Adalimumab mitigates ovarian ischemia–reperfusion injury in rats by regulating oxidative stress, apoptosis and resolution of inflammation

Aim: Ovarian torsion is a rare but an important reason of acute lower abdominal pain in women and associ- ated with serious morbidity and mortality, if not treated promptly. The aim of this study was to evaluate the effects of an antitumor necrosis factor- α antibody on ovarian torsion in a rat model of ischemia – reperfusion (I/R) injury.

Forty female Wistar Albino rats were used in the present study. The rats were randomly divided into four groups: group I (sham), group II (I/R), group III (I/R + isotonic saline) and group IV (I/R + adalimumab). The I/R model was induced by torsion of both ovaries. Immunohistochemical staining for interleukin-1 β (IL-1β), nuclear factor- κB (NF- κB), and inducible nitric oxide synthase was performed. Tis- sue and serum oxidative stress markers in conjunction with apoptotic index (AI) with the terminal deoxynu- cleotidyl transferase dUTP nick end labeling method were also calculated.

Tissue total oxidant status, oxidative stress index and nitric oxide values were signi ficantly decreased, and tissue total antioxidant status was found to be increased in group IV. In flammation, vascular congestion and hemorrhagia were signi ficantly lower in adalimumab-treated group. Serum oxidative stress markers and tissue malondialdehyde levels did not differ in study groups. The AI was signi ficantly increased in groups 2 and 3. Adalimumab treatment signi ficantly decreased the AI.

Adalimumab therapy in rats attenuated I/R induced ovarian injury, possibly suppressing inflammation, inhibiting oxidative stress, and altering apoptotic pathways. Key words: adalimumab, apoptosis, immunohistochemistry, ovarian torsion, oxidative stress.

The twisting or torsion of the ovary is a relatively common gynecological emergency especially in 1st three decades of life, which denotes the bending of the ovary and fallopian tube around the broad liga- ment. 1 Clinical presentation is sometimes confounded by non-speci fic symptoms such as abdominal pain, nausea and vomiting that leads to late or missed diag- nosis. 2 In order to prevent potential necrosis, infertility and life-threatening sequels of this entity, early diagnosis and prompt institution of adequate treatment may be life-saving. For this reason, in patients with suspected ovarian torsion, gynecologic consultation with subsequent surgical procedures are critical, irrespective of whether laboratory examina- tions and radiologic imaging yield normal results. Surgical intervention, either laparoscopic or laparot- omy commonly re-establish blood circulation never- theless, tissue atrophy or necrosis, may still occur and Received: May 16 2018. Accepted: October 2 2018. Correspondence: Dr Fatma Beyazit, Department of Obstetrics and Gynecology, Canakkale Onsekiz Mart University Medical Faculty, Sevim Buluç Street, Canakkale 17020, Turkey. Email: [email protected] 358 © 2018 Japan Society of Obstetrics and Gynecology doi:10.1111/jog.13846 J. Obstet. Gynaecol. Res. Vol. 45, No. 2: 358 –367, February 2019 is a common consequence. 3,4 Thus, it is important to develop alternative nonsurgical and speci fic therapeu- tic strategies against ovarian torsion in order to achieve short- and long-term preservation of ovarian function at a cellular level including protection from tissue ischemia caused by reperfusion damage from free radicals that are released after detorsion. Tumor necrosis factor- α (TNF-α) is one of the main mediators of in flammation and has been recognized as a target of therapeutic intervention in many in flam- matory disease states. 5 Clinical data advocate that TNF-α is released at the beginning of reperfusion, and its level increases during the early stages of ischemia and reperfusion (I/R). 6 Being a cell signaling circulat- ing protein, TNF- α is mainly involved in many harm- ful biological processes and diseases in the body during the course of I/R. 7,8 Adalimumab (ADA) is a recombinant human IgG1 monoclonal antibody spe- cific for human TNF- α and is traditionally used to treat psoriasis, psoriatic arthritis, and several other inflammatory conditions such as rheumatoid arthritis and in flammatory bowel diseases. The inhibition of TNF-α with ADA has been shown to decrease the severity of liver and intestinal I/R injuries in several animal models. 9,10 However, there have been no stud- ies of the protective effects of ADA against ovarian I/R injury. In this study, we aimed to evaluate the therapeutic effect of ADA on ovarian injury that is induced by I/R in an experimental rat model. Furthermore, we aimed to shed light on the possible mechanisms by which ADA could protect rat against I/R induced ovarian injury.

