{"paper_id":"fe1f23e5-7746-431b-bbe6-0ac444f6b20c","body_text":"Adalimumab mitigates ovarian ischemia –reperfusion injury\nin rats by regulating oxidative stress, apoptosis and\nresolution of in flammation\nFatma Beyazit1 , Bas¸ak Büyük 2, Hakan Turkon 3, Sait Elmas 4 and Metehan Uzun 5\n1Department of Obstetrics and Gynecology, 2Department of Histology and Embryology, 3Department of Biochemistry,\n4Experimental Research Application and Research Center and 5Department of Physiology, Çanakkale Onsekiz Mart University,\nÇanakkale, Turkey\nAbstract\nAim: Ovarian torsion is a rare but an important reason of acute lower abdominal pain in women and associ-\nated with serious morbidity and mortality, if not treated promptly. The aim of this study was to evaluate\nthe effects of an antitumor necrosis factor- α antibody on ovarian torsion in a rat model of ischemia –\nreperfusion (I/R) injury.\nMethods: Forty female Wistar Albino rats were used in the present study. The rats were randomly divided\ninto four groups: group I (sham), group II (I/R), group III (I/R + isotonic saline) and group IV\n(I/R + adalimumab). The I/R model was induced by torsion of both ovaries. Immunohistochemical staining\nfor interleukin-1 β (IL-1β), nuclear factor- κB (NF- κB), and inducible nitric oxide synthase was performed. Tis-\nsue and serum oxidative stress markers in conjunction with apoptotic index (AI) with the terminal deoxynu-\ncleotidyl transferase dUTP nick end labeling method were also calculated.\nResults: Tissue total oxidant status, oxidative stress index and nitric oxide values were signi ficantly\ndecreased, and tissue total antioxidant status was found to be increased in group IV. In flammation, vascular\ncongestion and hemorrhagia were signi ficantly lower in adalimumab-treated group. Serum oxidative stress\nmarkers and tissue malondialdehyde levels did not differ in study groups. The AI was signi ficantly\nincreased in groups 2 and 3. Adalimumab treatment signi ficantly decreased the AI.\nConclusion: Adalimumab therapy in rats attenuated I/R induced ovarian injury, possibly suppressing\ninflammation, inhibiting oxidative stress, and altering apoptotic pathways.\nKey words: adalimumab, apoptosis, immunohistochemistry, ovarian torsion, oxidative stress.\nIntroduction\nThe twisting or torsion of the ovary is a relatively\ncommon gynecological emergency especially in 1st\nthree decades of life, which denotes the bending of\nthe ovary and fallopian tube around the broad liga-\nment.\n1 Clinical presentation is sometimes confounded\nby non-speci fic symptoms such as abdominal pain,\nnausea and vomiting that leads to late or missed diag-\nnosis.\n2 In order to prevent potential necrosis,\ninfertility and life-threatening sequels of this entity,\nearly diagnosis and prompt institution of adequate\ntreatment may be life-saving. For this reason, in\npatients with suspected ovarian torsion, gynecologic\nconsultation with subsequent surgical procedures are\ncritical, irrespective of whether laboratory examina-\ntions and radiologic imaging yield normal results.\nSurgical intervention, either laparoscopic or laparot-\nomy commonly re-establish blood circulation never-\ntheless, tissue atrophy or necrosis, may still occur and\nReceived: May 16 2018.\nAccepted: October 2 2018.\nCorrespondence: Dr Fatma Beyazit, Department of Obstetrics and Gynecology, Canakkale Onsekiz Mart University Medical Faculty,\nSevim Buluç Street, Canakkale 17020, Turkey. Email: fatmabeyazit@yahoo.com\n358 © 2018 Japan Society of Obstetrics and Gynecology\ndoi:10.1111/jog.13846 J. Obstet. Gynaecol. Res. Vol. 45, No. 2: 358 –367, February 2019\n\n\nis a common consequence. 3,4 Thus, it is important to\ndevelop alternative nonsurgical and speci fic therapeu-\ntic strategies against ovarian torsion in order to\nachieve short- and long-term preservation of ovarian\nfunction at a cellular level including protection from\ntissue ischemia caused by reperfusion damage from\nfree radicals that are released after detorsion.