a microscope
in glandular epithelial cells and stromal cells
respectively.
Acinar Epithelial Score and Stromal Area Ratio
To evaluate glandular damage, acinar epithelial cells
were classified and scored, as follows; columnar (2 points), cuboidal (1 point), squamous-like
(0 point) shape. Three different pathologists without any information judged
the score. Using this scoring evaluation, 20 acinar
glands of each specimen were investigated. To assess stromal
proliferation, all areas of the specimen and the glandular area were measured
using a digitizer (Graph Tech, Tokyo, Japan) with photomicrographs. Using these
findings, the stromal ratio was calculated.
Statistical Analysis
All experiments were repeated at least twice. Each value was demonstrated as
the mean±SD. Dunnett's test
if in equal variance, or non-parametric Dunnett's
test if in unequal variance between treatment groups and Control group was
performed after 1-way ANOVA followed by Bartlett variance analysis test.
Mann-Whitney U test was performed between the Sham-ope
and Control groups.
Body and Prostate Weights
(Fig. 2)
In the Sham-ope group, the prostate was larger than
in other groups. Acinar glands were roundly shaped.
The acinar lumen was filled with eosinophilic
materials. Acinar epithelial cells were cylindrical
with a normally situated nucleus and the supranuclear
spaces of these cells contained secretory materials,
which were strongly stained with PAP antibody. A few fibrous tissues were found
in the stroma (Figs. 3A and 4A). Vimentin
positive cells were few, and the Vimentin positive
was small (data not shown).
In the Control group, the prostate was atrophic acinar
glands were irregularly shaped. The acinar lumen was
poor with pale stained eosinophilic materials and
filled with inflammatory cell infiltrations mainly characterized by neutrophils. Acinar epithelial
cells were flattened similar to a squamos cell. A few
secretory materials in the epithelial cells were
poorly reacted with PAP antibody. The stroma showed
severe proliferation with many lymphocyte and monocyte
infiltrations and marked fibrosis with fibroblasts (Figs. 3B and 4B). The stroma was stained very strongle
with Vimentin. The Vimentin
positive area was significantly increased (data not shown). In the CN-009 630
group, the findings were basically identical with the Control group (data not
shown).
In the CN-009 1260 group, acinar glands were more
roundly shaped than in the Control group. Acinar
epithelial cells were cuboidal, and the supranuclear spaces contained secretory
materials stained with anti-PAP that were much more abundant than the in
control group. Inflammatory cell infiltrations into the acinar
lumen were diminished. The stroma showed mild
proliferation with few lymphocytes, monocytes, and
mild fibrosis with fibroblasts (Figs. 3C and 4C). The Vimentin
positive area was much less than that of the Control group (data not shown).
In the GBW group, acinar epithelial cells were more cuboidal than in the Control group. Epithelial cells
contained secretory materials stained with anti-PAP,
which was basically identical with the CN-009 1260 group. Diminished call
infiltration into the lumen was found (Fig. 3E). However, the stroma showed a proliferative condition with many
lymphocyte and monocyte infiltrations and marked
fibrosis with many fibroblasts. The stroma was
stained strongly with Vimentin, and the positive area
was markedly increased (data not shown).
In the TS group, acinar glands were roundly shaped.
The acinar lumen was filled with eosinophilic
materials with a few cell infiltrations. Acinar
epithelial cells were cylindrical and the supranuclear
spaces contained many secretory materials with
reactive anti-PAP. However, the stroma was stained
strongly with Vimentin and showed mild proliferation
with fibroblasts (data not shown).