Materials and experimental design This experimental protocol was approved by the Insti- tutional Animal Use and Care Committee of Çanak- kale Onsekiz Mart University (COMU) (Approval No: 2017/06–15) and performed in accordance with the Helsinki Declaration of World Medical Association recommendations on animal studies. A total of 40 Wis- tar albino adult female rats were obtained from COMU Experimental Research Application and Research Center with a mean age of 4 months and a mean weight of 250 –300 g. The rats were housed in stainless steel cages in an animal room maintained at a standard humidity (50 –55%) and temperature 22 /C62 /C14C with 12-h light/dark cycles. All animals were fed standard food and water. Twelve hours before the study procedure feeding was stopped and the rats were only allowed to drink water. The entire experiment was conducted under half-sterile conditions. Experimental protocol Anesthesia Rats were anesthetized with intraperitoneal (i.p.) keta- mine hydrochloride (75 mg/kg) and xylazine (5–10 mg/kg), and if required anesthesia was main- tained with additional injections of ketamine hydrochloride. Ovarian I/R The ovarian I/R model was designed in a way paral- lel to previous trials. 11 In brief, the rats were supine positioned on a heated platform. The skin was pre- pared under aseptic conditions, and an approximately 2.5-cm longitudinal incision was performed in the midline area of the lower abdomen after shaving of abdominal area. Afterward peritoneal incision was done in order to locate the uterine horns and adnexa. After finding the adnexes, both ovaries were twisted and rotated 720 /C14clockwise and fixed to the abdomi- nal wall with a non-absorbable suture. Then abdomen wall was closed with continuous 1/0 silk suture in one layer. After 3 h, the fixed ovaries were freed by cutting the suture and both ovaries were detorsioned with a relaparotomy procedure and the abdomen was surgically closed again. Groups Wistar albino rats were randomly divided into four groups as follows: Group I (sham, n = 10): After sham operation at zero and 3-h point, both ovaries were surgically removed for assessment at 6-h point. Group II (torsion-detorsion group, n = 10): After torsion (at zero-point) and detorsion (at 3-h point), both ovaries surgically removed at the end of the study (6-h point). Group III (torsion-detorsion + isotonic saline group, n = 10): Rats underwent torsion and detorsion operation. Twenty minutes prior to the detorsion, 0.3-mL i.p. isotonic saline was applied. Both ovaries removed at the end of the study (6-h point). Group IV (torsion-detorsion + ADA group, n = 10): Rats underwent torsion and detorsion opera- tion. Twenty minutes prior to detorsion, 50-mg/kg ADA (Humira; Abbott Laboratories) applied 359© 2018 Japan Society of Obstetrics and Gynecology Adalimumab in ovarian torsion intraperitoneally. Both ovaries removed at the end of the study (6-h point). Evaluation Serum and tissue samples were used for evaluation purposes. Blood samples were collected at the begin- ning and at the end of the study (at 6-h point). In order to evaluate tissue samples, both of the ovaries were removed in all subjects. One of the ovaries was used for biochemical analysis, while the other was used for histopathological evaluation. Histopathological examinations In order to investigate histopathologic changes, ovar- ian tissue samples were consecutively numbered and fixated in 10% neutral buffered formalin for 48 h, dehydrated, cleared in xylene and embedded in paraf- fin and sent to the histology department of COMU. Evaluation of the pathology specimens was done by a histology specialist, who was blind to the four study groups. The paraf fin blocks were cut in 5- μm thick- ness on Rotary Microtome (Leica RM2125 RTS), and the sections were stained with hematoxylin and eosin (H&E). The histopathologic sections were evaluated under a light microscope (Zeiss AxioScope A1) for the presence of hemorrhagia, vascular congestion, edema and in flammation (neutrophil in filtration) and rated on a modi fied semiquantitative scale of 0 –3 as also stated by Bozkurt et al . 