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Introduction
Normal penile erection depends on vascular smooth
muscle relaxation in erectile tissue and penile arteries, with
the main mediator of relaxation being nitric oxide
(NO)[1]. Evidence suggests that oxidative stress mediated through
the superoxide radical (superoxide) and other reactive
oxygen species (ROS) may be central to impaired cavernosal
function in erectile dysfunction
(ED)[2]. The inactivation of NO by superoxide results in impaired penile NO
transmission and smooth muscle relaxation. Given our current
understanding of ED pathophysiology, antioxidants may be
beneficial in alleviating penile ED in the short and long term.
Berberine (Ber), an isoquinoline alkaloid, is a well-known
component of the Chinese herb Huanglian (Coptis chinensis
Franch) of the family Berberidaceae. The compound
exhibits pharmacological activities of antioxidant
action[3,4]. In our previous study, Ber was found to induce NO-mediated
relaxation of rabbit corpus
cavernosum[5]. Therefore, we aimed to observe the effects of Ber on rabbit corpus
cavenosum smooth muscle cells (CCSMC) injured by
hydrogen peroxide (H2O2) and to understand the role of Ber in
the prevention of ED.
Materials and methods
Animals Male New Zealand rabbits (22_26 weeks old,
3_4 kg body weight; supplied by the Animal Services Center,
Yunyang Medical College, Shiyan, China) were used. The
rabbits had free access to a standard diet (Altromin pellets)
and tap water until use. Fasting was achieved by depriving
food but not drinking water 16 h before surgery.
Isolation and culture of CCSMC The CCSMC were
isolated from male New Zealand rabbits as described
previously[6]. Briefly, the penises were excised at the point of adhesion to
the lower pubic bone. The organs were cleared from fat and
connective tissue. The facia penis, glans penis, corpus
spongiosum, and tunica albuginea were carefully removed.
The corpus cavernosum was washed in sterile
phosphate-buffered saline (PBS), cut into small strips, and placed for
12_18 h at 37 °C in a tube with 5 mL PBS-buffered minimum
essential medium (MEM) containing 0.1% collagenase (type
I). After incubation, the pieces were rinsed with
PBS-buffered MEM and then mechanically dispersed with a pipette
(tip inside diameter 0.5 mm) in the medium. Dissociated cells
were collected by centrifugation at 1000 r/min for 5 min and
resuspended in 5 mL complete MEM (supplemented with
20% fetal bovine serum, 2 mmol/L L-glutamine, 100 units/mL
penicillin G sodium, and 100 µg/mL streptomycin sulfate).
The cells were seeded in a 75 cm2 culture flask and cultured
undisturbed for 2_3 d in a humidified 5%
CO2_95% air incubator at 37 °C. After inoculation for 3 d, the
anchorage-dependent cells radially spread, and part of the area was
confluent. If the color of the medium became yellow (ie, pH
<7), the medium was changed immediately to discard the
suspension cells and broken fiber and cells. Afterward, the
medium was changed every 2_3 d according to the condition.
The cells were subcultured after attaining >90%
conflu-ency (at approximately 10_14 d). The previous medium was
decanted from the culture flask, PBS was added to wash the
cells, and the cells were detached with 0.25% trypsin for
30_60 s. When the most of cells became round and the
intracellular space became broader as seen on inverted microscopy,
complete Dulbecco's modified Eagle's medium (DMEM) was
added to stop the reaction of trypsin. Then the cells on the
wall of the culture flask were mechanically dispersed with a
pipette and subcultured at a ratio of 1:2 or 1:3 in the culture
flask. The cells from the second to fourth passage were
used.
Immunohistochemical staining for α-smooth muscle
actin The laboratory procedure for SP immunostaining
involved a streptomycin avidin-peroxidase
immunochemistry kit (Zhongshan Co, Beijing, China). Briefly, the
anchorage CCSMC were digested with 0.25% trypsin for 3_5 min
and suspended. The suspended cells were placed in 24-well
culture plates, some with coverslips, in complete growth
medium and cultured at 37 °C. After 2_3 d, the cells were
washed twice with PBS, fixed with 4% formaldehyde-PBS
for 10 min, and washed twice with PBS. The cells were
permeated with 0.5% Triton-X 100 for 10 min and washed
twice with PBS. After being blocked with 5% normal goat
serum for 30 min, the cells were incubated with a
monoclonal antibody against α-smooth muscle actin (1:100,
Chemicon International, Temecula, CA, USA) at 4 °C
over-night. After being washed 3 times with 0.01
mmol/L PBS, the cells were incubated with biotin-labeled goat anti-mouse
immunoglobulin G (IgG, 1:400) at 37 °C for 30 min. After 3
washings with 0.01 mmol/L PBS, the cells were incubated
with horseradish peroxidase-labeled streptomycin avidin
(1:400) at 37 °C for 20_30 min, and finally diaminobenzidine
(DAB) was added for staining. To ensure the reliability and
specificity of the results of the immunohistochemical
staining, goat serum and PBS were used to replace the first
antibody in our control test.
