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Introduction
Testicular torsion, which is called spermatic cord torsion, is a serious medical condition that occurs in 1 in 4000 men
younger than 25 years of age[1]. If testicular torsion is not treated within 4 to 6 h, spermatogenic cell loss will
occur[2,3]. However, even in men who have undergone surgical detorsion within this time period, the ipsilateral testis often becomes
permanently dysfunctional[3-5]. Ischemia/reperfusion injury following torsion/detorsion of the testes results in the
development of significant
pathology[2-4]. Sulfasalazine was first synthesized in 1942 by combining an antibiotic,
sulfapyridine, with an anti-inflammatory agent, 5-aminosalicylic acid (5-ASA). Sulfasalazine acts as a potent inhibitor of nuclear factor-kappa B
(NF-κB) by inhibiting I kappa B (IkB) phosphorylation, thereby preventing its translocation into
the nucleus and decreasing adhesion molecule
expression[6-8]. The aim of the present study was to determine if sulfasalazine can prevent the activation of
NF-κB in spermatogenic epithelium and prevent the apoptosis of spermatogenic cells after experimental testicular torsion.
Materials and methods
Animals Thirty-two young adult male Sprague-Dawley rats (bodyweight 240-280g) were purchased from the
Experimental Animal Center of the Medical Department of Wuhan University. The animals were housed in hanging wire mesh cages, 5
or 6 per cage, under controlled lighting conditions (14-h of light beginning at 6:00 AM, followed by 10-h of darkness) at a
temperature of 20-24 °C. They were handled daily for 1 week before the experiment. Rats were divided into 4 groups (A-D),
with 8 rats in each group. All drugs and reagents were purchased from Sigma (St Louis, MO, USA). Sulfasalazine,
administered at a dosage of 350 mg/kg per d, was dissolved in 15 mL 0.9% sodium chloride solution (SASP solution) for
intraperitoneal injection.
Experimental testicular torsion Animals were anesthetized with intraperitoneal injections of sodium pentobarbital (50
mg/kg of bodyweight) for surgery and the duration of ischemia. Testicular torsion was induced as described
elsewhere[9]. The testes were retracted through a low midline laparotomy. The left testis of rats in groups A and B were rotated 720° along the
longitudinal axis for 2 h. After the torsion was relieved by counter-rotation, the testis was replaced into the scrotum, and
blood flow return was observed. The rats of group A(T+SASP ) were given intraperitoneal injections of SASP solution just
before the incision was closed. After 5 h, when the rats were able to eat food, rats were intraperitoneally injected with the
SASP solution every 24 h. The rats of group B(T+SC) were treated as for group A, except that 15 mL 0.9% sodium chloride
solution was used instead of SASP. The left testis of rats in groups C and D underwent the same operation as rats in the other
two groups, except that testicular torsion was relieved immedi-ately. The rats of group C (SC) received
15 mL 0.9% sodium chloride solution via intraperitoneal injection just before closing cut. After 5 h, when the rats could eat food, we began to
intraperitoneally inject the rats with 15 mL 0.9% sodium chloride solution every 24 h. The rats of group D(SASP) were given
the SASP solution via intraperitoneal injection just before closing cut. After 5 h, when they all can eat food, we began to
intraperitoneally inject the rats with SASP solution every 24 h. At 72 h after repair of torsion, all animals were killed.
Western blotting The torsed/detorsed testes were dissected out and cleaned of adhering fatty and connective tissues,
washed with cold 0.9% sodium chloride solution, then cleaned with filter paper. We took out a 3 cm-long piece of seminiferous
tubule from the affected testis in order to analyze the change in
NF-κB with Western blotting. The rest of each testis was used
to measure the apoptosis of spermatogenic cells.
Nuclear proteins were prepared according to a procedure described
elsewhere[10]. Seminiferous tubules were
treated with ice-cold phosphate-buffered saline (PBS) and scraped. The cell suspension was
centrifuged at 1200×g at 4 °C for 8 min. The
cell pellets were combined and lysed by resuspension in 100 µL of lysis buffer
[10 mmol/L 4-(2-hydroxyethyl)-1-piperazine
ethanesulfonic acid (HEPES), pH 7.9, 60 mmol/L KCl, 1 mmol/L ethylenediaminetetracetic acid (EDTA), 1 mmol/L dithiothreitol
(DTT), 1 mmol/L phenyl-methylsulfonylfluoride
(PMSF), and 0.5% Nonidet P-40]. A 10 µL aliquot was removed and
mixed with an equal volume of trypan blue and examined under a light microscope (×40) to confirm the presence of round, intact nuclei.