12 Scoring scale was as follow; 0, no findings; 1, findings of 66%. Immunohistochemical staining Immunohistochemistry for IL-1 β (Cat #12242S, Cell Signaling Technology), NF- κB (Cat #RB-9034, Thermo Scientific, Lab vision) and inducible nitric oxide synthase (iNOS) (Cat #RB-9242, Thermo Scienti fic) was carried out with commercial kits according to the manufacturers’ instructions. Immunohistochemical (IHC) evaluation and scoring were done according to Jiang et al .13 According to this scoring, IL-1 β, NF- κB, and iNOS immunostaining scores were calculated by staining intensity (0, no staining; 1, weak but detectable staining; 2, moderate; and 3, strong staining). Apoptosis assessment with the TUNEL method Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was used to detect apopto- sis of the ovarian tissue. Prepared tissue was fixed with 4% neutral formaldehyde, embedded in paraf fin, and 4- μm-thick sections were cut from each paraf fin block. After dewaxing, hydration, and serum block- ing, ApopTag Peroxidase in situ Apoptosis Detection Kit (S7100, Millipore) was used according to the man- ufacturer’s protocol. The cells were observed and photographed under an optical microscope, and five high image fields of each sample were randomly selected, and 500 cells were counted in each field. Cells stained brown or black were judged TUNEL- positive apoptotic cells and presented in the form of apoptotic index (AI). Biochemical evaluation Ovarian tissue and blood samples were obtained from each animal at the end of the study. Blood samples were centrifuged at 3000 rpm for 15 min. The resultant serum samples were aliquoted into polypropylene tubes and stored at −80 /C14C until the biochemical analy- sis. The tissues were prepared at +4 /C14C in order to mea- sure malondialdehyde (MDA), nitric oxide (NO), total antioxidant status (TAS) and total oxidant status (TOS) levels. After washing with phosphate buffer solution (PBS), ovarian tissue samples were weighed and cut into small pieces and homogenized in radio immuno- precipitation assay (RIPA) (for measuring MDA) buffer and PBS (for measuring NO, TAS and TOS). Tissues were homogenized in all groups by using Mixer Mill MM 400 (Retsch, Haan, Germany). Assays were per- formed on the supernatant of the homogenate that was prepared by centrifugation at +4 /C14C. Blood and tissue biochemical markers were calcu- lated by spectrophotometric measurements. The pro- tein contents of the ovarian tissues were calculated according to the method described by Lowry et al . 14 Tissue MDA levels were determined by using spectro- photometric kit (Cat. No.:10009055, Cayman). The

are expressed as micromolar per gram protein. Tissue NO assay was determined via spectrophotom- etry at 540 nm by using a nitrate/nitrite colorimetric assay kit (Cat. No. 780001, Cayman). The results are expressed as micromolar per gram protein. Tissue TAS and TOS levels were determined at the end of the study, whereas serum TOS (Product Code: RL0024) and TAS (Product Code: RL0017) levels and oxidative stress index (OSI) measurement were car- ried out both at the beginning (at zero-point) and at the end of the study (at 6-h point). As described pre- viously by Jansen and Ruskovska, 15 serum TOS and TAS levels were calculated by using spectrophotomet- ric kits (Rel Assay Diagnostics, Gaziantep, Turkey). The results for TAS and TOS are expressed as μmol 360 © 2018 Japan Society of Obstetrics and Gynecology F. Beyazit et al. Trolox equivalent/L and μmol H2O2 equivalent/L, respectively. The ratio percentage of TOS to TAS was used to calculate the OSI. Speci fically, OSI (arbitrary unit) = ([TOS, μmol H2O2 equivalent/L]/[TAS, μmol Trolox equivalent/L]). Statistical analysis The SPSS 21.0 software ( SPSS for Windows) program was used for the statistical analysis. Both the histo- pathological and tissue IHC staining results were pre- sented as mean /C6standard deviation (SD) and median (min –max). Nonparametric tests were per- formed because the distribution of the data was not normal. The comparison of all groups was done using the Kruskal –Wallis test, and the Mann –Whitney U- test was used as the post-hoc test. Bonferroni correc- tion was carried out for the prevention of signi ficance inflation (0.05/total comparison number; P < 0.0083 was accepted as signi ficant). A P value <0.05 was accepted as statistically signi ficant.

