Invest Clin 64(3): 368 - 378, 2023 https://doi.org/10.54817/IC.v64n3a9
Corresponding author: Halis Suleyman, Department of Pharmacology, Faculty of Medicine, Erzincan Binali Yildirim
University, Erzincan, Turkey; Phone: +90 530 9211909; Fax: +90 446 2261819; E-mail: halis.suleyman@gmail.com
Effect of anakinra, tocilizumab, and the
combination thereof on bladder ischemia-
reperfusion damage in albino Wistar-type
rats.
Senol Bicer
1
, Bahadir Suleyman
2
,
Renad Mammadov
2
, Bulent Yavuzer
2
, Betul Cicek
3
,
Durdu Altuner
2
, Taha A. Coban
4
and Halis Suleyman
2
1
Department of Pediatric Surgery, Faculty of Medicine, Erzincan Binali Yildirim
University, Erzincan, Turkey.
2
Department of Pharmacology, Faculty of Medicine, Erzincan Binali Yildirim University,
Erzincan, Turkey.
3
Department of Physiology, Faculty of Medicine, Erzincan Binali Yildirim University,
Erzincan, Turkey.
4
Department of Medical Biochemistry, Faculty of Medicine, Erzincan Binali Yildirim
University, Erzincan, Turkey.
Keywords: anakinra; anakinra and tocilizumab combination; bladder; ischemia-reperfu-
sion damage; rats; tocilizumab.
Abstract. Several studies have reported that oxidative stress, and proinflam-
matory cytokines such as tumor necrosis factor-alpha (TNF-α), interleukin-one beta
(IL-1β), and interleukin-six (IL-6) are the main factors underlying bladder ischemia-
reperfusion (I/R) damage. Anakinra and tocilizumab are known to be antioxidants
and proinflammatory cytokine inhibitors. Our study aims to investigate if anakinra,
tocilizumab, and the combination (ATC) thereof have a protective effect against
oxidative and inflammatory bladder damage induced through the I/R procedure in
rats, and evaluate by comparing these compounds. Male rats were divided into five
groups: bladder sham-operation applied group (SG); bladder only I/R applied group
(IRG); anakinra+bladder I/R applied group (AIR); tocilizumab+bladder I/R applied
group (TIR); and ATC+bladder I/R applied group (ATIR). An atraumatic clamp was
placed on the abdominal aorta of animals in all groups (except SG), and one hour of
ischemia followed by two hours of reperfusion was performed. Our biochemical find-
ings showed that anakinra and tocilizumab significantly inhibited the increase of
oxidant malondialdehyde (MDA) and the decrease of antioxidants such as total glu-
tathione (tGSH), superoxide dismutase (SOD), and catalase (CAT) in bladder tissue
by I/R, both at the same levels. Furthermore, anakinra and tocilizumab significantly
suppressed the I/R-associated increase of TNF-α, IL-1β, and IL-6 in bladder tissue.
ATC was the one that best prevented the I/R-related increase in MDA, TNF-α, IL-1β,
and IL-6 and the decrease in tGSH, SOD, and CAT in the bladder tissue. ATC was
more beneficial than anakinra or tocilizumab alone in treating bladder I/R damage.
Effect of anakinra, tocilizumab, and their combination on bladder damage 369
Vol. 64(3): 368 - 378, 2023
Efecto de la anakinra, el tocilizumab y la combinación de
ambos sobre el daño por isquemia-reperfusión vesical en ratas
albinas tipo Wistar.
Invest Clin 2023; 64 (3): 368 – 378
Palabras clave: anakinra; combinación de anakinra y tocilizumab; vejiga; daño por
isquemia-reperfusión; ratas; tocilizumab.