The remainder of the suspension was centrifuged again. The nuclear
pellet was washed with lysis buffer without Nonidet
P-40 and centrifuged at
1200×g at 4 °C for 5 min. The pellet was
resuspended in 100 µL of nuclear resuspension buffer (25
mmol/L Tris-HCl, pH 8.0, 400 mmol/L KCl, 1 mmol/L DTT, 1 mmol/L PMSF, and 20% w/v glycerol), rapidly frozen and thawed 3 times,
and centrifuged at 4000×g at 4 °C for 12 min. The supernatant containing the
nuclear proteins was removed, and an aliquot
was used for protein determination adapted
for microtiter plates.
Ten micrograms of cytoplasmic protein extracts were dissolved in sodium dodecyl sulfate (SDS) buffer, boiled for 5 min,
and then subjected to polyacrylamide gel electrophoresis (PAGE) on 10% acrylamide gels. After SDS-PAGE, the gels were
transferred to nitrocellulose membranes
for 1 h at
4 °C. The blots were blocked with 5% nonfat dry
milk in PBS- 0.1% Tween 20 (PBS-T) overnight at 4 °C. Immunological
evaluations were then performed for 1 h in PBS-T containing 0.2 µg/mL
affinity-purified polyclonal antibodies against the p65
subunits of NF-κB. The blots were subsequently washed with
PBS-T and incubated for 1 h with goat anti-rabbit IgG antibody
conjugated to horseradish peroxidase (HRP) at a dilution of 1:1000 in PBS-T. After
extensive washing with PBS-T, HRP activity was visualized by applying a chemiluminescent substrate then exposing the membrane to an automatic image
analysis system. The relative amount (RA) of expression was taken as being proportional to the density of the relevant band
on the blot.
Immunohistochemical staining for NF-κB protein
Immunohistochemical staining for the NF-κB protein was performed
in cells of the seminiferous tubules by using the indirect immunoperoxidase method. The cells were incubated with rabbit
polyclonal antibody against the p65 subunit of
NF-κB at a dilution of 1:200 in PBS for 1 h at room temperature and then
incubated for 1 h with biotinylated goat anti-rabbit IgG at
a dilution of 1:500. The bound antibody was visualized with
avidin-biotin complexes. The slides were counterstained with hematoxylin. After being
mounted, every slide was viewed with an
Olympus microscope. For evaluation of
NF-κB activation, microscopic fields (×200) were selected at random. The cells that
were activated were stained yellow. The percentage of
NF-κB-positive nuclei was determined by counting 100 cells from the
spermatogenic epithelium.
Analysis of apoptosis of spermatogenic cells
The torsed testes were washed in cold 0.9% sodium chloride solution, and
then cleaned with filter paper. Tissue sections were stored in formaldehyde solution for 1 d, then embedded in paraffin.
Five-micrometer-thick sections were mounted on silan-coated glass slides and fixed for one day at 65 °C.
Endogenous peroxidase was inactivated by 0.3%
H2O2 for 30 min at room temperature. To make the sections permeable,
they were incubated with a permeabilization buffer for 5 min at 4 °C.
In situ end-labeling was performed by using an
In Situ Apoptosis Detection Kit (Boehringer Mannheim Corp, Mannheim, Germany) composed of nonradioactive
fluorescein-dideoxyuridine triphosphate. The sections were incubated with terminal deoxynucleotidyl transferase and
fluorescein-dideoxyuridine triphosphate at 37 °C for 90 min in a humidified chamber, and the 3¡¯-OH ends of the DNA fragments were tailed
with fluorescein. The sections were then washed 3 times in PBS. After being incubated with anti-fluorescein isothiocyanate
horseradish peroxidase conjugate for 30 min at 37 °C, the slides were washed 3 times in PBS, developed with 0.05%
diaminobenzidine, and stained for 15 min at room temperature. The specimens were then washed 3 times in distilled water,
counterstained with Mayer hematoxylin solution for 10 min, dehydrated, mounted, and viewed with an Olympus microscope.
For evaluation of apoptosis, microscopic fields (×200) were selected at random. Apoptotic spermatogenic cells were
stained yellow. The percentage of apoptotic spermatogenic cells, the apoptosis index (AI), was determined by counting 100
cells from the spermatogenic epithelium.
Statistical analysis Data are presented as mean±SD. Autoradiographic analyses were repeated 3 times. The significance
of the differences between means was determined by using Student¡¯s
t-test, ANOVA and the Dunnett t-test. Statistical
significance was set at P<0.05, and the marked statistical significance was set at
P<0.01.