\nTumor necrosis factor- α (TNF-α) is one of the main\nmediators of in flammation and has been recognized\nas a target of therapeutic intervention in many in flam-\nmatory disease states.\n5 Clinical data advocate that\nTNF-α is released at the beginning of reperfusion, and\nits level increases during the early stages of ischemia\nand reperfusion (I/R).\n6 Being a cell signaling circulat-\ning protein, TNF- α is mainly involved in many harm-\nful biological processes and diseases in the body\nduring the course of I/R.\n7,8 Adalimumab (ADA) is a\nrecombinant human IgG1 monoclonal antibody spe-\ncific for human TNF- α and is traditionally used to\ntreat psoriasis, psoriatic arthritis, and several other\ninflammatory conditions such as rheumatoid arthritis\nand in flammatory bowel diseases. The inhibition of\nTNF-α with ADA has been shown to decrease the\nseverity of liver and intestinal I/R injuries in several\nanimal models. 9,10 However, there have been no stud-\nies of the protective effects of ADA against ovarian\nI/R injury.\nIn this study, we aimed to evaluate the therapeutic\neffect of ADA on ovarian injury that is induced by\nI/R in an experimental rat model. Furthermore, we\naimed to shed light on the possible mechanisms by\nwhich ADA could protect rat against I/R induced\novarian injury.\nMaterials and Methods\nMaterials and experimental design\nThis experimental protocol was approved by the Insti-\ntutional Animal Use and Care Committee of Çanak-\nkale Onsekiz Mart University (COMU) (Approval No:\n2017/06–15) and performed in accordance with the\nHelsinki Declaration of World Medical Association\nrecommendations on animal studies. A total of 40 Wis-\ntar albino adult female rats were obtained from\nCOMU Experimental Research Application and\nResearch Center with a mean age of 4 months and a\nmean weight of 250 –300 g. The rats were housed in\nstainless steel cages in an animal room maintained at\na standard humidity (50 –55%) and temperature\n22 /C62\n/C14C with 12-h light/dark cycles. All animals\nwere fed standard food and water. Twelve hours\nbefore the study procedure feeding was stopped and\nthe rats were only allowed to drink water. The entire\nexperiment was conducted under half-sterile\nconditions.\nExperimental protocol\nAnesthesia\nRats were anesthetized with intraperitoneal (i.p.) keta-\nmine hydrochloride (75 mg/kg) and xylazine\n(5–10 mg/kg), and if required anesthesia was main-\ntained with additional injections of ketamine\nhydrochloride.\nOvarian I/R\nThe ovarian I/R model was designed in a way paral-\nlel to previous trials. 11 In brief, the rats were supine\npositioned on a heated platform. The skin was pre-\npared under aseptic conditions, and an approximately\n2.5-cm longitudinal incision was performed in the\nmidline area of the lower abdomen after shaving of\nabdominal area. Afterward peritoneal incision was\ndone in order to locate the uterine horns and adnexa.\nAfter finding the adnexes, both ovaries were twisted\nand rotated 720\n/C14clockwise and fixed to the abdomi-\nnal wall with a non-absorbable suture. Then abdomen\nwall was closed with continuous 1/0 silk suture in\none layer. After 3 h, the fixed ovaries were freed by\ncutting the suture and both ovaries were detorsioned\nwith a relaparotomy procedure and the abdomen was\nsurgically closed again.\nGroups\nWistar albino rats were randomly divided into four\ngroups as follows:\nGroup I (sham, n = 10): After sham operation at\nzero and 3-h point, both ovaries were surgically\nremoved for assessment at 6-h point.\nGroup II (torsion-detorsion group, n = 10): After\ntorsion (at zero-point) and detorsion (at 3-h point),\nboth ovaries surgically removed at the end of the\nstudy (6-h point).\nGroup III (torsion-detorsion + isotonic saline\ngroup, n = 10): Rats underwent torsion and detorsion\noperation. Twenty minutes prior to the detorsion,\n0.3-mL i.p. isotonic saline was applied. Both ovaries\nremoved at the end of the study (6-h point).