Resumen. Varios estudios han demostrado que el estrés oxidativo, y las ci-
tocinas proinflamatorias tales como el factor de necrosis tumoral alfa (TNF-α),
la interleucina uno beta (IL-1β) y la interleucina seis (IL-6) son los principales
factores subyacentes al daño por isquemia-reperfusión (I/R) vesical. Se sabe
que la anakinra y el tocilizumab son antioxidante e inhibidores de las citocina
proinflamatorias. Nuestro estudio pretende investigar si la anakinra, el tocili-
zumab y la combinación (ATC) de ambos tienen un efecto protector contra el
daño oxidativo e inflamatorio de la vejiga inducido mediante el procedimiento
de I/R en ratas, y evaluarlo mediante la comparación de estos compuestos. Se
dividieron a ratas macho en cinco grupos: un grupo sometido a la operación
simulada (SG); un grupo al cual solo se aplicó I/R a la vejiga (IRG); un grupo
al cual se aplica el procedimiento I/R a la vejiga + anakinra (AIR); un grupo
en el que se aplica el procedimiento I/R a la vejiga + tocilizumab (TIR); y un
grupo en el que se aplica el procedimiento I/R a la vejiga + ATC (ATIR). Se
colocó una pinza atraumática en la aorta abdominal de los animales de todos
los grupos (excepto SG), y se realizó una hora de isquemia seguida de dos horas
de reperfusión. Nuestros hallazgos bioquímicos mostraron que la anakinra y el
tocilizumab inhibieron significativamente el aumento del malondialdehído oxi-
dante (MDA) y la disminución de antioxidantes como el glutatión total (tGSH),
la superóxido dismutasa (SOD) y la catalasa (CAT) en el tejido de la vejiga por
I/R, ambos a los mismos niveles. Además, la anakinra y el tocilizumab suprimie-
ron significativamente el aumento de TNF-α, IL-1β e IL-6 asociado a la I/R en
el tejido vesical. La combinación ATC fue la que mejor previno el aumento de
MDA, TNF-α, IL-1β e IL-6 relacionado con la I/R y la disminución de tGSH, SOD
y CAT en el tejido vesical. La ATC resultó más beneficiosa que la anakinra o el
tocilizumab solos en el tratamiento del daño por I/R de la vejiga.
Received: 17-04-2023 Accepted: 06-07-2023
INTRODUCTION
As known, the bladder’s function is to
store urine and empty it at an appropriate
time. An adequate amount of blood flow,
oxygen, and nutrient support is needed to
maintain this function at a normal level
1
.
In disorders such as urinary retention, ath-
erosclerosis, vasospasm, embolization, and
thrombosis, the bladder cannot be supplied
adequately with blood, and ischemia de-
velops
2
. Clinical and experimental studies
have shown that reperfusion contributes to
the impairment of bladder function in case
of re-blooding of the ischemic bladder
3,4
.
It has been reported in the literature that
370 Bicer et al.
Investigación Clínica 64(3): 2023
the factors underlying bladder I/R damage
are reactive oxygen species (ROSs)
5
. It has
been reported that I/R procedure-induced
increase in ROSs production in the blad-
der leads to accelerated lipid peroxidation
(LPO)
6
. In addition, there are also studies
linking bladder I/R damage with an increase
in proinflammatory cytokines
5
. In particu-
lar, proinflammatory cytokines such as inter-
leukin one beta (IL-1β) and interleukin six
(IL-6) are thought to be the main factors in
the pathogenesis of bladder I/R damage
7,8
.
This information obtained from the litera-
ture suggests that IL-1β and IL-6 cytokine
antagonists with antioxidant effects may be
helpful in the treatment of bladder I/R.
Anakinra, which we will investigate its
effect against bladder I/R damage in our
study, is a recombinant antagonist of the
IL-1β receptor
9
. Anakinra is known as an
anti-inflammatory agent
10
. Since it blocks
both IL-1α and IL-1β receptors, it is used
in treating various inflammatory diseases
11
. Anakinra has been shown to protect tes-
ticular tissue from I/R damage by inhibit-
ing the increased production of IL-1β and
malondialdehyde (MDA), a toxic product of
LPO
12
. In addition, it has been reported that
anakinra protects ovarian tissue from oxida-
tive and inflammatory damage of I/R
13
.
Tocilizumab, which we plan to investi-
gate its effect against bladder I/R damage,
is a monoclonal antibody drug that is an
IL-6 receptor antagonist
14
. Tocilizumab has
been approved for the pediatric treatment of
rheumatoid arthritis and polyarticular and
systemic juvenile idiopathic arthritis
15
. Er-
dem KTO et al. reported that tocilizumab
protects kidney tissue from inflammatory
and oxidative damage of I/R by inhibiting
the increase of IL-6 and other cytokines
16
.
