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Journal of Reproductive Immunology 100 (2013) 87– 92

Contents lists available at ScienceDirect

Journal of Reproductive Immunology

j o ur na l ho me pag e: www.elsev ier .com/ locate / j repr imm

on-steroidal anti-inflammatory drug modulates oxidative stress andalcium ion levels in the neutrophils of patients with primaryysmenorrhea

nder Kaplana, Mustafa Nazıroglub,∗, Mehmet Güneya, Mehmet Aykurb

Department of Obstetrics and Gynecology, Faculty of Medicine, Suleyman Demirel University, Isparta, TurkeyDepartment of Biophysics, Faculty of Medicine, Suleyman Demirel University, Isparta, Turkey

a r t i c l e i n f o

rticle history:eceived 14 July 2013ccepted 1 October 2013

eywords:ntioxidantrimary dysmenorrheaSAIDalcium ioneutrophilxidative stress

a b s t r a c t

Primary dysmenorrhea is a common inflammatory disease with an uncertain pathogenesis,although one consistent finding is increased neutrophil activity. We aimed to investigate theeffects of a non-steroidal anti-inflammatory drug (NSAID) on oxidative stress and Ca2+ levelsin neutrophils from patients with primary dysmenorrhea. Blood samples were obtained forneutrophil isolation from six female patients with primary dysmenorrhea (patients) and sixhealthy female subjects. The NSAID (diclofenac) was taken daily by the patient group for 6weeks before a second blood sample was taken. Neutrophils isolated after diclofenac treat-ment were investigated in three settings: (1) after incubation with verapamil and diltiazem(V + D), (2) after incubation with 2-aminoethoxydiphenyl borate (2-APB), and (3) with nei-ther exposure. Neutrophil lipid peroxidation and stimulated intracellular Ca2+ levels werehigher in the patients than in the controls, although their levels were reduced after sixweeks of treatment with diclofenac. Ca2+ levels from neutrophils obtained after diclofenactreatment were further decreased after incubation with V + D or 2-APB, compared withthose exposed to neither agent. Neutrophil glutathione peroxidase and total antioxidantstatus were lower in the patients than in the controls and higher post-treatment with

diclofenac. Reduced glutathione levels were similar in the control, patient, and treatmentgroups. In conclusion, we observed the importance of Ca2+ influx into the neutrophils andoxidative stress in the pathogenesis of the patients with primary dysmenorrhea. The NSAIDdiclofenac appeared to provide a protective effect against oxidative stress and Ca2+ entrythrough modulation of neutrophil VGCC and TRP calcium channels.

Abbreviations: 2-APB, 2-aminoethoxydiphenyl borate; fMLP,-formyl-l-methionyl-l-leucyl-l-phenylalanine; GSH, reduced glu-

athione; GSH-Px, glutathione peroxidase; MDA, malondialdehyde;PO, myeloperoxidase; NSAID, non-steroidal anti-inflammatory drug;

OS, reactive oxygen species; SOD, superoxide dismutase; V + D,erapamil + diltiazem; VGCC, voltage-gated calcium channels.∗ Corresponding author at: Department of Biophysics, Faculty ofedicine, Suleyman Demirel University, TR-32260 Isparta, Turkey.

el.: +90 246 2113641; fax: +90 246 2371165.E-mail address: mustafanaziroglu@sdu.edu.tr (M. Nazıroglu).

165-0378/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved.ttp://dx.doi.org/10.1016/j.jri.2013.10.004

© 2013 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Dysmenorrhea is characterized by abdominal or lowerback pain that lasts for at least two days during themenstrual cycle and is very common in young women(20–24 years of age), with a prevalence of 45–95% (Moore,2007). Dysmenorrhea is categorized as primary dysmen-orrhea, which is menstrual pain in the absence of any

apparent organic disorder, and secondary dysmenorrhea,which occurs in association with an identifiable illness(Harel, 2012). Although primary dysmenorrhea is verycommon, its causes are unclear.

