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- Original Article -
Effects of icariin on erectile function and expression of nitric oxide synthase isoforms in castrated rats
Wu-Jiang Liu, Zhong-Cheng Xin, Hua Xin, Yi-Ming Yuan, Long Tian, Ying-Lu Guo
Andrology Center of Peking University First Hospital, Beijing 100009, China
Aim: To investigate the effect of icariin on erectile function and the expression of nitric oxide synthase (NOS)
isoforms in castrated rats. Methods: Thirty-two adult male Wistar rats were randomly divided into one
sham-operated group (A) and three castrated groups (B, C and D). One week after surgery, rats were treated with normal
saline (groups A and B) or oral icariin (1 mg/[kg·day] for group C and 5 mg/[kg·day] for group D) for 4 weeks. One
week after treatment, the erectile function of the rats was assessed by measuring intracavernosal pressure (ICP)
during electrostimulation of the cavernosal nerve. The serum testosterone (ST) levels, the percent of smooth muscle
(PSM) in trabecular tissue, and the expression of mRNA and proteins of neuronal nitric oxide synthase (nNOS),
inducible nitric oxide synthase (iNOS), endothelial nitric oxide synthase (eNOS) and phosphodiesterase V (PDE5) in
corpus cavernosum (CC) were also evaluated.
Results: ICP, PSM, ST and the expression of nNOS, iNOS, eNOS
and PDE5 were significantly decreased in group B compared with those in group A
(P < 0.01). However, ICP, PSM and the expression of nNOS and iNOS were increased in groups C and D compared with those in group B
(P < 0.05). Changes in ST and the expression of eNOS and PDE5 were not significant
(P > 0.05) in groups C and D compared with those in group B.
Conclusion: Oral treatment with icariin (> 98.6 % purity) for 4 weeks potentially improves
erectile function. This effect is correlated with an increase in PSM and the expression of certain NOS in the CC of
castrated rats. These results suggest that icariin may have a therapeutic effect on erectile dysfunction.
(Asian J Androl 2005 Dec; 7: 381-388)
Keywords: icariin; corpus cavernosm; intracavernosal pressure; nitric oxide synthase; erectile dysfunction
Corresponence to: Dr Zhong-Cheng Xin, Andrology Center of
Peking University First Hospital, Beijing 100009, China.
Received 2004-11-12 Accepted 2005-03-11
Specific phosphodiesterase V (PDE5) inhibitors have
been demonstrated to be safe and effective for erectile
dysfunction (ED) management. These agents are commonly considered as the first line therapy for this disease.
However, the clinical effect of a PDE5 inhibitor requires
at least a minimal nitric oxide (NO) signal triggered by
sexual stimulation. This, in turn, causes the induction of
cGMP turnover. Hence, without sexual stimulation, the
PDE5 inhibitor itself is unable to increase intracellular
cGMP synthesis and induce erection [1, 2].
The pathological conditions of NO synthesis involve
cavernosal nerve injury during radical prostatectomy or
severe diabetic neuropathy [3, 4], which can lead to ED;
treatment with PDE5 inhibitors has had limited success
even under sexual stimulation. Therefore, direct
enhancement of NO synthesis may be an effective approach for
the treatment of ED [5, 6].
The nitric oxide synthase (NOS) family includes two
"constitutive", Ca2+-dependent enzymes. They are
originally discovered in neurons (nNOS) and endothelial cells
(eNOS) and are also present in the corpus cavernosum
(CC) . The third, a Ca2+-independent enzyme, an
"inducible" isoform (iNOS) was also found in human and
rat penis . Previous studies have demonstrated that
the expressions and activities of NOS isoforms in the
CC are well correlated with erectile function [5, 6].
Epimedii herba has been used as a tonic drug in
traditional Chinese medicine (TCM) for centuries. This
medicinal herb is belived to be a potent enhancer of
erectile function. However, it is difficult to perform a
prospective, placebo-controlled study to determine its
safety and efficacy as well as to investigate its
mechanism on ED because of multiple compounds in this herb.
(C33H40O15, molecular weight: 676.67) has been
determined to be the main active component of Epimedii
In our previous study, we observed that icariin
induced relaxation of the isolated CC of rabbits in a
dose-dependent manner . Such relaxation could be
inhibited by NOS specific inhibitor,
Nw-Nitro-L-arginine (L-NNA). We also observed that icariin increased
intraca-vernosal pressure (ICP) significantly, which could be
conversely inhibited by L-NNA, without any influence
on the mean arterial pressure (MAP) in another model
Icariin showed a stronger inhibitory effect on cGMP-specific PDE5 than on cAMP-specific PDE4. The inhibition of icariin on PDE5 (0.43 mol/L) was not
significantly different from that of papaverine (0.68 mol/L).