All this information obtained from the liter-
ature shows that anakinra and tocilizumab
may be effective in treating bladder I/R dam-
age. In particular, it suggests that the com-
bination of anakinra and tocilizumab (ATC)
may be more effective in the treatment of
bladder I/R damage. There was no infor-
mation in the literature investigating the
effects of anakinra, tocilizumab, and ATC
against bladder I/R damage. Therefore, our
study aims to investigate whether anakinra,
tocilizumab, and ATC have a protective effect
against I/R-induced oxidative and inflamma-
tory bladder damage in rats, and evaluate by
comparing both compounds.
MATERIALS AND METHODS
Animals
A total of 30 albino Wistar-type male rats
weighing between 270-290 g were utilized in
the experiment. Erzincan Binali Yildirim Uni-
versity Experimental Animals Application and
Research Center provided all animals. The an-
imals were fed with animal food in groups at
average room temperature (22°C) and hosted
in 12 hours of light, and 12 hours of darkness
environment, under appropriate conditions
before the experiment. The experiments were
conducted following the Turkey Regulation
of Animal Research Ethics. In addition, this
study was carried out under the principles
of the Declaration of Helsinki. The protocols
and procedures were approved by the local
Animal Experimentation Ethics Committee
of Erzincan Binali Yildirim University (Meet-
ing Date: 26.01.2023; Meeting No: 2023/01;
Decision No: 01).
Chemicals
A Pfizer Turkey representative provid-
ed ketamine used in this experiment while
anakinra was obtained from Sobi-Sweden,
and a Roche Mustahzarları Turkey represen-
tative provided tocilizumab (80 mg/4 mL
concentrated solution for infusion).
Experimental Groups
Animals were divided into five groups:
sham-operation applied group (SG);
bladder only I/R applied group (IRG);
anakinra+bladder I/R applied group (AIR);
tocilizumab+bladder I/R applied group
(TIR); and ATC+bladder I/R applied group
(ATIR).
Effect of anakinra, tocilizumab, and their combination on bladder damage 371
Vol. 64(3): 368 - 378, 2023
Anesthesia procedure
Surgical procedures were carried out
under sterile conditions by intraperitoneal
(IP) ketamine administration at a dose of 60
mg/kg. After the ketamine injection, the rats
were kept waiting for the appearance of the
appropriate anesthesia period during which
the surgical procedure would be performed.
When the animals remained immobile in the
supine position was considered a suitable an-
esthesia period for surgical intervention
17
.
Experimental procedure
One hour before anesthesia, anakinra
was injected IP at a dose of 50 mg/kg in the
AIR group of animals (n=6), and tocilizumab
at a dose of 8 mg/kg in the TIR group (n=6).
The ATIR (n=6) group of animals was admin-
istered 50 mg/kg anakinra + 8 mg/kg to-
cilizumab IP. The SG (n=6), and IRG (n=6)
groups of animals were given distilled water
as a solvent by the same route. One hour fol-
lowing the administration of drugs and dis-
tilled water, laparotomy was carried out with
a 2.5 cm long midline incision applied to the
abdomen of the rats under sterile conditions.
Thereupon, an atraumatic clamp was placed
on the abdominal aorta of the animals in all
groups (except the SG group), and ischemia
was induced for one hour, followed by two
hours of reperfusion. At the end of this pe-
riod, all animal groups were sacrificed with
high doses (120 mg/kg) of ketamine anes-
thesia. Bladder tissues were removed from
the sacrificed animals and were analyzed bio-
chemically. The biochemical experimental re-
sults obtained from all groups were evaluated
by comparing the groups.
Biochemical analysis
Preparation of samples
Tissue samples were washed with physi-
ological saline and placed in Petri dishes.
The tissues were pulverized by grinding in
the presence of liquid nitrogen. In addition,
tissue samples were homogenized. The su-
pernatants were used for MDA, tGSH, SOD,
CAT, and protein analyses.
Determination of MDA, tGSH, SOD, CAT,
and protein
Determination of MDA, tGSH, and SOD
in bladder tissues was carried out by en-
zyme-linked immunosorbent assay (ELISA)
kits which are produced for experimental
animals, and each analysis was performed
according to the kit instructions (MDA:
Catalog no. 10009055; tGSH: Catalog no.
703002; SOD: Catalog no. 706002; Cayman
Chemical Company). CAT determination
was performed in line with the method pro-
posed by Goth
18
. Protein determination was
determined spectrophotometrically at 595
nm following the Bradford method
19
.