All rats survived and were included in the final analysis. Histopathologic findings All rats were evaluated for tissue damage by asses- sing related parameters, such as hemorrhagia, congestion, edema and polymorphonuclear leukocyte (PMNL) in filtration. Table 1 shows total histopatho- logic scores of these parameters expressed as mean (SD) and median (min-max). There were no histopath- ological changes in the sham group (Group I). The tis- sues in the I/R and I/R + saline group showed histopathological changes of condensed hemorrhagia, increased edema, signs of vascular congestion and infiltration of in flammatory cells along with degenera- tive and apoptotic cells. Histopathologic changes were similar in both I/R group and I/R+ isotonic saline group, whereas after i.p. ADA application tissue injury scores were reduced, and comparable with those in the sham group: Figure 1 shows histopatho- logical findings for each group using H&E. Effect of ADA on apoptosis induced by I/R In order to evaluate the apoptotic cells in the ovarian tissue, we analyzed the AI of ovarian cells by using the TUNEL technique. Quantitative assessment of AI in ovarian tissue using TUNEL technique also con- firmed histopathological findings. Ovarian tissue in the sham-operated group showed less TUNEL- positive cells (21.8 /C67.85), whereas I/R (53.8 /C617.13) and I/R + saline (58.1 /C617.41) groups had signi ficantly elevated TUNEL-positive cells (P = 0.05 for both). Intraperitoneal ADA application significantly decreased the AI (33.0 /C611.94) com- pared with I/R and I/R + saline groups ( P < 0.05 for both) (Fig. 2). Table 1 Comparison of ovarian damage scores between all groups Hemorrhagia Congestion Edema In flammation Group I ( n = 10) Mean (/C6SD) 0.1 /C60.10 0.3 /C60.48 0.10 /C60.31 0.1 /C60.10 Median (min-max) 0 (0 –1) 0 (0 –1) 0 (0 –1) 0 (0 –1) Group II ( n = 10) Mean (/C6SD) 2.8 /C60.42 1.6 /C61.07 2.70 /C60.48 2.8 /C60.42 Median (min-max) 3 (2 –3) 1 (0 –3) 3 (2 –3) 3 (2 –3) Group III ( n = 10) Mean (/C6SD) 2.8 /C60.42 2.8 /C60.42 2.7 /C60.48 2.8 /C60.42 Median (min-max) 3 (2 –3) 2 (2 –3) 3 (2 –3) 3 (2 –3) Group IV ( n = 10) Mean (/C6SD) 1.7 /C60.94 1.6 /C61.07 0.6 /C61.07 1.3 /C61.25 Median (min-max) 1 (1 –3) 1 (1 –3) 0 (0 –3) 1 (0 –3) P 0.001* 0.019† 0.007‡ 0.015§ *Comparison of Group 1/Group 2, Group 1/Group 3, Group 1/Group 4, Group 2/Group 4, Group 3/Group 4 ( P = 0.001, P = 0.001, P = 0.001, P = 0.004 and P = 0.007 §, respectively); †Comparison of Group 1/Group 2, Group 1/Group 3, Group 1/Group 4, Group 2/Group 4, Group 3/Group 4 ( P = 0.001, P = 0.001, P = 0.004, P = 0.002 and P = 0.002, respectively); ‡Comparison of Group 1/Group 2, Group 1/Group 3, Group 2/Group 4, Group 3/Group 4 ( P = 0.001, P = 0.001, P = 0.001 and P = 0.001, respectively); §Comparison of Group 1/Group 2, Group 1/Group 3, Group 1/Group 4, Group 2/Group 4, Group 3/Group 4 ( P = 0.001, P = 0.001, P = 0.002, P = 0.008 and P = 0.008, respectively). SD, standard deviation. 361© 2018 Japan Society of Obstetrics and Gynecology Adalimumab in ovarian torsion IHC findings I/R (group II) caused a signi ficant increase in ovarian expression of IL-1 β (2.40 /C60.69), NF- κB (2.40 /C60.69), and iNOS (2.80 /C60.42) compared with the sham group. Signi ficant reductions in these parameters were observed in the ADA-treated group in compari- son with both group II (I/R) and group III (I/R + saline group) ( P < 0.05) (Table 2). Biochemical findings in ovarian tissue Ovarian tissue oxidative stress markers were mea- sured after the study. Tissue NO levels were similar in groups I (5.87 /C62.16) and IV (5.46 /C61.77), whereas tissue NO levels were found to be statisti- cally increased in groups II (10.03 /C64.12) and III (9.78 /C63.68). Tissue MDA levels were found to be similar among all four groups ( P = 0.447). Tissue TOS (1.14 /C60.57) and OSI (6.67 /C65.41) levels were found to be decreased in group IV compared with group II (2.20 /C61.09 for TOS and 19.87 /C67.0 for OSI) and group III (2.18 /C60.76 for TOS and 22.37 /C65.85 for OSI) ( P < 0.05 for both). Total antioxidant status levels in the ADA group were found to be compara- ble with the sham group, whereas tissue TAS levels were found to be decreased in groups II and III (Fig. 3). Oxidative stress markers in serum Serum oxidative stress markers were measured both before and after the experiment. There were no signif- icant relationships among groups for TOS, TAS and OSI. Although serum TOS levels were in a decreasing trend after ADA application (from 12.72 /C64.11 to 10.65 /C64.07), this was found to be statistically insig- nificant (P = 0.112) (Table 3).