\nGroup IV (torsion-detorsion + ADA group,\nn = 10): Rats underwent torsion and detorsion opera-\ntion. Twenty minutes prior to detorsion, 50-mg/kg\nADA (Humira; Abbott Laboratories) applied\n359© 2018 Japan Society of Obstetrics and Gynecology\nAdalimumab in ovarian torsion\n\nintraperitoneally. Both ovaries removed at the end of\nthe study (6-h point).\nEvaluation\nSerum and tissue samples were used for evaluation\npurposes. Blood samples were collected at the begin-\nning and at the end of the study (at 6-h point). In\norder to evaluate tissue samples, both of the ovaries\nwere removed in all subjects. One of the ovaries was\nused for biochemical analysis, while the other was\nused for histopathological evaluation.\nHistopathological examinations\nIn order to investigate histopathologic changes, ovar-\nian tissue samples were consecutively numbered and\nfixated in 10% neutral buffered formalin for 48 h,\ndehydrated, cleared in xylene and embedded in paraf-\nfin and sent to the histology department of COMU.\nEvaluation of the pathology specimens was done by a\nhistology specialist, who was blind to the four study\ngroups. The paraf fin blocks were cut in 5- μm thick-\nness on Rotary Microtome (Leica RM2125 RTS), and\nthe sections were stained with hematoxylin and eosin\n(H&E). The histopathologic sections were evaluated\nunder a light microscope (Zeiss AxioScope A1) for the\npresence of hemorrhagia, vascular congestion, edema\nand in flammation (neutrophil in filtration) and rated\non a modi fied semiquantitative scale of 0 –3 as also\nstated by Bozkurt et al .\n12 Scoring scale was as follow;\n0, no findings; 1, findings of <33%; 2, findings of\n33–66%; 3, findings of >66%.\nImmunohistochemical staining\nImmunohistochemistry for IL-1 β (Cat #12242S, Cell\nSignaling Technology), NF- κB (Cat #RB-9034, Thermo\nScientific, Lab vision) and inducible nitric oxide\nsynthase (iNOS) (Cat #RB-9242, Thermo Scienti fic)\nwas carried out with commercial kits according to the\nmanufacturers’ instructions.\nImmunohistochemical (IHC) evaluation and scoring\nwere done according to Jiang et al .13 According to this\nscoring, IL-1 β, NF- κB, and iNOS immunostaining\nscores were calculated by staining intensity (0, no\nstaining; 1, weak but detectable staining; 2, moderate;\nand 3, strong staining).\nApoptosis assessment with the TUNEL method\nTerminal deoxynucleotidyl transferase dUTP nick end\nlabeling (TUNEL) staining was used to detect apopto-\nsis of the ovarian tissue. Prepared tissue was fixed\nwith 4% neutral formaldehyde, embedded in paraf fin,\nand 4- μm-thick sections were cut from each paraf fin\nblock. After dewaxing, hydration, and serum block-\ning, ApopTag Peroxidase in situ Apoptosis Detection\nKit (S7100, Millipore) was used according to the man-\nufacturer’s protocol. The cells were observed and\nphotographed under an optical microscope, and five\nhigh image fields of each sample were randomly\nselected, and 500 cells were counted in each field.\nCells stained brown or black were judged TUNEL-\npositive apoptotic cells and presented in the form of\napoptotic index (AI).\nBiochemical evaluation\nOvarian tissue and blood samples were obtained from\neach animal at the end of the study. Blood samples\nwere centrifuged at 3000 rpm for 15 min. The resultant\nserum samples were aliquoted into polypropylene\ntubes and stored at −80\n/C14C until the biochemical analy-\nsis. The tissues were prepared at +4 /C14C in order to mea-\nsure malondialdehyde (MDA), nitric oxide (NO), total\nantioxidant status (TAS) and total oxidant status (TOS)\nlevels. After washing with phosphate buffer solution\n(PBS), ovarian tissue samples were weighed and cut\ninto small pieces and homogenized in radio immuno-\nprecipitation assay (RIPA) (for measuring MDA) buffer\nand PBS (for measuring NO, TAS and TOS). Tissues\nwere homogenized in all groups by using Mixer Mill\nMM 400 (Retsch, Haan, Germany). Assays were per-\nformed on the supernatant of the homogenate that was\nprepared by centrifugation at +4\n/C14C.