However, the selective activity of icariin on PDE5
(PDE4/PDE5 of IC50) was 36.93-fold higher than that of
papaverine . These results suggested that icariin could
increase the activity of NOS and/or PDE5 and thus could
ultimately lead to increased cGMP to enhance erectile
function. However, the long-term effects of icariin on
ED and its exact mechanism remain unknown. Our previous study with a 4-week oral administration of
icariin-enriched fraction (51 % purity) on castrated rats showed
that this preparation could improve erectile function and
increase nNOS and iNOS expression in CC .
In the present study, we further purified icarrin and
investigated its effect on ED by analyzing the serum
testosterone (ST) level, cavernous tissue histology and the
mRNA and protein expression of NOS isoforms in castrated rats. We selected castrated rats as our ED animal
model because castration may decrease substantially the
erectile responsesto various stimulations. This
castration model has been widely used for the assessment of a
variety of agents in ED treatment [11-13].
2 Materials and methods
Thirty-two adult male Wistar rats weighing 250
g-300 g (Grade A, certificate No. scxk11-00-0006) were
obtained from the Animal Breeding Center of Beijing
Medical University (Beijing, China). The rats were
randomly divided into four groups with eight rats each: one
sham operated group (A) and three castrated groups (B,
C and D). Underanesthesia, rats from groups B, C and D
underwent bilateral orchiectomy through a 2-cm midline
scrotal incision and both testes were removed. Rats in
group A were sham operated. Seven days after the operation, rats from groups A and B were intragastricly
administrated with normal saline per day for 4 weeks,
whereas rats from groups C and D were similarly administered with icariin
1 mg/(kg·day) and 5 mg/(kg·day),
respectively for 4 weeks.
2.2 Drugs and reagents
Icariin was purified from the extract of Epimedii
herba by high-speed counter-current chromatography
with a two-phase solvent system composed of n-hexane-n-buta nol-methanol-distilled water (1:4:2:6, v/v) as
described previously [7, 9]. The purity was higher than
98.6 %. Trizol (Gibco/BRL, Carisbad, USA) is used to
extract RNA from the tissue. Reagents for the reverse
transcriptase polymerase chain reaction (RT-PCR) were
obtained from Sigma (St. Louis, MO, USA). The PCR primers were synthesized by SBS (Beijing, China).
Specific antibodies against nNOS, iNOS and eNOS were
products of Santa Cruz (Santa Cruz, CA, USA) and
purchased from Zhongshan Biochemical Company (Beijing,
China). The antibody against phosphodiesterase type 5A
was a product of Fab Gennix (No. PD5A-101AP, Shre-veport, LA, USA). The testosterone ELISA assay kit was
purchased from DRG. Inc. (Marbug, Germany).
2.3 ICP measurement
After orally administered with icariin for 4 weeks,
the rats were kept for 7 days for voiding to avoid the
residual effect of icariin. The rats were then anesthetized
with intramuscular sodium pentobarbital (50 mg/kg) and
the operation was carried out according to Adachi
et al. . Briefly, the pelvic nerve and the main penile nerve
(cavernous nerve) were surgically exposed and
stimulated with a square pulse stimulator connected to a
platinum bipolar electrode positioned on the erectile nerve.
Two 25-gauge butterfly needles were inserted into the
CC and carotid artery and the ICP and MAP were recorded with a pressure transducer integrated into a
computerized data acquisition system (BioPac system, Goleta,
CA, USA) that was calibrated with a manometer to
express the response in millimeters of mercury. The
unilateral electrical field stimulation of the cavernous nerve
was undertaken for 60 s using a square wave stimulator
( 5 V at a frequency of 15 Hz with square wave duration
of 1 ms and the stimulating intervals were 1 min with at
least 15 min between stimulations). The effects of icariin
on erectile function were evaluated by changes in the
ratio of ICP to MAP and the area under the curve (AUC)
2.4 Serum testosterone assay
After the assessment of erectile function was
com-pleted, a 2-mL blood sample was obtained from each rat
and stored at 4 °C. Analysis was carried out according to
the user¡¯s manual of testosterone ELISA assay kit.
2.5 Smooth muscle staining
After assessing the erectile function and blood
sampling, the entire penis including the penile shaft and caudal were excised. The skeletal muscles and the
connective tissue were removed. Vertical sections of the
middle part of the penile shaft were harvested for tissue
histology study with paraffin sections and with Masson¡¯s
trichrome staining. CC was isolated from the remaining
parts of the penis and stored in a liquid nitrogen tank as
samples for RT-PCR and Western blot.