Results
Investigation of NF-κB protein levels To elucidate the mechanisms responsible for altered susceptibility of
spermatogenic cells to apoptosis, we investigated the
NF-κB protein levels in nuclear protein and cytoplasmic protein. There was no
significant difference (P>0.05) in NF-κB protein levels not only in nuclear protein
(t=1.405), but also in cytoplasmic protein
(t=1.294) between the spermatogenic epithelium of groups C and D; and likewise, there was no significant difference
(P>0.05) in NF-κB protein levels not only in nuclear protein
(t=1.615) but also in cytoplasmic protein
(t=1.144) between the spermatogenic epithelium of groups A and C. However, a markedly significant increase
(F=9.56, P<0.01) in NF-κB protein levels was
noted in nuclear protein in the spermatogenic epithelium of group B compared with groups A and C, but no significant
difference (F=1.92, P>0.05) in
NF-κB protein levels was noted in cytoplasmic protein in the spermatogenic epithelium of
group C compared with group D (Figure 1, Table 1).
We obtained NF-κB coefficients (C) according to the method previously described by Thiele
et al[11], using the equation C=NU/CY, where NU is the
NF-κB protein level in nuclear protein and CY is the
NF-κB protein level in cytoplasmic protein. There was no significant difference
(t=
0.995, P>0.05) in the NF-κB coefficient for the spermatogenic epithelium of groups C and D, and no significant difference
(t=1.182, P>0.05) in the NF-κB coefficient for spermatogenic epithelium of groups A and C. There was, however, a markedly
significant increase (F=10.27, P<0.01) in the
NF-κB coefficient for nuclear protein in the spermatogenic epithelium of group
B compared with groups A and C (Table 1).
Immunohistochemical staining for NF-κB
protein To elucidate the role of NF-κB protein in the apoptotic process, we
investigated its level of expression and cellular distribu-tion. We found that almost all
NF-κB protein was in the cytoplasm in groups A, C and D. The
NF-κB protein levels in the nuclear protein were much greater than those in the cytoplasmic protein
in group B (Figures 2 and 3). There was no significant difference
(t=0.834, P>0.05) in the proportion of cells with
NF-κB-positive nuclei in the spermatogenic epithelium of groups C and D, and there was no significant difference
(t=1.711, P>0.05) in the proportion of cells with
NF-κB-positive nuclei in the spermatogenic epithelium of groups A and C. There was, however,
a markedly significant increase (F=7.19,
P<0.01) in the proportion of cells with
NF-κB-positive nuclei in the spermatogenic epithelium of group B compared with groups A and C (Table 2). We found that cells with
NF-κB-positive nuclei were always spermatogonia or spermatocytes, and few Sertoli cells, Leydig cells, or endothelial cells changed.
Apoptosis of spermatogenic cells In order to evaluate the apoptosis of spermatogenic cells in the spermatogenic
epithelium, we analyzed the AI of spermatogenic cells by using the TUNEL technique. There was no significant
difference (t=0.962, P>0.05) in AI in the spermatogenic cells of groups C and D, and there was no significant difference
(t=
1.699, P>0.05) in AI in the spermatogenic cells of groups A and C. There was a markedly significant increase
(F=8.41,
P<0.01) in AI in the spermatogenic cells of group B compared with group A and C (Figures 4 and 5, Table 2). Apoptotic
spermatogenic cells were always spermatogonia or spermato-cytes, and not Sertoli cells, Leydig cells or endothelial cells.
Discussion
Testicular torsion results from an inadequate fixation of the testicular mesorchium, leading to hypermobility of the
testis[1,3,4,12]. The degree of tissue injury is directly proportional to the degree of testicular
ischemia[3,4,13]. Experimental testicular
torsion induces testicular ischemia during torsion. Relief of torsion allows reperfusion of the tissue. Testicular torsion causes
ischemia and reperfusion injury that results in spermatogenic cell loss. Loss of spermatogenic cells after
reperfusion is caused by spermatogenic-cell-specific apoptosis. The spermatogenic-cell-specific apoptosis ultimately
results in male infertility[3,14-16].
According to Weins and Lucchesi[17], reperfusion injury is induced by ROS, which arise either from activation of the
xanthine oxidase system in parenchymal cells or from leukocytes that first adhere to the reperfusing venule wall before
undergoing diapedesis into the tissue itself. Torsed testes torsion undergo ischemia and reperfusion injury that results
in increased adhesion molecule expression, leukocyte migration, and damage to the spermatogenic
epithelium[14-16]. Following reperfusion, ROS are released from ischemic tissues and produce local damage, resulting in an upregulation of endothelial cell
surface proteins. The increase in endothelial proteins and local vasodilation promotes the binding of neutrophils and
infiltration of the damaged
tissue[16,17]. The neutrophils promote necrosis and additional injury with the release of more ROS,
thereby propagating an ongoing cycle of reperfusion
injury[2,4,14,17].