TNF-α, IL-1β, and IL-6 analysis
We measured the weight of the sam-
ples. After that, all the tissues were cut, rap-
idly frozen with liquid nitrogen, and homog-
enized by pestle and mortar; we maintained
samples at 2-8°C after melting. We added
PBS (pH 7.4), 1/10 (w/v), and then vortex
for 10 seconds, centrifuged 20 min at 10000
x g, and collected the supernatants carefully.
Tumor necrosis factor alpha (TNF-α; ng/L)
was assayed using a TNF-α ELISA rat kit (Cat
no: YHB1098Ra, Shanghai LZ, Shanghai,
China), interleukin one beta (IL-1β; pg/L)
was assayed using an IL-1β ELISA rat kit
(Cat no: YHB0616Ra, Shanghai LZ, Shang-
hai, China), and interleukin-6 (IL-6; ng/L)
was assayed using a commercial kit supplied
by Eastbiopharm Co Ltd (Hangzhou, China).
Statistical analysis
The experiment results were expressed
as means ± standard errors of the mean
(Mean±SEM). The statistical analyses were
performed with the SPSS Statistics Program
for Windows (IBM Corp., released 2013, Ver-
sion 22.0, Armonk, NY, USA). The normality
of the distribution for continuous variables
in the biochemical test results was checked
by the Shapiro‒Wilk test. The significance of
differences between groups was determined
using one-way ANOVA, as the distribution
was normal. Levene’s test was performed to
372 Bicer et al.
Investigación Clínica 64(3): 2023
determine whether the homogeneity of vari-
ances was achieved. Afterward, Tukey HSD
(honestly significant difference) or Games-
Howell was applied as a posthoc test depend-
ing on the assumption whether the variances
were homogeneous or not. The probability
value of p<0.05 was regarded to indicate
statistical significance.
RESULTS
MDA analysis results of bladder tissue
As shown in Fig. 1A, MDA levels in the
bladder tissue of the I/R procedure-applied
group were higher than in the bladder tissue
of the sham operation-applied group. The
difference in the levels of MDA in the bladder
tissue of the sham-operation-applied group
and the I/R procedure-applied group was
statistically significant (p<0.001). Anakin-
ra (p=0.007), tocilizumab (p=0.003), and
ATC (p<0.001) significantly suppressed
the increase in MDA levels induced by the
I/R procedure in bladder tissue. Anakinra
(p=0.002) and tocilizumab (p=0.001) were
found to approximate the MDA levels in the
bladder tissue to the control group values.
The closest MDA value to the control group
that underwent sham operation was found in
the ATIR group (p=0.014).
tGSH analysis results of bladder tissue
The tGSH levels in the bladder tissue of
the I/R procedure-applied group were found
to be lower than that in the bladder tissue
of the sham operation-applied group (Fig.
1B). The difference in the levels of tGSH in
the bladder tissue of the sham-operation-
applied group and the I/R procedure-applied
group was statistically significant (p<0.001).
Anakinra (p=0.004), tocilizumab (p=0.003),
and ATC (p<0.001) significantly suppressed
the decrease in tGSH levels induced by the
I/R procedure in bladder tissue. A statistically
significant difference was found in the tGSH
levels in the bladder tissue of the anakinra
(p<0.001) and tocilizumab (p<0.001)-treat-
ed groups compared to the values of the con-
trol group that underwent sham operation.
The closest tGSH value to the control group
that underwent sham operation was found in
the ATIR group (p=0.015).
SOD analysis results of bladder tissue
As shown in Fig. 1C, SOD activity in the
bladder tissue of the I/R procedure-applied
group was lower than that in the bladder
tissue of the sham operation-applied group.
The difference in activity of the SOD in the
bladder tissue of the sham operation-applied
group and the I/R procedure-applied group
was statistically significant (p<0.001).
Anakinra (p=0.012), tocilizumab (p=0.011),
and ATC (p<0.001) significantly suppressed
the decrease in SOD activity caused by the
I/R procedure in bladder tissue. A statistical-
ly significant difference was found in the SOD
activities in the bladder tissue of the anakinra
(p<0.001) and tocilizumab (p<0.001) ap-
plied groups compared to the control group
values that underwent sham operation. The
closest SOD activity to the control group that
underwent sham operation was found in the
ATIR group (p=0.043).