To evaluate percent of smooth muscle (PSM) changes, 12 areas were randomly selected from the same
tissue section and observed by computerized photomi-
crographs. The results were analyzed by the ratio of
smooth muscle and collagen using Adobe Photoshop 7.0
software (Adobe, San Jose, CA, USA) and presented as
the number of pixel counts of muscle or reddish brown
areas divided by the pixel count of collagen or green areas.
For statistical analysis, the mean of the calculations was
used per animal. The hematoxylin and eosin staining was
also performed to make the selected regions comparable.
Total RNA of the CC was isolated by means of TRIZOL. Three micrograms of total RNA were used for
the amplification of nNOS, eNOS and iNOS by RT-PCR.
b-actin was used as an internal control. The PCR
primers were designed with computer software as follows:
forward 5¡¯-CAACACAAAGGAATGAATCC-3¡¯, reverse 5¡¯-GCTGAAAACCTCATCTGC-3¡¯ for nNOS
(NM-052799.1, gi|16258810, corresponding to 4487
bp-4005 bp); forward 5¡¯-GGAAATAGAAACAACA GGAACC-3¡¯, reverse 5¡¯-AGCGCATACCACTTCAGC-3¡¯
for iNOS (NM-012611.2, gi|31377499, corresponding to 689 bp-1222 bp); forward 5¡¯-CCTAACTTGCC
TTGCATCC-3¡¯, reverse 5¡¯-GCTTTACCACTGAGCT AAATCC-3¡¯ for eNOS (XM-342615.1, gi|34853989,
corresponding to 3010 bp-3769 bp); forward 5¡¯-CATG
TTTGAGACCTTCAACACC-3¡¯, reverse 5¡¯- TAATGTC ACGCACGATTTCC-3¡¯ for
b-actin (BC063166.1, gi|38648901, corresponding to 447 bp-718 bp). The
amplification was initiated with incubation at 95
°C for 10 min, followed by 27 cycles consisting of 94
°C for 45 s, 58 °C for 50 s and 72
°C for 60 s, and ended with 72 °C for 10 min. PCR products were detected by
electrophoresis on 1 % agarose/ErBr gel. Specificity of RT-PCR
products was confirmed by sequencing. The density was
measured at five points for each band. Data were
presented as the mean density of the mRNA band in
question relative to that of the b-actin band in the same sample.
2.7 Western blot
Tissues of the CC were homogenized in ice-cold
lyses buffer (25 mmol/L HEPES, pH 8.0, 5 mmol/L EDTA,
1 % sodium deoxycholate, 1 % Triton X-100, 0.1 % SDS,
and protease inhibitors). Homogenated tissues (25 %
w/v) were centrifuged at
10 000 × g for 20 min at 4
°C to remove tissue debris without precipitating plasma
membrane fragments that contained eNOS. Protein
concentration was determined by the Lowry method
. Twenty micrograms of total protein were used in SDS-PAGE.
Anti-NOS (nNOS, iNOS and eNOS) serum and anti-PDE5
serum were diluted at 1:1000 in Tris-buffered saline (TBS)
and used as primary antibodies. A 1:8000 dilution of
horseradish peroxidase-labeled rabbit anti-rat IgG fragment was
used as a secondary antibody; a-actin was used as an
internal control. Exposure of luminol-reacted membrane
onto X-ray films was performed. The films were scanned
with a BioRad GS-670 densitometer and the band
intensity was semi-quantified with the UTHSCSA Image Tool
for Windows (2.0) (Triad Scientific Inc., Swarthmore
Ave, Lakewood, New Jersey, USA). The density was measured at five points for each band. Data were
presented as the mean density of the protein band in
question relative to that of the b-actin band in the same sample.
2.8 Statistic analysis
The software SPSS version 11.0 for Windows (SPSS
Inc., Chicago, IL, USA) was used for the statistical
analysis including the Student-Neumann-Keul¡¯s test and the
paired t-test. The values were presented as the mean ±
SEM. P < 0.05 was considered to be statistically significant.
3.1 Effects of icariin on ICP and MAP
The values of the peak ICP after cavernous nerve
stimulation were showed in Figure1. No obvious changes
were observed for MAP ([102.00 ± 3.22] mmHg). The
increase of ICP in castrated rats (group B) was
significantly lower than that in the sham-operation group (group
A) (P < 0.05). Castrated rats treated with icariin (groups
C and D) significantly increased the erectile response
(ICP) to cavernous nerve stimulation compared with
group B, as measured by ICP/MAP and total ICP values.
3.2 Effects of icariin on serum ST level
The mean serum ST levels in groups A, B, C and D
were (2.41 ± 0.91) pmol/mL, (0.38 ± 0.15) pmol/mL,
(0.38 ± 0.10) pmol/mL and (0.38 ± 0.11)
pmol/mL, respectively. The mean ST level was significantly lower in all
castrated rats (groups B, C and D) regardless of icariin
treatment (P < 0.05). No significant differences were seen
in the icariin-treated rats (groups C and D) compared
with untreated rats (group B) (P > 0.05).