In this experimental model of ovarian I/R, we aimed to evaluate the antioxidant, anti-in flammatory and antiapoptotic effects of ADA with both histopatho- logic and biochemical examinations. And as a result, we demonstrated that treatment with ADA reduced I/R injury via anti-in flammatory and antioxidant pathways at cellular tissue level. These findings sug- gest that ADA with its immunomodulatory properties could be regarded as an excellent bridge to surgical intervention in selected cases not immediately suitable for definitive therapy. The present study demonstrated that IL-1 β expres- sions are signi ficantly down-regulated in the ADA group compared with the I/R group and the I/R + saline group. Although, our study is the first that Figure 1 Microscopic comparison of inflammation, vascular conges- tion, and edema in all four groups. Ischemia–reperfusion (I/R) and I/R + saline-treated group showed significantly greater histological damage including increased cellu- lar in flammation (black arrow), vascular congestion (white arrow), hemorrhage (white arrowhead) and edema (black arrowhead) compared to adalimumab (ADA)- treated group and sham. 362 © 2018 Japan Society of Obstetrics and Gynecology F. Beyazit et al. investigated the expression of IL-1 β in ADA-treated ovarian I/R, in a study by Cure et al .16 IL-1β, IL-6 and TNF-α expressions were analyzed after ADA treat- ment in a rat model of abdominal aorta cross-clamp- ing. IL-1 β and TNF- α levels were found to be decreased in the ADA-treated group compared with the I/R and control group. Because IL-1 β is such a prominent proin flammatory cytokine in a multitude of systemic in flammatory states, prolonged IL-1 β overproduction in the in flammatory response, may

in enhanced tissue damage due to the immune cells overactivation and the production of prote- ases.17,18 Moreover, in some certain instances, IL-1 β overproduction may be the cause, or be linked with more or less severe in flammatory conditions several of which have recently been classi fied as autoin flam- matory diseases. 19 In this context, ADA could exert its beneficial effects in I/R injury by regulating IL-1 β- induced vascular permeability, neutrophil recruitment and maturation in the early phases of in flammation. Therefore, histopathologic improvements including marked decreases in in flammatory cell in filtrate, Figure 2 (a) Apoptosis assess- ment by terminal deoxynu- cleotidyl transferase dUTP nick end labeling (TUNEL) technique within each group. Ischemia–reperfusion (I/R) and I/R + saline groups exhibited signi fi- cantly elevated TUNEL -positive cells (brown stained cells) compared with the sham and adalimumab (ADA)-treated group (# P < 0.05 between ADA vs I/R and I/R + saline groups). (b) Apoptotic index in each group. 363© 2018 Japan Society of Obstetrics and Gynecology Adalimumab in ovarian torsion Table 2 Comparison of ovarian immunohischemistochemical staining results between groups IL-1β NF-κB iNOS Group I ( n = 10) Mean (/C6SD) 0.70 /C60.48 1.20 /C60.78 2.20 /C60.63 Median (min-max) 0 (0 –1) 1 (0 –2) 2 (1 –3) Group II ( n = 10) Mean (/C6SD) 2.40 /C60.69 2.40 /C60.69 2.80 /C60.42 Median (min-max) 2.5 (1 –3) 2.5 (1 –3) 3 (2 –3) Group III ( n = 10) Mean (/C6SD) 2.20 /C60.63 2.60 /C60.69 2.70 /C60.49 Median (min-max) 2 (1 –3) 3 (1 –3) 3 (1 –3) Group IV ( n = 10) Mean (/C6SD) 1.40 /C60.51 1.60 /C60.69 2.40 /C60.51 Median (min-max) 1 (1 –2) 1.5 (1 –3) 2 (2 –3) P 0.000* 0.001† 0.003‡ *Comparison of Group 1/Group 2, Group 1/Group 3, Group 2/Group 4 ( P = 0.001, P = 0.001 and P = 0.005, respectively); †Comparison of Group 1/Group 2, Group 1/Group 3, Group 2/Group 4, Group 3/Group 4 ( P = 0.004, P = 0.002, 0.004 and P = 0.008, respectively); ‡Comparison of Group 1/Group 2, Group 1/Group 3, Group 2/Group 4 ( P =0 . 0 0 2 ,P =0 . 0 0 2 a n dP = 0.004, respectively). iNOS, inducible nitric oxide synthase; SD, standard deviation. Figure 3 Expressions of oxidative stress markers and antioxidant enzymes in the ovarian tissue ( *P < 0.05 between adali- mumab [ADA] vs ischemia –reperfusion [I/R] and I/R + saline groups). 