Previous studies have demonstrated that NF-κB played a vital role in ischemia-reperfusion injury
events[18-21]. NF-κB is a transcription factor associated with immunomodula-tion of the immune response. In its quiescent state,
NF-κB is a heterodimer consisting of the p50 and p65 (Rel A) subunits that remain in the cytoplasm bound to a family of inhibitory proteins that are
collectively termed IkB. NF-κB is activated through phosphorylation and subsequent degradation of
IkB. Many agents, including ROS, interleukin-1 (IL-1), and tumor necrosis
factor-α (TNF-α) are sufficient to cause IkB
phosphorylation[22-25]. Once activated, the nuclear localization signal of
NF-κB is exposed and NF-κB is translocated into the nucleus.
NF-κB transcriptionally activates many genes involved in the
inflammatory process, including intercellular adhesion molecule 1
(ICAM-1), vascular cellular adhesion molecule 1 (VCAM-1), and E-selectin (ELAM-1), which are important mediators of the
inflammatory process in reperfusion
injury[25-29]. Apoptosis always occurs in the ischemic-reperfused tissue, and the
functions of the tissue never remain
unchanged[14-16]. Our study was designed to investigate the importance of
NF-κB in testis ischemia-reperfusion injury. We hypothesized that the increase in free radical production (in particular ROS) during testicular
torsion activated NF-κB as well as increased the host response, leading to increase in the apoptosis of spermatogenic cells.
Our data suggest the importance of NF-κB in testis ischemia-reperfusion injury, and further suggest the novel use of
sulfasalazine as an inhibitor of NF-κB following testes torsion. Sulfasalazine is currently used to treat inflammatory bowel
disease and rheumatoid arthritis[6,7]. However, despite being in common use for the past 50 years, its mechanism of action
remains undefined. Recently, sulfasalazine has been found to have numerous biological effects, including
immunosuppressive and modulatory actions on lymphocytes and leukocyte function. Sulfasalazine inhibits IL-2 synthesis and IL-1
production in lymphocytes[6,8,29]. Other studies have demonstrated that salicylates inhibited
IkB phosphorylation to block inflammatory
effects[6,8]. Wahl et
al[6] found that sulfasalazine acted as a potent inhibitor of
NF-κB by inhibiting IkB phosphorylation, thereby preventing its translocation into the nucleus and decreasing adhesion molecule expression. In addition, sulfasalazine
has been shown to act as a free radical
scavenger[7,29]. We compared the NF-κB coefficient and AI of the 4 groups, and found
that there was no significant difference in the
NF-κB coefficient or AI in the spermatogenic epithelium between groups C and
D, or between groups A and C, but a markedly significant increase in both was noted in the spermatogenic epithelium of group
B compared with groups A and C. This finding shows that administration of sulfasalazine when no testicular torsion has
occurred does not cause increases in the NF-κB coefficient or AI; however, when testicular torsion has occurred,
administration of sulfasalazine can prevent increases in the
NF-κB coefficient and AI. We also investigated the
NF-κB protein levels in the nuclear protein and cytoplasmic protein of the spermatogenic epithelium of the 4 groups, and found that when no
testicular torsion occurred, NF-κB protein was not translocated from the cytoplasm into the nucleus, and administration of
sulfasalazine did not cause this transloca-tion. However, when testicular torsion occurred,
NF-κB protein was activated; after torsion, NF-κB protein levels increased, particularly in the nuclear protein, then led to increases in the apoptosis of
spermatogenic cells. Administration of sulfasalazine can prevent the activation of
NF-κB protein, and then also prevent increases in AI. We also found that cells with
NF-κB-positive nuclei were always spermatogonia or spermatocytes, which were also
always the apoptotic cells. Conversely, the cells that were seldom activated, for example Sertoli cells, Leydig cells, and
endothelial cells, were seldom apoptotic. This finding demonstrates that
NF-κB activation is important in the apoptosis process. Treatment with sulfasalazine prior to detorsion decreases the AI close to the levels of the negative controls,
suggesting that sulfasalazine can protect against results that stimulate the
NF-κB pathway and decrease the concentration of ROS present in testicular torsion, which would also decrease adhesion molecule expression. In conclusion, we found that
NF-κB played a vital role in the ischemia-reperfusion immunology of testicular torsion and we discovered a novel use for the
anti-inflammatory agent sulfasalazine. Treatment with sulfasalazine was able to decrease reperfusion injury by preventing
increases in AI in the testicular torsion model. Sulfasalazine inhibits the activation of
NF-κB, and is an inexpensive and safe agent that could have an important role in the ischemia-reperfusion drug regimen to decrease episodes of apoptosis.
Sulfasalazine may be beneficial to decrease morbidity and infertility following testis torsion.
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