ductive

88 Ö. Kaplan et al. / Journal of Repro

Increased levels of proinflammatory cytokines, inter-leukins (Yeh et al., 2004), and tumor necrosis factor-�(TNF-�), neutrophil hyperfunction (Marchini et al., 1995),and excessive reactive oxygen species (ROS) produc-tion (Marchini et al., 1995; Dikensoy et al., 2008) havebeen reported in patients with primary dysmenorrhea.Neutrophils are cells that play an important role inimmune responses (Ayub and Hallett, 2004). In primarydysmenorrhea, there is an increase in neutrophil function-dependent inflammatory metabolites, such as interleukinsand prostaglandins, in the peripheral blood (Harel, 2012).Hence, understanding the physiological mechanisms inneutrophils from patients with primary dysmenorrheamay help to clarify the disease etiology.

Ischemia is induced during uterine contraction becauseof decreased blood flow to the myometrium (Buhimschiet al., 1995). This can trigger the accumulation of free radi-cals, such as ROS (Sirmali et al., 2007). Free radicals arethe products of biological reduction reactions (Nazıroglu,2007), and overproduction of ROS has been implicated inthe pathogenesis of dysmenorrhea (Dikensoy et al., 2008).ROS can cause disease and cell damage by damaging othermolecules, including proteins, lipids, and DNA. Antioxi-dants such as superoxide dismutase (SOD), glutathioneperoxidase (GSH-Px), and catalase protect cells from ROSdamage (Nazıroglu, 2007, 2012; Kovacic and Somanathan,2008). ROS-induced changes to proteins and DNA can leadto altered cellular function or activation of proteolytic cas-cades that ultimately result in endometrial damage andinflammation (Güney et al., 2008; Güney, 2012).

Ca2+ is an important ion that controls several intracellu-lar processes, such as exocytosis, secretion, and apoptosis(Nazıroglu, 2007; Ayub and Hallett, 2004). In neutrophils,intracellular free Ca2+ ions control chemotaxis and adhe-sion (Korkmaz et al., 2011). Ca2+ entry also plays animportant role in the regulation of superoxide radical pro-duction by neutrophils (S ahin et al., 2011). Therefore, achange in intracellular Ca2+ levels in neutrophils directlyaffects the neutrophil response (Yamazaki et al., 2006).

Since there is no specific treatment for primary dysmen-orrhea, symptomatic and empirical treatment methodsare used. Non-steroidal anti-inflammatory drugs (NSAIDs)such as diclofenac are frequently used for the treatmentof inflammation and pain in a wide variety of disorders,including primary dysmenorrhea (Yamazaki et al., 2006;Harel, 2012). Diclofenac has been shown to be effec-tive in the treatment of primary dysmenorrhea, and itsuse is widespread. Although the mechanisms of actionof NSAIDs in patients with primary dysmenorrhea arenot yet fully understood, they have been shown to haveanti-inflammatory, antioxidant, and inhibitory effects oncardiac and neuronal cells (Yamazaki et al., 2006; Yarishkinet al., 2009), and diclofenac has been reported to inhibitvoltage-gated calcium channels (VGCC) in neonatal rat ven-tricular cardiomyocytes (Yarishkin et al., 2009).

In the present study, we investigated the mechanismsinvolved in neutrophil activation and inflammation in

patients with primary dysmenorrhea. Our first aim wasto research the importance of Ca2+ in the neutrophil cyto-sol in patients with primary dysmenorrhea and the effectof NSAID (diclofenac) treatment on neutrophil cytosolic

Immunology 100 (2013) 87– 92

Ca2+ release from intracellular stores evoked by N-formyl-l-methionyl-l-leucyl-l-phenylalanine (fMLP). Our secondaim was to investigate the effects of NSAID treatment onneutrophil lipid peroxidation and antioxidant status.