\nBlood and tissue biochemical markers were calcu-\nlated by spectrophotometric measurements. The pro-\ntein contents of the ovarian tissues were calculated\naccording to the method described by Lowry et al .\n14\nTissue MDA levels were determined by using spectro-\nphotometric kit (Cat. No.:10009055, Cayman). The\nresults are expressed as micromolar per gram protein.\nTissue NO assay was determined via spectrophotom-\netry at 540 nm by using a nitrate/nitrite colorimetric\nassay kit (Cat. No. 780001, Cayman). The results are\nexpressed as micromolar per gram protein.\nTissue TAS and TOS levels were determined at the\nend of the study, whereas serum TOS (Product Code:\nRL0024) and TAS (Product Code: RL0017) levels and\noxidative stress index (OSI) measurement were car-\nried out both at the beginning (at zero-point) and at\nthe end of the study (at 6-h point). As described pre-\nviously by Jansen and Ruskovska,\n15 serum TOS and\nTAS levels were calculated by using spectrophotomet-\nric kits (Rel Assay Diagnostics, Gaziantep, Turkey).\nThe results for TAS and TOS are expressed as μmol\n360 © 2018 Japan Society of Obstetrics and Gynecology\nF. Beyazit et al.\n\nTrolox equivalent/L and μmol H2O2 equivalent/L,\nrespectively. The ratio percentage of TOS to TAS was\nused to calculate the OSI. Speci fically, OSI (arbitrary\nunit) = ([TOS, μmol H2O2 equivalent/L]/[TAS, μmol\nTrolox equivalent/L]).\nStatistical analysis\nThe SPSS 21.0 software ( SPSS for Windows) program\nwas used for the statistical analysis. Both the histo-\npathological and tissue IHC staining results were pre-\nsented as mean /C6standard deviation (SD) and\nmedian (min –max). Nonparametric tests were per-\nformed because the distribution of the data was not\nnormal. The comparison of all groups was done using\nthe Kruskal –Wallis test, and the Mann –Whitney U-\ntest was used as the post-hoc test. Bonferroni correc-\ntion was carried out for the prevention of signi ficance\ninflation (0.05/total comparison number; P < 0.0083\nwas accepted as signi ficant). A P value <0.05 was\naccepted as statistically signi ficant.\nResults\nAll rats survived and were included in the final\nanalysis.\nHistopathologic findings\nAll rats were evaluated for tissue damage by asses-\nsing related parameters, such as hemorrhagia,\ncongestion, edema and polymorphonuclear leukocyte\n(PMNL) in filtration. Table 1 shows total histopatho-\nlogic scores of these parameters expressed as mean\n(SD) and median (min-max). There were no histopath-\nological changes in the sham group (Group I). The tis-\nsues in the I/R and I/R + saline group showed\nhistopathological changes of condensed hemorrhagia,\nincreased edema, signs of vascular congestion and\ninfiltration of in flammatory cells along with degenera-\ntive and apoptotic cells. Histopathologic changes were\nsimilar in both I/R group and I/R+ isotonic saline\ngroup, whereas after i.p. ADA application tissue\ninjury scores were reduced, and comparable with\nthose in the sham group: Figure 1 shows histopatho-\nlogical findings for each group using H&E.\nEffect of ADA on apoptosis induced by I/R\nIn order to evaluate the apoptotic cells in the ovarian\ntissue, we analyzed the AI of ovarian cells by using\nthe TUNEL technique. Quantitative assessment of AI\nin ovarian tissue using TUNEL technique also con-\nfirmed histopathological findings. Ovarian tissue in\nthe sham-operated group showed less TUNEL-\npositive cells (21.8 /C67.85), whereas I/R\n(53.8 /C617.13) and I/R + saline (58.1 /C617.41) groups\nhad signi ficantly elevated TUNEL-positive cells\n(P = 0.05 for both). Intraperitoneal ADA application\nsignificantly decreased the AI (33.0 /C611.94) com-\npared with I/R and I/R + saline groups ( P < 0.05 for\nboth) (Fig. 2).