Cell Proliferation and
Apoptosis
(PCNA and TUNEL Positive Cell Counts (Fig. 5))
No significant differences among the groups were observed in the PCNA positive
cell counts in epithelial cells (Fig. 6) or in stromal
calles (Fig. 7). In the Sham-ope
group, a few TUNEL positive cells were found (Fig. 5A). The findings of the
Control group were basically identical with the Sham-ope
group (Fig. 5B). In the CN-009 1260 group, TUNEL positive cells in the stroma were more abundant than in the Sham-ope and Control groups (Fig. 5C). In the TUNEL positive
cell counts, no significant differences were observed in acinar
epithelial cells (Fig. 8). However, in the stroma,
TUNEL positive cells were significantly (P< 0.05)
Fig. 3. HE staining of the prostate in the experimental nonbacterial prostatitis rat. (A) Sham-ope
group: The acinar lumen is filled with eosinophilic materials without any cells. Acinar epithelial cells are cylindrical. A few fibrous
tissues are found in the stroma. (B) Control group:
The acinar lumen is filled with induced inflammatory
cells mainly characterized by neutrophilis. Acinar epithelial cells are flattened similar to squamous cells. The stroma shows
severe proliferation with many lymphocyte and monocyte
infiltrations and remarkable fibrosis with fibroblasts. (C) CN-009 1260 group: Acinar epithelial cells are cuboidal.
Inflammatory cell infiltrations into the acinar lumen
are diminished. The stroma shows mild proliferation
with a few lymphocytes, monocytes, and fibroblasts.
(D) PolliAQ group: Stromal
proliferation is relatively mild without severe inflammatory cells. (E) PolliLIP group: Acinar epithelial
cells are cuboidal, and diminished inflammatory cell
infiltrations are shown. 400 The bar indicates 10 mm. Increased in the CN-009
1260 group or PolliAQ group compared with the control
group (Fig. 9).
Acinar Epithelial Score
(Fig. 10)
In the Control group, acinar epithelial score was
significantly lower (P > 0.01) than that of the Sham-ope
group. In comparison with the Control group (Fig. 10), the acinar
epithelial score was significantly higher (P < 0.01) in the CN-009 1260, PolliLIP and TS groups.
Stromal Area Ratio (Fig. 11)
In the Control group, the stroma area ratio was
significantly higher (P < 0.01) than that of Sham-ope
group.
Fig. 4. Immunohistochemical findings (PAP
Staining) of the prostate in experimental nonbacterial prostatitis
rats. (A) Sham-ope group: Supranuclear
spaces of acinar epithelial cells contain secretory materials which are stained with anti-PAP. (B)
Control group: Acinar epithelial cells are flattened
similar to squamous cell. Secretory
materials are poorly reactive with anti-PAP. (C) CN-009 1260 group: Supranuclear spaces contained secretory
materials with PAP staining, which are significantly more abundant than in the
Control group. «400 the bar indicates 10 mm.
In comparison with the Control group (Fig. 11) the stromal
area ratio of the CN-009 1260 was significantly (P < 0.01) lower. The PolliAQ group was also significantly (P < 0.05) lower than
the Control group. However, there was no difference between other groups.
Fig. 5. Immunohisochemical findings
(TUNEL) of the prostate in rats. (A) Sham-ope group:
A few TUNEL positive cells are shown. (B) Control group: The findings are
basically identical to these of the Sham-ope group.
(C) CN-009 1260 group: TUNEL positive cells in the stroma
are more abundant compared with the Sham-ope and
Control groups. «400 the bar indicates 10 mm
Discussion
Although chronic prostatitis is a common disease, it
is very difficult to treat effectively. Typical clinical findings are decreased
potentia, perineal or
scrotal pain, urethral discharge and lower urinary tract irritative
symptoms. The prostate gland is irregularly indurated
and the numbers of leukocytes in expressed prostatic secretion are increased
[15]. Pathological findings of this disease are chronic inflammation
characterized by aggregates of lymphocytes in the stroma
and acute inflammation characterized by the presence of neutrphic
polymorphonnuclear leukocytes in the lumen of acinar glands [15 - 17]. Pathological definition of chronic
prostatitis is different from the clinical definition
for the urologists. Clinical definition has been the combination of a clinical
symptom and inflammatory cells in expressed prostatic secretion. The
pathological inflammation of the prostate was repoted
to be not frequent in the patients with symptoms of chronic prostatitis/chronic
pelvic pain symdrome [16].