2. Subjects and methods

2.1. Patients and controls

The study was conducted at the Biophysics ResearchLaboratory, Suleyman Demirel University, Turkey. Thepatients enrolled in the study were selected from theGynecology Department of Suleyman Demirel Univer-sity, and they fulfilled the diagnostic criteria for primarydysmenorrhea (Proctor and Farquhar, 2006). Their maincomplaint was dysmenorrhea, and each patient underwenta detailed gynecological examination, pelvic ultrasound,and laboratory tests. The medical history of each patientwas recorded, and their pain score was monitored forthree ovulation cycles. The exclusion criteria for thepatients were the presence of inflammatory disease,fibromyalgia, premature coronary artery disease, diabetesmellitus, or hypertension. Six healthy controls were alsoincluded, and informed consent was obtained from allthe study participants. The patients and controls werewomen who were not undergoing hormone replacementtherapy and had not taken vitamin or mineral supple-ments for 6 months. They were non-smokers and didnot drink alcohol. Demographic characteristics, clinicalinformation, physical examination findings, and laboratorytests were recorded for all the subjects included in thestudy.

2.2. Study groups

Baseline blood samples were obtained from the patientand control groups (n = 6 in each). The patient group wasthen administered a 50-mg diclofenac potassium tabletdaily for 6 weeks (Dolerex; Abdi Ibrahim Medicine Inc.,Vefa, Istanbul, Turkey), and blood samples were obtained.

2.3. Isolation of neutrophils

After fasting overnight, 35-mL blood samples from theantecubital vein were drawn into tubes with an anticoagu-lant. Peripheral whole blood was obtained, and neutrophilswere isolated by centrifugation using Ficoll, as describedpreviously (S ahin et al., 2011).

2.4. Measurement of intracellular calcium concentration([Ca2+]i)

Neutrophils were loaded with fura-2 acetoxymethylester (fura-2/AM) by using a previously described method(Uguz et al., 2009). The neutrophils (5 × 106 cells/mL) wereincubated with 4 �M fura-2/AM in a loading extracel-

lular buffer for 45 min at 37 ◦C in the dark. They werethen washed twice, incubated for an additional 30 minat 37 ◦C to complete probe de-esterification, and resus-pended in loading buffer at a density of 3 × 106 cells/mL.

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groups. Ca2+ entry into the cytosol was also significantlylower in neutrophils post-diclofenac treatment exposed toV + D than in the control (p < 0.001), patient (p < 0.001), and

Ö. Kaplan et al. / Journal of Repro

he neutrophils were exposed to 1 �M fMLP to stimu-ate intracellular calcium (Ca2+

i) release. Fluorescence wasecorded from 2-mL aliquots of a magnetically stirredeutrophil suspension at 37 ◦C by using a spectrofluo-ometer (Carry Eclipsys; Varian Inc, Sydney, Australia)ith excitation wavelengths of 340 and 380 nm and an

mission wavelength of 505 nm. The intracellular calciumoncentration ([Ca2+]i) was monitored using the fura-240/380 nm fluorescence ratio, calibrated according to theethod described by Grynkiewicz et al. (1985). Ca2+ release

nanomolar) was estimated using the integral of the risen [Ca2+]i for 160 s, taking a sample every second after theddition of 1 �M fMLP, as previously described (Heemskerkt al., 1997; Espino et al., 2009).

In some experiments, neutrophils from the diclofenacreatment group were incubated with 0.01 mM of theGCC blockers verapamil and diltiazem (V + D) or the

ransient receptor potential (TRP) cation channel blocker 2-minoethoxydiphenyl borate (2-APB) at 0.1 mM for 30 minrior to the measurement of [Ca2+]i (Nazıroglu et al.,011a,b).

.5. Determination of lipid peroxidation

Thiobarbituric acid reactive substances were quan-ified by comparing the absorption with the standardurve of malondialdehyde (MDA) equivalents generated bycid-catalyzed hydrolysis of 1,1,3,3 tetramethoxypropanePlacer et al., 1966). The pink-colored chromogen formedy the reaction of thiobarbituric acid with lipid peroxida-ion (LP) breakdown products was measured spectropho-ometrically (Shimadzu UV-1800; Shimadzu Corp., Kyoto,apan) at a wavelength of 535 nm. The levels of LP in theeutrophil samples were expressed as �mol/g protein.

