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

, Bahadir Suleyman

Renad Mammadov

, Bulent Yavuzer

, Betul Cicek

Durdu Altuner

, Taha A. Coban

and Halis Suleyman

Department of Pediatric Surgery, Faculty of Medicine, Erzincan Binali Yildirim

University, Erzincan, Turkey.

Department of Pharmacology, Faculty of Medicine, Erzincan Binali Yildirim University,

Erzincan, Turkey.

Department of Physiology, Faculty of Medicine, Erzincan Binali Yildirim University,

Erzincan, Turkey.

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

In disorders such as urinary retention, ath-

erosclerosis, vasospasm, embolization, and

thrombosis, the bladder cannot be supplied

adequately with blood, and ischemia de-

velops

. 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)

. It has

been reported that I/R procedure-induced

increase in ROSs production in the blad-

der leads to accelerated lipid peroxidation

(LPO)

. In addition, there are also studies

linking bladder I/R damage with an increase

in proinflammatory cytokines

. 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

. Anakinra is known as an

anti-inflammatory agent

. Since it blocks

both IL-1α and IL-1β receptors, it is used

in treating various inflammatory diseases

. 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

. In addition, it has been reported that

anakinra protects ovarian tissue from oxida-

tive and inflammatory damage of I/R

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

. Tocilizumab has

been approved for the pediatric treatment of

rheumatoid arthritis and polyarticular and

systemic juvenile idiopathic arthritis

. 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

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).