In experimental animals, Lewis, Wistar and Copenhagen
rats have a high incidence of spontaneous nonbacterial prostatitis
[14]. Administration of exogenous 17b-estradiol can induce 100% of the
incidence on prostatitis in old Wistar
rats [18] and castration also has a similar effect [13, 18]. Naslund et al. [13] reported that histopathological
findings were very similar between spontaneous nonbacterial prostatitis
and estradiol-induced prostatitis
in rats [13]. These histopathological findings in rat
spontaneous age-dependant prostatitis demonstrated
several similarities to pathological defined chronic prostatitis
in humans [19, 20]. These findings suggested that this rat model would be a
useful model for the study of the treatment of human chronic prostatitis. Therefore, we decided to investigate the
effects and mechanisms of CN-009 using a nonbacterial prostatitis
rat model [13, 14] induced by 17b-estradiol injection and castration.
No differences in the prostate weight were found in CN-009 630, CN-009 1260, PolliAQ and PolliLIP groups
compared with the Control group. Since the weight of the prostate is mostly
determined by the amount of residual secretory fluid,
these findings may indicate that CN-009 cannot prevent the reduction of secretory prostatic fluid.
In the CN-009 1260 group, we observed roundly shaped acinar
glands, cuboidal acinar
epithelial cells containing secretory materials with
positive PAP staining and diminished cell infiltrations into the lumen compared
with the Control group. The acinar epithelial score
was significantly increased. CN-009 could protect acinar
epithelial function and cell shape against nonbacterial inflammation. PolliLIP had a similar effect to CN-009 in the acinar glands. PolliAQ was not
effective in the acinar epithelial function of this
rat model. Therefore, PolliLIP may play a role for
the protection of epithelial damage in NBP. The effect of PolliLIP
is discriminated from TS effect. In an in vitro study, PolliLIP
was reported to inhibit the cyclooxygenases and
5-lipooxygenases in the biosynthesis of the prostaglandins and leucotriens enhance inflammatory cell infiltrations, PolliLIP may protect against inflammation into the acinar lumen by inhibition of these enzymes. Furthermore,
CN-009 showed an inhibition on the heat-induced hemolysis,
which is correlated to lysosomal membrane stability
[11]. CN-009 appears to stabalize a lysosomal membrane, recover cell function and protect
against degeneration of the acinar epithelium.
In addition, PolliAQ was shown to inhibit the growth
of an immortal prostate cancer cell line in vitro [22]. However, their
mechanisms are unknown. In the present study, the ratio of stromal
area was significantly decreased in the CN-009 1260 and PolliAQ
groups. Stromal TUNEL positive cell counts were
increased in these groups. Therefore, CN-009 and PolliAQ
may inhibit stromal cell proliferations by enhanced
apoptosis. Although the exact mechanism of this process is unclear, several
speculations are possible such as the direct effect by the apoptosis of
fibroblast, and the indirect effect by the apoptosis of lymphocytes through the
inhibition of several cytokines, such as several interleukins. Further
laboratory studies are necessary to elucidate the exact mechanisms of this compund.
Since no toxicological effects have been shown even in long-term administration,
CN-009 is thought to be a safe drug [6, 23]. Here we reported the effects and
mechanisms of CN-009 on rat experimental nonbacterial prostatitis
model. CN-009 will also be a safe and effective agent against human
nonbacterial chronic prostatitis.
In conclusion, CN-009 can work as a potent anti-inflammatory agent against
chronic prostatitis. These present findings suggest
that PolliLIP, a fat-soluble fraction of CN-009,
protects the function and shape of acinar glandular
epithelium and PolliAQ, a water-soluble fraction of
CN-009, inhibits stromal cell proliferations in
association with enhanced apoptosis.
Acknowledgments
We thank M. Komukai, M. Ishii, F. Kimura and E. Higaki for their technical assistance.
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