364 © 2018 Japan Society of Obstetrics and Gynecology F. Beyazit et al. along with decreased vascular congestion and edema in the ADA-treated experimental group are an impor- tant finding of the present study. Various physiopathological mechanisms have been put forward to explain the tissue damage that occurs during ovarian torsion and detorsion including ovar- ian lipid peroxidation, vascular leucocyte margination and apoptosis. 20 The detorsion involves the produc- tion of toxic reactive oxygen species (ROS) with the return of blood flow following ischemia. 21 Overpro- duction of ROS could lead to a signi ficant elevation in lipid peroxidation, thereby generating MDA and destroying the antioxidant defense systems of the human body. 16,22 It has been demonstrated that I/R of a tissue is strictly linked with lipid peroxidation which causes oxidative demolition of the cellular membranes by autocatalytic processes leading to harmful accumulation of toxic metabolites and cellu- lar death 23,24 In this context, MDA, being an end product of peroxidative decomposition of polyenoic fatty acids, is generally used as a reliable indicator of tissue damage. 25 In the present study, we found no significant alterations after i.p. ADA injection in tissue MDA levels indicating lack of lipid peroxidation in our study groups. The most logical explanation for the failure to document unaltered MDA levels is that the concentration of free radicals generated is not enough to activate lipid peroxidation or other antioxi- dant systems were able to compensate ongoing oxida- tive state. Besides MDA, assessment of NO, TOS, TAS and OSI are also reliable indicators of oxidative stress caused by insuf ficient blood flow and possibly as pre- dictive or prognostic markers in I/R conditions. 26 We assayed oxidative status as TOS, TAS and OSI in both tissue and serum samples. In addition, tissue NO mea- surement was done in order to provide a comprehen- sive suite of oxidative status indicators and markers of reperfusion injury. The present study is therefore con- sidered to give a more complete overview of the effects of ADA in an I/R injury. We found increased TAS and decreased TOS, OSI and NO levels in ovarian tissue samples after i.p. ADA injection. Due to an increase in these oxidants and a decrease in total anti- oxidants, the oxidant/antioxidant balance shifted toward oxidative stress in the ovarian tissues of the I/R rats. Adalimumab treatment in these rats signi fi- cantly decreased TOS and NO and increased TAS levels compared to untreated and isotonic saline applied groups. Although there is no study in the liter- ature exploring the effects of ADA in an ovarian I/R injury, the salubrious effect of ADA in I/R injuries have also been demonstrated in several other studies. In an experimental model of liver injury by Cure et al ., 16 it has been demonstrated that during I/R injury ADA decreases cytokines, prevents the increase of NO by maintaining the balance among arginase, carbamoyl phosphate synthetase-1, and NOS, and consequently protects the cells from death. Similarly, in a study by Kurt et al ., 27 it has been reported that with decreasing the release of cytokines and prevent- ing the generation of ROS via blocking TNF- α, ADA could diminish lung damage during I/R process. In our study, oxidative stress was only present in ovarian tissues, and no difference was found between groups in terms of serum oxidant/antioxidant param- eters before and after the study. As we reported no effects of I/R on serum oxidative stress markers irre- spective from the treatment protocols, this may be attributed to the short treatment time and I/R periods. The importance of treatment intervals on tis- sue and plasma oxidative stress levels was empha- sized in several studies. Auberval et al . 