CAT analysis results of bladder tissue
The CAT activity in the I/R procedure
applied group’s bladder tissue was lower than
that in the bladder tissue of the sham opera-
tion applied group (Fig. 1D). The difference
in activity of the CAT in the bladder tissue
of the sham operation-applied group and the
I/R procedure-applied group was statistically
significant (p<0.001). Anakinra (p=0.009),
tocilizumab (p=0.002), and ATC (p<0.001)
significantly suppressed the decrease in CAT
activity in bladder tissue caused by the I/R
procedure. A statistically significant differ-
ence was found in the CAT activities in the
bladder tissue of the anakinra (p<0.001)
and tocilizumab (p<0.001) applied groups
compared to the control group values that
underwent sham operation. The closest CAT
activity to the control group that underwent
sham operation was found in the ATIR group
(p=0.015).
Effect of anakinra, tocilizumab, and their combination on bladder damage 373
Vol. 64(3): 368 - 378, 2023
TNF-α analysis results of bladder tissue
As shown in Fig. 2A, TNF-α levels in the
bladder tissue of the I/R procedure applied
group were higher than in the bladder tissue
of the sham operation applied group. The dif-
ference in the levels of TNF-α in the bladder
tissue of the sham operation-applied group
and the I/R procedure-applied group was
statistically significant (p<0.001). Anakinra
(p=0.002), tocilizumab (p=0.002), and ATC
(p<0.001) significantly suppressed the in-
crease in TNF-α levels caused by the I/R pro-
cedure in bladder tissue. Anakinra (p=0.003)
and tocilizumab (p=0.003) were found to
approximate TNF-α levels in the bladder tis-
sue to the control group values. There was no
significant difference in TNF-α levels between
the control group that underwent the sham
operation and the group that applied ATC
(p=0.140).
Fig. 1. Oxidative stress in bladder tissues of experimental groups. A) Malondialdehyde (MDA) levels: Bars are
means ± SEM.
a
means
p<0.05 when all groups were compared with the SG group.
b
means
p<0.05
when the other drug treatment groups were compared with the IRG group.
c
means
p<0.05 when drug
treatment groups were alone compared with the ATIR combined treatment group. B)
Total glutathio-
ne (tGSH) levels: Bars are mean ± SEM.
a
means
p<0.05 when all groups were compared with the SG
group.
b
means
p<0.05 when the other drug treatment groups were compared with the IRG group.
c
means
p<0.001 when drug treatment groups were alone compared with the ATIR combined treatment
group. C) Superoxide dismutase (SOD) levels: Bars are mean ± SEM.
a
means
p<0.05 when all groups
were compared with the SG group.
b
means
p<0.05 when the other drug treatment groups were com-
pared with the IRG group.
c
means
p<0.05 when drug treatment groups were alone compared with the
ATIR combined treatment group. n=6 per group. D) Catalase (CAT) levels: Bars are mean ± SEM.
a
means
p<0.05 when all groups were compared with the SG group.
b
means
p<0.05 when the other drug
treatment groups were compared with the IRG group.
c
means
p<0.05 when drug treatment groups
were alone compared with the ATIR combined treatment group. Group. n=6 per group. SG: sham-
operation group; IRG: ischemia-reperfusion group; AIR: anakinra+ischemia-reperfusion group; TIR:
tocilizumab+ischemia-reperfusion group; ATIR: anakinra+tocilizumab+ischemia-reperfusion group.
374 Bicer et al.
Investigación Clínica 64(3): 2023
IL-1β analysis results of bladder tissue
IL-1β level in the bladder tissue of the
I/R procedure applied group was found to be
higher than the bladder tissue of the sham
operation applied group (Fig. 2B). The dif-
ference in the levels of IL-1β in the bladder
tissue of the sham operation-applied group
and the I/R procedure-applied group was
statistically significant (p<0.001). Anakin-
ra (p=0.002), tocilizumab (p=0.003), and
ATC (p<0.001) significantly suppressed
the increase in IL-1β levels caused by the
I/R procedure in bladder tissue. Anakinra
(p=0.001) and tocilizumab (p<0.001) were
found to approximate IL-1β levels in the
bladder tissue to the control group values.
The closest IL-1β value to the control group
that underwent sham operation was found in
the ATIR group (p=0.028).