.6. Reduced glutathione, glutathione peroxidase, androtein assay

The glutathione (GSH) content of the neutrophil sam-les was measured at 412 nm by using the method of Sedlaknd Lindsay (1968) and expressed as �mol/g protein. Quan-ification of glutathione peroxidase (GSH-Px) activity waserformed using the method described by Lawrence andurk (1971), and the data were expressed as internationalnits (IU)/g protein. The protein content in neutrophil sam-les was measured by the method of Lowry et al. (1951),ith bovine serum albumin as the standard.

.7. Total antioxidant status determination

The neutrophil total antioxidant status (TAS) levels wereeasured colorimetrically by using the TAS kit (Mega

ip Inc., Gaziantep, Turkey) (Erel, 2004). The results werexpressed as �mol H2O2 equivalent/g protein (�mol H2O2quiv/g protein).

.8. Statistical analyses

All results are expressed as means ± SD. Data werenalyzed using the SPSS statistical program (version 17.0oftware; SPSS Inc., Chicago, IL, USA). P values less than

Immunology 100 (2013) 87– 92 89

0.05, determined using the Mann–Whitney U test, wereregarded as statistically significant.

3. Results

3.1. Results of the demographic values

The present study included 6 patients with primarydysmenorrhea and 6 healthy controls. The mean age ofthe study subjects was 23.3 ± 2.34 years in the primarydysmenorrhea group and 22.6 ± 2.39 years in the controlgroup. There was no statistically significant differencebetween the ages of these groups.

3.2. LP and antioxidant results

The neutrophil levels of LP, TAS, GSH, and GSH-Px areshown in Table 1. LP levels were significantly higher inthe neutrophils of the dysmenorrhea patients than in theneutrophils of the controls (p < 0.01). There was also a sig-nificant difference in the LP levels of the patients before andafter treatment with diclofenac, with significantly less LPpost-treatment (p < 0.01). Neutrophil TAS level and GSH-Pxactivity were significantly lower in the patient group thanin the control group (p < 0.05). These antioxidant meas-ures were also significantly lower in the patient groupafter treatment with diclofenac (p < 0.05 for TAS, p < 0.001for GSH-Px activity). GSH-Px activity was also significantlyhigher after diclofenac treatment than in the control group(p < 0.001). No significant differences in GSH levels werefound among patients, controls, or patients after diclofenactreatment.

3.3. Effects of diclofenac on fMLP-stimulated [Ca2+]iconcentration in neutrophils

The effects of diclofenac on cytosolic [Ca2+]i in neu-trophils are shown in Figs. 1 and 2. The increase inneutrophil [Ca2+]i was significantly higher in the patientgroup than in the control group (p < 0.001). The increase inneutrophil [Ca2+]i was significantly lower post-diclofenactreatment (p < 0.001).

3.4. Effects of VGCC and TRP channel blockers onfMLP-stimulated Ca2+

i release in neutrophils

The effects of VGCC and TRP channel blockers on [Ca2+]iin neutrophils are also shown in Figs. 1 and 2. The increasein [Ca2+]i was significantly lower in the neutrophils post-diclofenac treatment exposed to 2-APB than in the control(p < 0.001), patient (p < 0.001), and treatment (p < 0.05)

treatment (p < 0.001) groups. The effect of V + D on cytosolicCa2+ was greater than that of 2-APB (p < 0.001), indicat-ing that VGCC had a larger modulatory effect than TRPchannels.

90 Ö. Kaplan et al. / Journal of Reproductive Immunology 100 (2013) 87– 92

Table 1Neutrophil lipid peroxidation (LP), reduced glutathione (GSH), glutathione peroxidase (GSH-Px), and total antioxidant status (TAS) values in controls andpatients with primary dysmenorrhea before and after NSAID treatment (mean ± SD, n = 6).