28 demonstrated that although short-term high-fat diet (HFD) exhibits oxidative stress on pancreatic and hepatic tissues, this effect was not valid for plasma oxidative stress markers. However, longer period of treatment with HFD led to the detection of oxidative stress in plasma due to increases in the levels of oxi- dized proteins and lipids. Table 3 Serum TOS, TAS and OSI measurements among groups before and after the study Serum TOS Serum TAS Serum OSI Before After P Before After P Before After P Group I ( n = 10) 11.8 /C61.5 10.1 /C61.7 0.981 1.1 /C60.2 0.8 /C60.2 0.051 11.2 /C63.2 13.9 /C63.7 0.051 Group II ( n = 10) 12.4 /C64.2 10.7 /C62.3 0.299 1.1 /C60.1 0.9 /C60.3 0.107 11.35 /C63.6 12.6 /C63.9 0.455 Group III ( n = 10) 12.9 /C62.3 12.5 /C62.5 0.216 1.1 /C60.2 1.1 /C60.3 0.848 12.1 /C62.9 10.6 /C63.4 0.327 Group IV ( n = 10) 12.7 /C64.1 10.6 /C64.1 0.112 1.0 /C60.3 0.8 /C60.1 0.106 15.2 /C65.2 12.7 /C65.2 0.292 P NS NS — NS N S — NS NS — NS, not signi ficant; OSI, oxidative stress index; TAS, total antioxidant status; TOS, total oxidant status. 365© 2018 Japan Society of Obstetrics and Gynecology Adalimumab in ovarian torsion A number of studies have con firmed the pivotal role of NF- κB in I/R injury cases. 29,30 NF-κB, which is a transcription factor that regulates the expression of multiple in flammatory and immune genes, plays a crucial role in host defense, chronic in flammatory dis- eases and even tumoural conditions. 31 It is activated through phosphorylation and subsequent degradation of I κB. Several agents including ROS, IL-1 β and TNF- α are capable to phosphorylate I κB. 32 After activation of I κB, NF- κB triggers key mediators of in flammation responsible from I/R injury, including intercellular adhesion molecule 1, iNOS, cyclooxygenase-2, interleukin-1ß, interleukin-6, and vascular cellular adhesion molecule 1 that leads to the development of apoptosis. 33,34 In the present study, we found that ADA partially rescued the effects of I/R injury via multiple mechanisms, including the down-expression of NF- κB and inhibiting ovarian cell apoptosis. Adali- mumab has signi ficantly improved the postischemic recovery of rat ovaries, which was paralleled by sig- nificant alterations of ovarian cell apoptosis. Terminal deoxynucleotidyl transferase dUTP nick end labeling assay demonstrated that rats in our I/R and I/R plus saline groups showed a signi ficantly increased num- ber of TUNEL-positive cells compared to that in sham-operated and ADA applied rats. This finding of the study with down-expression of NF- κB suggests that NF- κB inhibiton is important in the antiapoptotic process during I/R injury. Increased tissue NO levels through activation of iNOS is one of the key mechanisms involved in I/R injuries. 35 Overproduction of NO by iNOS interacts quickly with the superoxide radicals that are pro- duced during reperfusion injuries to form peroxyni- trite, which induces protein damage by forming nitrotyrosine. 34,36 In a rat model of experimental kid- ney injury, Wang et al .36 demonstrated elevated levels of NO metabolites and the mRNA of iNOS in ischemia-reperfused kidneys in which tyrosol treat- ment attenuated iNOS-mediated NO production, which in turn reduced oxidative stress and minimized the extent of renal injury induced by I/R. Similarly, in a study by Ozcan and colleagues, 35 authors deter- mined the ef ficacy of a selective NF- κB inhibitor, pyr- rolidium dithiocarbamate (PDTC), on long-term histological damage in testicular I/R injuries. In rats administered PDTC, iNOS and p65 expressions were found to be signi ficantly reduced compared with the torsion group suggesting a major role of iNOS and NF-κB in reperfusion injuries. Immunohistochemical analysis of the present study revealed severe staining in iNOS and the NF- κB in the torsion and torsion +saline group, whereas a weak staining in the sham and ADA-treated group suggesting an activation of NF-κB and iNOS by I/R and resulting with increased intraovarian NO levels and subsequent ovarian injury. In conclusion, ADA therapy attenuated I/R induced ovarian injury, possibly due to suppression of in flammation, blockade of oxidative stress, and alteration of apoptotic pathways. Our results indicate substantial new aspects of this field and highlight the therapeutic potential of ADA for treating ovarian damage induced by I/R injury with providing the rationale for its use. Acknowledgments This research was supported by The Scienti fic Research Projects Coordination Unit of COMU as ‘Independent Research Project ’ (Project ID: TSA- 2017-1324). Disclosure All of the authors declare that there is no con flict of interest regarding to this article.

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