3.3 Effects of icariin on PSM
The mean PSM in the castrated rats (group B) was
significantly decreased when compared with the
sham-operated rats (P < 0.05); however, oral icariin treatment
(groups C and D) showed an obvious increase in PSM
compared with the untreated rats (group B)
(P < 0.05) (Figure 2).
3.4 Effects of icariin on mRNA expression of NOS
isoforms and PDE5
The RT-PCR results (Figure 3) showed that the mRNA
of nNOS, iNOS, eNOS and PDE5 were significantly decreased in the castrated rats in group B compared with
that in the sham-operated rats (group A)
(P < 0.05). However, the nNOS and iNOS mRNA in groups C and
D were significantly increased in both icariin-treated rats
(P < 0.05, P < 0.01); however, no obvious changes in
the expression of eNOS and PDE5 were observed
(P > 0.05).
3.5 Effects of icariin on protein expression of NOS
isoforms and PDE5
The Western blot results (Figure 4). The protein
expression of nNOS, iNOS, eNOS and PDE5 was
significantly decreased in castrated rats in group B compared
with that in the sham-operated rats (group A)
(P < 0.05). However, the nNOS and iNOS protein expression in
groups C and D were significantly increased in both
icariin-treated rats (P < 0.05,
P < 0.01); however, no obvious changes in the expression of eNOS or PDE5
were observed (P > 0.05).
In the present study, the castrated rats showed
significantly reduced erectile function that was paralleled
with the decreased peak and total ICP. These
observations were consistent with the previous studies [12, 13,
15]. In our previous study, we showed that a 4-week
oral administration of icariin-enriched fraction (51%
purity; 1 mg/[kg·day] and
5 mg/[kg·day]) improved erectile function and increased nNOS and iNOS expression
in CC of castrated rats . In this study, we further
demonstrated that 4-week oral treatment with pure icariin
(> 98.6 %); 1 mg/(kg·day) and
5 mg/(kg·day) significantly increased the peak and total ICP (area under the
erectile curve) in castrated rats compared with controls
but without influence on ST levels. These results
suggested that long-term oral treatment with icariin-enriched
fraction and/or pure icariin might improve the erectile
function of castrated rats.
It has been known that the trabecular smooth muscle
content is the key structure in normal erectile function
and the degree of loss of corporeal smooth muscle
content correlates with the extent of impairment in
corporeal veno-occlusive function . Since icarrin
administration increases smooth muscle content in the CC, this
medicinal herb active component may have an effect on
reversing ED. Whether icariin could affect smooth muscle
apoptosis and/or collagen synthesis remains to be studied.
In the present study, icariin was found to increase
the mRNA and protein expression of nNOS and iNOS in
the CC of castrated rats, which was well correlated with
the changes of PSM and ICP. In addition, the ST levels
of the castrated rats (group B) were significantly
decreased compared with those of the control rats (group
A) and were not altered by icariin administration (groups
C and D). These results suggested that the mechanisms
of icariin on ED and NOS mRNA and protein expressions are not associated with testosterone synthesis.
Whether icariin can directly affect the activity of NOS
promoter and/or the related nuclear transcription factors
in smooth muscle cells is currently under investigation.
The levels of NOS proteins were known to be
influenced by several signals via transcriptional or
translational mechanisms and the changes in the expression of
NOS isoforms after several chemical and physical
stimulations have been reported . For example, nNOS and
eNOS levels in rat cavernosal tissue were observed to
increase after repeated PGE1 treatment . Similarly,
changes in nNOS and eNOS protein content were observed following orchiectomy and androgen replacement
[12, 13]. iNOS and eNOS content in rat cavernosal
tissue can also be upregulated by single reagent VEGF .
In our previous study, chronic oral treatment with Icariin
increases the erectile function (ICP) and restores the
eNOS expression in corpus cavernosum of arteriogenic
erectile dysfunction rats .
Studies noted that icariin could produce two
metabolic products of icarinoside II and icaritin after oral
administration . Our own studies showed that oral icariin
specifically inhibited PDE5 and upregulated NOS expression. These observed effects could be associated
with the icariin metabolic products, however, this needs
to be further investigated.
In summary, the 4-week oral administration
of icariin to castrated rats can promote erectile function, which is
correlated with the increased PSM and the mRNA and protein expression of NOS but with no influence on ST
levels. Although its mechanism remains to be explored,
this study suggests that icariin may have long-term
therapeutic effects on ED.
The authors would like to acknowledge the National
Nature Science Foundation of China (No. 30070927) and
Beijing National Nature Science Foundation (No. 7012023) for grants for this work.
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