\nTable 1 Comparison of ovarian damage scores between all groups\nHemorrhagia Congestion Edema In flammation\nGroup I ( n = 10)\nMean (/C6SD) 0.1 /C60.10 0.3 /C60.48 0.10 /C60.31 0.1 /C60.10\nMedian (min-max) 0 (0 –1) 0 (0 –1) 0 (0 –1) 0 (0 –1)\nGroup II ( n = 10)\nMean (/C6SD) 2.8 /C60.42 1.6 /C61.07 2.70 /C60.48 2.8 /C60.42\nMedian (min-max) 3 (2 –3) 1 (0 –3) 3 (2 –3) 3 (2 –3)\nGroup III ( n = 10)\nMean (/C6SD) 2.8 /C60.42 2.8 /C60.42 2.7 /C60.48 2.8 /C60.42\nMedian (min-max) 3 (2 –3) 2 (2 –3) 3 (2 –3) 3 (2 –3)\nGroup IV ( n = 10)\nMean (/C6SD) 1.7 /C60.94 1.6 /C61.07 0.6 /C61.07 1.3 /C61.25\nMedian (min-max) 1 (1 –3) 1 (1 –3) 0 (0 –3) 1 (0 –3)\nP 0.001* 0.019† 0.007‡ 0.015§\n*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,\nP = 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\n2/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\n2, 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\nGroup 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\nand P = 0.008, respectively). SD, standard deviation.\n361© 2018 Japan Society of Obstetrics and Gynecology\nAdalimumab in ovarian torsion\n\nIHC findings\nI/R (group II) caused a signi ficant increase in ovarian\nexpression of IL-1 β (2.40 /C60.69), NF- κB (2.40 /C60.69),\nand iNOS (2.80 /C60.42) compared with the sham\ngroup. Signi ficant reductions in these parameters\nwere observed in the ADA-treated group in compari-\nson with both group II (I/R) and group III\n(I/R + saline group) ( P < 0.05) (Table 2).\nBiochemical findings in ovarian tissue\nOvarian tissue oxidative stress markers were mea-\nsured after the study. Tissue NO levels were similar\nin groups I (5.87 /C62.16) and IV (5.46 /C61.77),\nwhereas tissue NO levels were found to be statisti-\ncally increased in groups II (10.03 /C64.12) and III\n(9.78 /C63.68). Tissue MDA levels were found to be\nsimilar among all four groups ( P = 0.447). Tissue TOS\n(1.14 /C60.57) and OSI (6.67 /C65.41) levels were found\nto be decreased in group IV compared with group II\n(2.20 /C61.09 for TOS and 19.87 /C67.0 for OSI) and\ngroup III (2.18 /C60.76 for TOS and 22.37 /C65.85 for\nOSI) ( P < 0.05 for both). Total antioxidant status\nlevels in the ADA group were found to be compara-\nble with the sham group, whereas tissue TAS levels\nwere found to be decreased in groups II and\nIII (Fig. 3).\nOxidative stress markers in serum\nSerum oxidative stress markers were measured both\nbefore and after the experiment. There were no signif-\nicant relationships among groups for TOS, TAS and\nOSI. Although serum TOS levels were in a decreasing\ntrend after ADA application (from 12.72 /C64.11 to\n10.65 /C64.07), this was found to be statistically insig-\nnificant (P = 0.112) (Table 3).\nDiscussion\nIn this experimental model of ovarian I/R, we aimed\nto evaluate the antioxidant, anti-in flammatory and\nantiapoptotic effects of ADA with both histopatho-\nlogic and biochemical examinations. And as a result,\nwe demonstrated that treatment with ADA reduced\nI/R injury via anti-in flammatory and antioxidant\npathways at cellular tissue level. These findings sug-\ngest that ADA with its immunomodulatory properties\ncould be regarded as an excellent bridge to surgical\nintervention in selected cases not immediately suitable\nfor definitive therapy.\nThe present study demonstrated that IL-1 β expres-\nsions are signi ficantly down-regulated in the ADA\ngroup compared with the I/R group and the I/R +\nsaline group. Although, our study is the first that\nFigure 1 Microscopic comparison of\ninflammation, vascular conges-\ntion, and edema in all four groups.\nIschemia–reperfusion (I/R) and\nI/R + saline-treated group showed\nsignificantly greater histological\ndamage including increased cellu-\nlar in flammation (black arrow),\nvascular congestion (white arrow),\nhemorrhage (white arrowhead)\nand edema (black arrowhead)\ncompared to adalimumab (ADA)-\ntreated group and sham.\n362 © 2018 Japan Society of Obstetrics and Gynecology\nF. Beyazit et al.