IL-6 analysis results of bladder tissue
As shown in Fig. 2C, IL-6 levels in the
bladder tissue of the I/R procedure applied
group were higher than in the bladder tissue
of the sham operation applied group. The
difference in the levels of IL-6 in the bladder
tissue of the sham operation-applied group
and the I/R procedure-applied group was
statistically significant (p<0.001). Anakin-
ra (p=0.002), tocilizumab (p=0.001), and
ATC (p<0.001) significantly suppressed the
increase in IL-6 levels caused by I/R in blad-
der tissue. A significant difference was found
between the IL-6 levels in the bladder tissue
Fig. 2. Cytokine expressions in bladder tissues of experimental groups. A) Tumor necrosis factor-alpha
(TNF-α) levels: Bars are mean ± SEM.
a
means
p<0.05 when all groups were compared with the SG
group.
b
means
p<0.05 when the other drug treatment groups were compared with the IRG group.
c
means
p<0.05 when drug treatment groups were alone compared with the ATIR combined treatment
group. B) Interleukin- 1 beta (IL-1β) levels: Bars are mean ± SEM.
a
means
p<0.05 when all groups
were compared with the SG group.
b
means
p<0.05 when the other drug treatment groups were
compared with the IRG group.
c
means
p<0.05 when drug treatment groups were alone compared
with the ATIR combined treatment group. C) Interleukin- 6 (IL-6) levels: Bars are mean ± SEM.
a
means
p<0.001 when all groups were compared with the SG group.
b
means
p<0.05 when the other
drug treatment groups were compared with the IRG group.
c
means
p<0.05 when drug treatment
groups were alone compared with the ATIR combined treatment group. n=6 per group. SG: sham-
operation group; IRG: ischemia-reperfusion group; AIR: anakinra+ischemia-reperfusion group; TIR:
tocilizumab+ischemia-reperfusion group; ATIR: anakinra+tocilizumab+ischemia-reperfusion group.
Effect of anakinra, tocilizumab, and their combination on bladder damage 375
Vol. 64(3): 368 - 378, 2023
of the anakinra-applied group and the con-
trol group values (p<0.001). The difference
between the IL-6 levels in the bladder tissue
of the tocilizumab-applied group and the
control group was statistically insignificant
(p=0.102). The closest IL-6 value to the
control group who underwent sham opera-
tion was found in the ATIR group (p=0.986).
DISCUSSION
This study investigated the effects
of anakinra, tocilizumab, and ATC on ex-
perimentally induced bladder I/R injury in
rats. It has been reported in the literature
that during the bladder I/R process, the
increased production of ROSs disrupts the
antioxidant balance and ultimately leads to
oxidative stress
5,6
. Oxidative stress is known
to first affect lipids in the cell membrane.
When ROSs induce the cell membrane’s
LPO reaction, MDA emerges as an end prod-
uct
6
. MDA resulting from LPO is also itself
toxic and may cause further destruction by
disrupting the structure and functions of
the membrane
20
. This series of events oc-
curring in bladder cells is known to be one
of the most accused factors in the formation
of bladder damage
5
. Therefore, MDA, a toxic
product of LPO and an essential indicator of
oxidative stress, was measured in our study.
It has been reported that the MDA level is
significantly increased in bladder I/R dam-
age models, and this increase in MDA level is
consistent with a significant decrease in the
contractile ability of the bladder
5,21,22
. The
fact that the MDA level was high in the blad-
der I/R group in our study findings shows
that our experimental results coincide with
the literature information.
However, our experimental I/R model
found that anakinra, tocilizumab, and ATC
significantly suppressed the increase in MDA
level caused by the I/R procedure. At the
same time, it was detected that anakinra
and tocilizumab showed a synergistic effect
together, reducing the severity of the LPO
reaction at the highest level and bringing
MDA levels closer to the values of the con-
trol group. There was no information in the
literature showing the effect of anakinra and
tocilizumab on bladder I/R damage. Howev-
er, previous studies reported that anakinra
significantly suppressed the increase of MDA
level in intestinal tissue and tocilizumab in
renal tissue by I/R and showed a protective
effect
16,23
.