Effect of Ber on cultured CCSMC injured by
H2O2
Cell viability assays In this experiment, the groups of
cells included the control group (cultured in medium only),
model group (cultured in 1 mmol/L
H2O2), and test groups (cultured in different concentrations of Ber and 1 mmol/L
H2O2).
A methyl thiazolyl tetrazolium (MTT) assay (Duchefa,
The Netherlands) to measure cell viability was performed
as described previously[7]. The CCSMC were digested with
0.25% trypsin and seeded at 2×104 cells/well in
200 µL in 96-well culture plates. The cells were first cultured for
2_3 d in MEM medium supplemented with 10% fetal bovine
serum under normal conditions, then 24 h in medium
containing 5% fetal bovine serum. Subsequently, the cells
were incubated with Ber (0.1_1000 µmol/L) for 24 h,
followed by 4 h stimulation with 1 mmol/L
H2O2, and then incubated for an additional 4 h at 37 °C with 20 µL 0.5
mg/mL MTT. The medium was removed and 150 µL/well
DMSO was added to resolve the formazan salts. The plates
were shaken for 10 min to allow formazan dissolution and
then absorbance was measured on a microplate reader at
490 nm. The cell survival rate was calculated: (absorbance
of treated group_absorbance of blanks)/(absorbance of
control_absorbance of blanks)×100 as a percentage.
Measurement of NO products NO undergoes a series
of reactions with several molecules present in biological fluid,
leading to the accumulation of the final products, nitrate,
and nitrite (NO2_ and
NO3_). Thus, the index of total
production is the sum of both
NO2_ and
NO3_ accumulated in the tissue samples. The CCSMC were seeded at
2×104 cells/well in 96-well culture plates and treated as described above. The
cell supernatant was collected and the NO products were
determined according to the manufacturer's (Nanjing
Jian-cheng Bioengineering Institute, Nanjing, China)
recommendation on a microplate reader at 550 nm.
Lactate dehydrogenase release Cell injury induced
by H2O2 was quantitatively assessed by the measurement of
lactate dehydrogenase (LDH) released from damaged or
destroyed treated cells, and an aliquot of bathing media was
combined with NADH and pyruvate solutions. LDH
activity proportional to the rate of pyruvate loss was assayed
according to the manufacturer's (Nanjing Jiancheng
Bioengineering Institute, China) recommendation on a microplate at
440 nm.
Determination of superoxide dismutase
activity The superoxide dismutase (SOD) activity of the treated cell
supernatant was determined on a microplate at 550 nm. One
unit of SOD was defined as the amount required to inhibit
the rate of reduction of cytochrome c by 50%.
Assessment of malondialdehyde content
Malon-dialdehyde (MDA), an end product of lipid peroxidation,
reacts with thiobarbituric acid to form a colored substance.
The CCSMC were seeded at 2×104 cells/well in 96-well
culture plates and treated. Then the medium was discarded
and the cells were washed twice with PBS. Subsequently, a
solution of 1 mL 0.1 mmol/L PBS-0.05 mmol/L EDTA (pH 8.0)
and 50 µL 1% Triton-X 100 was added into the well to lyse
the cells. The MDA content of the cells was determined on
a microplate reader at 532 nm. The protein concentrations
were determined by Coomassie brilliant blue method.
Drugs and chemicals Ber (chemical purification>98%)
was purchased from Sigma (St Louis, MO, USA). DMEM
(high glucose) medium and collagenase type I were from
Gibco (USA). Trypsin (1:250) and L-glutamine were from
Amresco (Solon, Ohio, USA). MTT was from Duchefa
(The Netherlands). The detection kits for SOD, MDA, NO, LDH,
and Coomassie brilliant blue G250 were from Nanjing
Jiancheng Bioengineering Institute (China). Fetal bovine
serum was from Hangzhou Sijiqing Biological Engineering
Materials (Hangzhou, China).