\n\ninvestigated the expression of IL-1 β in ADA-treated\novarian I/R, in a study by Cure et al .16 IL-1β, IL-6 and\nTNF-α expressions were analyzed after ADA treat-\nment in a rat model of abdominal aorta cross-clamp-\ning. IL-1 β and TNF- α levels were found to be\ndecreased in the ADA-treated group compared with\nthe I/R and control group. Because IL-1 β is such a\nprominent proin flammatory cytokine in a multitude\nof systemic in flammatory states, prolonged IL-1 β\noverproduction in the in flammatory response, may\nresult in enhanced tissue damage due to the immune\ncells overactivation and the production of prote-\nases.17,18 Moreover, in some certain instances, IL-1 β\noverproduction may be the cause, or be linked with\nmore or less severe in flammatory conditions several\nof which have recently been classi fied as autoin flam-\nmatory diseases. 19 In this context, ADA could exert its\nbeneficial effects in I/R injury by regulating IL-1 β-\ninduced vascular permeability, neutrophil recruitment\nand maturation in the early phases of in flammation.\nTherefore, histopathologic improvements including\nmarked decreases in in flammatory cell in filtrate,\nFigure 2 (a) Apoptosis assess-\nment by terminal deoxynu-\ncleotidyl transferase dUTP\nnick end labeling (TUNEL)\ntechnique within each\ngroup. Ischemia–reperfusion\n(I/R) and I/R + saline\ngroups exhibited signi fi-\ncantly elevated TUNEL\n-positive cells (brown\nstained cells) compared with\nthe sham and adalimumab\n(ADA)-treated group (# P\n< 0.05 between ADA vs I/R\nand I/R + saline groups).\n(b) Apoptotic index in each\ngroup.\n363© 2018 Japan Society of Obstetrics and Gynecology\nAdalimumab in ovarian torsion\n\nTable 2 Comparison of ovarian immunohischemistochemical staining results between groups\nIL-1β NF-κB iNOS\nGroup I ( n = 10)\nMean (/C6SD) 0.70 /C60.48 1.20 /C60.78 2.20 /C60.63\nMedian (min-max) 0 (0 –1) 1 (0 –2) 2 (1 –3)\nGroup II ( n = 10)\nMean (/C6SD) 2.40 /C60.69 2.40 /C60.69 2.80 /C60.42\nMedian (min-max) 2.5 (1 –3) 2.5 (1 –3) 3 (2 –3)\nGroup III ( n = 10)\nMean (/C6SD) 2.20 /C60.63 2.60 /C60.69 2.70 /C60.49\nMedian (min-max) 2 (1 –3) 3 (1 –3) 3 (1 –3)\nGroup IV ( n = 10)\nMean (/C6SD) 1.40 /C60.51 1.60 /C60.69 2.40 /C60.51\nMedian (min-max) 1 (1 –2) 1.5 (1 –3) 2 (2 –3)\nP 0.000* 0.001† 0.003‡\n*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\nof 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);\n‡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,\ninducible nitric oxide synthase; SD, standard deviation.\nFigure 3 Expressions of oxidative stress markers and antioxidant enzymes in the ovarian tissue ( *P < 0.05 between adali-\nmumab [ADA] vs ischemia –reperfusion [I/R] and I/R + saline groups).\n364 © 2018 Japan Society of Obstetrics and Gynecology\nF. Beyazit et al.\n\nalong with decreased vascular congestion and edema\nin the ADA-treated experimental group are an impor-\ntant finding of the present study.\nVarious physiopathological mechanisms have been\nput forward to explain the tissue damage that occurs\nduring ovarian torsion and detorsion including ovar-\nian lipid peroxidation, vascular leucocyte margination\nand apoptosis.\n20 The detorsion involves the produc-\ntion of toxic reactive oxygen species (ROS) with the\nreturn of blood flow following ischemia. 21 Overpro-\nduction of ROS could lead to a signi ficant elevation in\nlipid peroxidation, thereby generating MDA and\ndestroying the antioxidant defense systems of the\nhuman body. 16,22 It has been demonstrated that I/R\nof a tissue is strictly linked with lipid peroxidation\nwhich causes oxidative demolition of the cellular\nmembranes by autocatalytic processes leading to\nharmful accumulation of toxic metabolites and cellu-\nlar death\n23,24 In this context, MDA, being an end\nproduct of peroxidative decomposition of polyenoic\nfatty acids, is generally used as a reliable indicator of\ntissue damage. 25 In the present study, we found no\nsignificant alterations after i.p. ADA injection in tissue\nMDA levels indicating lack of lipid peroxidation in\nour study groups. The most logical explanation for\nthe failure to document unaltered MDA levels is that\nthe concentration of free radicals generated is not\nenough to activate lipid peroxidation or other antioxi-\ndant systems were able to compensate ongoing oxida-\ntive state.