Antioxidant parameters Control group Patients without diclofenac treatment Patients post-diclofenac treatment

LP (�mol/g protein) 5.63 ± 0.73 6.97 ± 0.51b 6.18 ± 0.31e

TAS (�mol H2O2 equiv/g protein) 4.98 ± 1.19 3.77 ± 0.55a 4.92 ± 0.85d

GSH (�mol/g protein) 1.73 ± 0.09 1.78 ± 0.21 1.79 ± 0.08GSH-Px (IU/g protein) 4.74 ± 0.99 4.07 ± 0.62a 7.09 ± 0.94c,f

ap < 0.05, bp < 0.01, and cp < 0.001 versus control group.dp < 0.05, ep < 0.01, and fp < 0.001 versus patient group.

Fig. 1. Effects of NSAID treatment on intracellular Ca2+ release in neutrophils from controls and patients with primary dysmenorrhea. Stimulation wasperformed using 1 �M fMLP. ap < 0.001 versus controls; bp < 0.001 versus patients; cp < 0.05 and dp < 0.001 versus treatment group; ep < 0.001 treatment + 2-APB.

nd pati

Fig. 2. Effects of NSAID treatment on [Ca2+]i in neutrophils from controls afMLP.

4. Discussion

It is well known that increased neutrophil activation

induces the release of inflammatory factors. Although it isknown that there is increased inflammation through neu-trophil activation in patients with primary dysmenorrhea,the mechanisms underlying this neutrophil activation are

ents with primary dysmenorrhea. Stimulation was performed using 1 �M

unclear. In this study, we aimed to investigate these mech-anisms to improve understanding of the etiopathogenesisof dysmenorrhea.

When a bacterial infection occurs, increased [Ca2+]idue to release from intracellular stores and entry intoneutrophils via VGCC and TRP channels is a key initia-tor of activation, leading to increased ROS production and

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roviding a means of attacking the bacteria (Dikensoy et al.,008; Bréchard and Tschirhart, 2008). In chronic inflam-atory disease, however, this mechanism contributes to

he development of oxidative damage. Increased produc-ion of ROS through NADPH oxidase activation and releasef myeloperoxidase (MPO) enzyme occur at the site ofndometrial damage. MPO aggravates ROS toxicity by pro-ucing hypochlorous acid, a more potent oxidant thatontributes to neutrophil and tissue oxidation (Ayub andallett, 2004). Increased ROS in the neutrophils causes DNAamage (Bréchard and Tschirhart, 2008).

To our knowledge, there are no previous reports ofeutrophil [Ca2+]i in patients with primary dysmenor-hea. However, previous studies of neutrophil activation inatients with inflammatory familial Mediterranean fevernd Behcet’s disease (Korkmaz et al., 2011; S ahin et al.,011) have reported higher neutrophil [Ca2+]i in patientroups than in the controls. Therefore, it is thought that anncrease in neutrophil [Ca2+]i may have an effect on diseasetiopathogenesis.

Non-steroidal anti-inflammatory drugs are frequentlysed to treat primary dysmenorrhea because of their anal-esic and anti-inflammatory properties. However, little isnown about the effects of NSAIDs on neutrophil func-ion in this condition. In the present study, we foundhat treatment with the NSAID diclofenac decreased theeutrophil [Ca2+]i response to fMLP in patients with pri-ary dysmenorrhea. This response was decreased further

n neutrophils from diclofenac-treated patients analyzedn the presence of V + D or 2-APB. Both types of calciumhannel antagonists were used to investigate which washe most important in this context. The high and lowGCCs were blocked using V + D (Shima et al., 2008), and-APB was used as a non-specific TRP cation channellocker (Togashi et al., 2008; Nazıroglu et al., 2011a,b).he data from this study indicated that blockade of eitherGCC or TRP had significant effects on neutrophil [Ca2+]iesponse to fMLP, although VGCC blockade produced theargest reduction. A previous study also reported thaticlofenac, with or without nifedipine, inhibited VGCC

n rat ventricular cardiomyocytes in a whole-cell voltagelamp technique study (Yarishkin et al., 2009). Yamazakit al. (2006) reported that the anti-apoptotic effectsf diclofenac occurred independently of its effects onOX-2 activity, via modulation of endoplasmic reticulumtress.