Statistical analysis Data are expressed as mean±SEM.
Differences between groups were assessed by the Student's
t-test. P<0.05 was considered significant.
Results
Morphology and verification of CCSMC After culture
for approximately 10_14 d, the CCSMC grew radially and a
group of cells grew in parallel along their longitudinal axis,
which showed obvious orientation (Figure 1). The
CCSMC were verified as spindle-shaped cells by
anti-α-actin monoclonal antibody immunohistochemical staining
(Figure 2).
Effects of Ber on CCSMC injured by
H2O2 Pretreatment with 1
mmol/L H2O2 decreased the cell viability from 100% for
control cells to 48.57%±4.1% (P<0.01), and treatment with
Ber (10_1000 µmol/L) inhibited the effect of
H2O2 (P<0.05 or P<0.01; Figure 3).
The NO level in the CCSMC was far lower
(66.8±16.3 µmol/L) than that in the endothelial cells (569±20.3
µmol/L). Treatment with 1 mmol/L
H2O2 decreased the level of NO
from 66.8±16.3 to 6.7±2.1 (P<0.01). However, incubation with
1_1000 µmol/L Ber in a concentration dependent manner
increased the level of NO (n=8, P<0.01; Figure 4).
H2O2 treatment increased LDH release, as compared
with the control CCSMC, from 497.6±69.5 to
1100.5±56.3 U/L (P<0.01), and treatment with 0.1_1000 µmol/L Ber inhibited this
effect (n=8, P<0.05 or P<0.01; Figure 5). SOD activity was
decreased with H2O2 treatment from 49.5±1.8 to 30.1±2.6
U/mL (P<0.01), and treatment with 1_1000 µmol/L Ber
restored the activity (n=8, P<0.05 or
P<0.01; Figure 6).
However, 1 mmol/L
H2O2 treatment significantly
increased the MDA content from 3.7±1.3 to 78.4±2.9
nmol/mg protein (P<0.01), and treatment with 0.1_1000 µmol/L Ber could inhibit
this effect (n=8, P<0.01; Figure 7).
Discussion
Oxidative stress, resulting from a disturbance in the
balance between the formation of free radicals in the body and
their scavenging, is believed to affect the development of
endothelial dysfunction and neuropathy within erectile
tissues. High levels of ROS in both blood and tissues act as
profibrotic factors that stimulate collagen deposition and
reduce the ratio of smooth muscle to collagen, thus
impairing tissue compliance to relaxation by "hardening" the
arterial media[8]. Oxygen-free radicals exert their cytoplasmic
effect by peroxidation of membrane phospholipids, which
leads to a change in permeability and loss of membrane
integrity[9]. Drugs with multiple mechanisms of protective
action, including antioxidant properties, may be one way to
minimize tissue injury in human diseases.
Our study provides evidence supporting the paradigm
of antioxidant therapy for the prevention of ED. We
established a H2O2 injured model with cultured rabbit CCSMC and
measured cell viability, NO production, and SOD activity;
then MDA, a lipid peroxidation product, as an index of
oxidative stress; and LDH release, to assess the change in
membrane permeability induced by radicals. We found that 1
mmol/L H2O2 significantly increased MDA content and LDH
release and decreased cell viability, NO production, and SOD
activity. However, treatment with 10_1000 µmol/L Ber
inhibited the damage induced by
H2O2 by improving cell viability
(P<0.05 or P<0.01) and decreasing MDA content
(P<0.01) and LDH release (P<0.01).
NO is a mediator of penile erection, and reduced NO
levels in the penile corpora is associated with
ED[1,9]. With excessive oxidative stress, the reaction of ROS with NO to
form peroxynitrite reduces NO concentrations in tissues,
which would lead to ED by impairing NO-dependent smooth
muscle cell relaxation. Therefore, the effect of Ber on
increasing NO production would be beneficial for the
prevention of penile ED.
SOD, an antioxidant enzyme, also has an important role
in protecting against free radicals by reducing ROS level.
We showed that SOD activity was significantly decreased
after H2O2 exposure. However, treatment with Ber restored
SOD activity.
Our results show that Ber has antioxidant action in
CCSMC injured by H2O2 through increasing cell viability,
NO production, and SOD activity and decreasing MDA
content and LDH release. These effects of Ber can be of benefit
in the prevention of ED.
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