\nBesides MDA, assessment of NO, TOS, TAS and\nOSI are also reliable indicators of oxidative stress\ncaused by insuf ficient blood flow and possibly as pre-\ndictive or prognostic markers in I/R conditions.\n26 We\nassayed oxidative status as TOS, TAS and OSI in both\ntissue and serum samples. In addition, tissue NO mea-\nsurement was done in order to provide a comprehen-\nsive suite of oxidative status indicators and markers of\nreperfusion injury. The present study is therefore con-\nsidered to give a more complete overview of the\neffects of ADA in an I/R injury. We found increased\nTAS and decreased TOS, OSI and NO levels in ovarian\ntissue samples after i.p. ADA injection. Due to an\nincrease in these oxidants and a decrease in total anti-\noxidants, the oxidant/antioxidant balance shifted\ntoward oxidative stress in the ovarian tissues of the\nI/R rats. Adalimumab treatment in these rats signi fi-\ncantly decreased TOS and NO and increased TAS\nlevels compared to untreated and isotonic saline\napplied groups. Although there is no study in the liter-\nature exploring the effects of ADA in an ovarian I/R\ninjury, the salubrious effect of ADA in I/R injuries\nhave also been demonstrated in several other studies.\nIn an experimental model of liver injury by Cure\net al .,\n16 it has been demonstrated that during I/R\ninjury ADA decreases cytokines, prevents the increase\nof NO by maintaining the balance among arginase,\ncarbamoyl phosphate synthetase-1, and NOS, and\nconsequently protects the cells from death. Similarly,\nin a study by Kurt et al .,\n27 it has been reported that\nwith decreasing the release of cytokines and prevent-\ning the generation of ROS via blocking TNF- α, ADA\ncould diminish lung damage during I/R process.\nIn our study, oxidative stress was only present in\novarian tissues, and no difference was found between\ngroups in terms of serum oxidant/antioxidant param-\neters before and after the study. As we reported no\neffects of I/R on serum oxidative stress markers irre-\nspective from the treatment protocols, this may be\nattributed to the short treatment time and I/R\nperiods. The importance of treatment intervals on tis-\nsue and plasma oxidative stress levels was empha-\nsized in several studies. Auberval et al .\n28\ndemonstrated that although short-term high-fat diet\n(HFD) exhibits oxidative stress on pancreatic and\nhepatic tissues, this effect was not valid for plasma\noxidative stress markers. However, longer period of\ntreatment with HFD led to the detection of oxidative\nstress in plasma due to increases in the levels of oxi-\ndized proteins and lipids.\nTable 3 Serum TOS, TAS and OSI measurements among groups before and after the study\nSerum TOS Serum TAS Serum OSI\nBefore After P Before After P Before After P\nGroup 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\nGroup 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\nGroup 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\nGroup 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\nP NS NS — NS N S — NS NS —\nNS, not signi ficant; OSI, oxidative stress index; TAS, total antioxidant status; TOS, total oxidant status.\n365© 2018 Japan Society of Obstetrics and Gynecology\nAdalimumab in ovarian torsion\n\nA number of studies have con firmed the pivotal\nrole of NF- κB in I/R injury cases. 29,30 NF-κB, which is\na transcription factor that regulates the expression of\nmultiple in flammatory and immune genes, plays a\ncrucial role in host defense, chronic in flammatory dis-\neases and even tumoural conditions. 31 It is activated\nthrough phosphorylation and subsequent degradation\nof I κB. Several agents including ROS, IL-1 β and TNF-\nα are capable to phosphorylate I κB.\n32 After activation\nof I κB, NF- κB triggers key mediators of in flammation\nresponsible from I/R injury, including intercellular\nadhesion molecule 1, iNOS, cyclooxygenase-2,\ninterleukin-1ß, interleukin-6, and vascular cellular\nadhesion molecule 1 that leads to the development of\napoptosis.