We found that neutrophil LP levels in patients with pri-ary dysmenorrhea were higher than those in the controls,hereas the levels of TAS and GSH-Px activity were low.

o our knowledge, LP, GSH, TAS, and GSH-Px have not pre-iously been studied in the neutrophils of patients withrimary dysmenorrhea. In a study by Dikensoy et al. (2008),erum LP, nitric oxide, and adrenomedullin levels werehown to be significantly increased in patients with pri-ary dysmenorrhea. In another study, the levels of MDA

nd interleukin-6 were elevated in patients with primaryysmenorrhea (Yeh et al., 2004). In a study by Akdemir

t al. (2010), levels of serum nitric oxide and asymmetricimethylarginine (as a marker of endothelial dysfunctionnd nitrogen radicals) were observed to be significantlyigher in patients with primary dysmenorrhea than in

Immunology 100 (2013) 87– 92 91

the control group. Taken together, these results indicatedreduced antioxidant activity in primary dysmenorrhea.

Although oxidative stress and inflammation play impor-tant roles in patients with primary dysmenorrhea, preciselyhow these are connected with neutrophil activation in thedisease pathogenesis is not known. In the present study,we aimed to produce more robust results than those ofprevious studies by examining multiple indicators of oxida-tive stress (LP, TAS, and GSH-Px) in neutrophils, whichplay an important role in disease pathogenesis, rather thanin sera, erythrocytes, or tissues that can be affected byother factors. As neutrophils are nucleated cells, an increasein [Ca2+]i and oxidative stress leading to DNA damagemay affect neutrophil activation (Bréchard and Tschirhart,2008; Korkmaz et al., 2011). Selenium-dependent GSH-Pxis a major antioxidant enzyme involved in the eliminationof free radicals. GSH-Px detoxifies hydrogen peroxide toform water (Nazıroglu, 2009). If antioxidants are incapableof eliminating ROS, lipid peroxidation (LP) begins (Kovacicand Somanathan, 2008). The values of neutrophil GSH-Pxand TAS were lower in patients with primary dysmenor-rhea than in the controls, in the absence of any significantdifference in GSH level; this supported the suggestion thatan imbalance between ROS production and the antioxi-dant system may play a role in disease pathogenesis. Inthe current study, the LP levels of the patient group werealso higher than those of the control group. Since therewas a significant difference in the LP levels between thetreatment group and the control group, it is thought thatthe increase in oxidative stress may be responsible for theetiology of primary dysmenorrhea.

In conclusion, we found that the NSAID diclofenac mod-ulated oxidative stress and Ca2+ levels in neutrophils frompatients with primary dysmenorrhea. This modulatory rolewas exerted through VGCC and TRP cation channels. This isthe first report of the protective effect of NSAID admin-istration against excess oxidative stress and Ca2+ levels.These data indicate the possibility of developing a newapplication for NSAIDs as modulators of oxidative stress inneutrophils. Future studies could target TRP channels andVGCC for the development of new treatment approaches toprimary dysmenorrhea.

Authorship

MN formulated the present hypothesis and was respon-sible for writing the report. ÖK and MA were responsiblefor data analyses. MG made critical revisions to themanuscript.

Conflict of interest statement

The authors declare that there are no conflicts of inter-est.

Acknowledgments

The study was partially supported by the ScientificResearch Unit of Suleyman Demirel University (ProtocolNumber: 3055-TU-12). An abstract of the study was sub-mitted as a poster presentation to the “4th International

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neonatal rat ventricular cardiomyocytes. Korean J. Physiol. Pharmacol.

92 Ö. Kaplan et al. / Journal of Repro

Congress on Cell Membranes and Oxidative Stress Focus on:Calcium Signaling and TRP Channels,” June 26–29, 2012,Isparta, Turkey (www.cmos.org.tr).

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