It is known from the literature that the
impaired redox balance is closely associated
with the I/R event
5,24
. As such, our study
investigated the effects of anakinra, tocili-
zumab, and ATC on tGSH, SOD, and CAT
levels in the bladder tissue of rats applied
with the I/R procedure. The cited parame-
ters are significant indicators of antioxidant
capacity and are known to protect tissues
against oxidative stress
25
. Our results show
that a significant decrease was detected in
tGSH, SOD, and CAT levels in parallel with
the increasing MDA concentration following
the I/R procedure. Our study’s stated results
are similar to previous studies showing a de-
crease in antioxidant enzymes following an
increase in LPO in experimentally created
bladder I/R
24,26,27
. ATC, whose effect we in-
vestigated on oxidative damage, prevented
the reduction of tGSH, SOD, and CAT in
I/R-induced bladder tissue more significant-
ly than anakinra and tocilizumab adminis-
tered alone. Even though there are no stud-
ies in the literature examining the effects of
anakinra and tocilizumab on antioxidant en-
zyme levels in rats with formed experimental
bladder I/R, it has been reported that they
significantly suppress the decrease in anti-
oxidant enzyme levels in intestinal and re-
nal I/R damage, respectively
16,23
. Our find-
ings indicate that anakinra and tocilizumab
support antioxidant defense mechanisms by
showing additive, synergistic effects in blad-
der tissue.
Many experimental studies have shown
that ROSs increase proinflammatory cyto-
kine production in bladder I/R-damaged
cells
7,8
. TNF-α, IL-1β, and IL-6 are the most
emphasized cytokines in bladder inflamma-
376 Bicer et al.
Investigación Clínica 64(3): 2023
tory response
5,7,28
. As can be understood
from our findings, it was detected that there
was a significant increase in TNF-α, IL-1β,
and IL-6 levels in the bladder tissue of rats
after the I/R procedure compared to the
control group. Our findings correspond with
previous studies such as Shin et al., Kanno et
al., and Altunkaynak et al.
5,7,28
. In our study,
we found that anakinra and tocilizumab
alone and ATC significantly inhibited the in-
crease of TNF-α, IL-1β, and IL-6 in bladder
tissue, and this effect was more significant
in the ATC group. In the available litera-
ture, we found no information on the effect
of anakinra and tocilizumab on the inflam-
mation in I/R-induced bladder tissue. How-
ever, Butler et al. reported that the anakinra
suppressed inflammation by regulating neu-
ropeptide levels in an experimental cystitis
model and protected the bladder by reduc-
ing the number of bacteria and neutrophils
in the urine
29
. However, in the literature,
anakinra and tocilizumab have significantly
inhibited inflammation caused by testicular,
ovarian, and renal I/R damage
12,13,16
.
In conclusion, the I/R procedure has led
to oxidative stress and inflammation in blad-
der tissue. Anakinra and tocilizumab alone
suppressed I/R-induced oxidative and inflam-
matory bladder damage to almost the same
extent. ATC was the best suppressor of I/R-
induced bladder oxidative and inflammatory
damage. This effect appeared due to anakinra
and tocilizumab’s additive, synergistic effect.
Our results suggest that ATC may be more
useful than anakinra and tocilizumab alone in
treating bladder I/R damage. We think that
histopathologic studies may be helpful in the
detailed elucidation of this issue.
ACKNOWLEDGMENTS
The authors do not intend to thank any
person or institution for this study.
Funding
None
Conflict of interest
None
Authors ORCID
• Senol Bicer (SB):
0000-0002-6380-4861
• Bahadir Suleyman (BS):
0000-0001-5795-3177
• Renad Mammadov (RM):
0000-0002-5785-1960
• Bulent Yavuzer (BY):
0000-0001-7576-0678
• Betul Cicek (BC):
0000-0003-1395-1326
• Durdu Altuner (DA):
0000-0002-5756-3459
• Taha Abdulkadir Coban (TAC):
0000-0003-1711-5499
• Halis Suleyman (HS):
0000-0002-9239-4099
Authors’ participation
Concept and design of the study: SB,
BS, DA and HS. Acquisition of data: BS,
RM, BY, BC and TAC. Data analysis and in-
terpretation: SB, BS, RM, BC, DA, TAC and
HS. Writing the manuscript: SB, DA, TAC
and HS. Critical revision of the manuscript:
RM, BY, BC, DA and HS. Approval of the final
version: SB, BS, RM, BY, BC, DA, TAC, and
HS. Statistical advice: BY and DA. Ethical
or administrative advice: HS.
All authors of this paper have read and
approved the final version of the submitted
manuscript.
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