\n33,34 In the present study, we found that\nADA partially rescued the effects of I/R injury via\nmultiple mechanisms, including the down-expression\nof NF- κB and inhibiting ovarian cell apoptosis. Adali-\nmumab has signi ficantly improved the postischemic\nrecovery of rat ovaries, which was paralleled by sig-\nnificant alterations of ovarian cell apoptosis. Terminal\ndeoxynucleotidyl transferase dUTP nick end labeling\nassay demonstrated that rats in our I/R and I/R plus\nsaline groups showed a signi ficantly increased num-\nber of TUNEL-positive cells compared to that in\nsham-operated and ADA applied rats. This finding of\nthe study with down-expression of NF- κB suggests\nthat NF- κB inhibiton is important in the antiapoptotic\nprocess during I/R injury.\nIncreased tissue NO levels through activation of\niNOS is one of the key mechanisms involved in I/R\ninjuries.\n35 Overproduction of NO by iNOS interacts\nquickly with the superoxide radicals that are pro-\nduced during reperfusion injuries to form peroxyni-\ntrite, which induces protein damage by forming\nnitrotyrosine.\n34,36 In a rat model of experimental kid-\nney injury, Wang et al .36 demonstrated elevated levels\nof NO metabolites and the mRNA of iNOS in\nischemia-reperfused kidneys in which tyrosol treat-\nment attenuated iNOS-mediated NO production,\nwhich in turn reduced oxidative stress and minimized\nthe extent of renal injury induced by I/R. Similarly, in\na study by Ozcan and colleagues,\n35 authors deter-\nmined the ef ficacy of a selective NF- κB inhibitor, pyr-\nrolidium dithiocarbamate (PDTC), on long-term\nhistological damage in testicular I/R injuries. In rats\nadministered PDTC, iNOS and p65 expressions were\nfound to be signi ficantly reduced compared with the\ntorsion group suggesting a major role of iNOS and\nNF-κB in reperfusion injuries. Immunohistochemical\nanalysis of the present study revealed severe staining\nin iNOS and the NF- κB in the torsion and torsion\n+saline group, whereas a weak staining in the sham\nand ADA-treated group suggesting an activation of\nNF-κB and iNOS by I/R and resulting with increased\nintraovarian NO levels and subsequent ovarian\ninjury.\nIn conclusion, ADA therapy attenuated I/R\ninduced ovarian injury, possibly due to suppression\nof in flammation, blockade of oxidative stress, and\nalteration of apoptotic pathways. Our results indicate\nsubstantial new aspects of this field and highlight the\ntherapeutic potential of ADA for treating ovarian\ndamage induced by I/R injury with providing the\nrationale for its use.\nAcknowledgments\nThis research was supported by The Scienti fic\nResearch Projects Coordination Unit of COMU as\n‘Independent Research Project ’ (Project ID: TSA-\n2017-1324).\nDisclosure\nAll of the authors declare that there is no con flict of\ninterest regarding to this article.\nReferences\n1. Nayki C, Nayki U, Keskin Cimen F et al . The effect of rutin\non ovarian ischemia-reperfusion injury in a rat model. Gyne-\ncol Endocrinol 2018; 34 (9): 809–814.\n2. Yurtcu E, Togrul C, Ozyer S et al. Dose dependent protective\neffects of vardena fil on ischemia-reperfusion injury with bio-\nchemical and histopathologic evaluation in rat ovary.\nJ Pediatr Surg 2015; 50: 205–209.\n3. Huchon C, Fauconnier A. Adnexal torsion: A literature\nreview. 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Selective nuclear factor kappa b (NFkB) inhibitor,\nPyrrolidiumDithiocarbamate prevents, long-term histologic\ndamage in ischemia-reperfusion injuries after delayed testic-\nular torsion. Urol J 2016; 13: 2702–2706.\n36. Wang P, Zhu Q, Wu N, Siow YL, Aukema H, O K. Tyrosol\nattenuates ischemia-reperfusion-induced kidney injury via\ninhibition of inducible nitric oxide synthase. J Agric Food\nChem 2013; 61: 3669–3675. 367© 2018 Japan Society of Obstetrics and Gynecology\nAdalimumab in ovarian torsion","source_license":"CC0","license_restricted":false}