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Original Article

Gynecol Obstet Invest 2011;72:37–42

DOI: 10.1159/000322393

Effects of 4-Hydroxy-2-Nonenal, a MajorLipid Peroxidation-Derived Aldehyde, andN-Acetylcysteine on the Cyclooxygenase-2Expression in Human Uterine Myometrium

Kumiko Temma-Asano a Ekaterine Tskitishvilia Takeshi Kanagawaa

Takuji Tomimatsua Tateki Tsutsuia Tadashi Kimuraa Yang Sil Changb

Takafumi Nakamurab

Yuichiro Nakaib

Koichiro Shimoyab

a Department of Obstetrics and Gynecology, Osaka University Graduate School of Medicine, Osaka, andb Department of Obstetrics and Gynecology, Kawasaki Medical School, Kurashiki City , Japan

was measured by ELISA. Results: 4-HNE induced the COX-2

mRNA expression and PGE2 production in the uterine myo-

metrial tissue culture in a dose-dependent and time-depen-

dent manner. NAC inhibited 4-HNE-induced COX-2 expres-

sion. Conclusion: 4-HNE may play an important role in pre-

term labor. NAC might be protective against preterm labor

under oxidative stress. Copyright © 2011 S. Karger AG, Basel

Introduction

Preterm delivery remains the most important cause ofperinatal morbidity and mortality despite recent advanc-es in tocolytic treatment and neonatal intensive care.Most of spontaneous preterm deliveries are associated

with intrauterine infection of bacteria that passagesthrough the cervix from the vagina. The bacteria appearto ascend first into the choriodecidual space, then crossthe chorioamniotic membranes into the amniotic fluid[1, 2]. Bacterial endotoxins activate the decidua and fetalmembranes to produce a number of cytokines. Variouscytokines such as interleukin (IL)-1, IL-6 and IL-8 areproduced by the placenta [3–5]. Such elevated placentalcytokines result in elevation of fetal and amniotic fluid

Key Words

Prostaglandin Preterm labor Oxidative stress

Myometrium N-Acetylcysteine 4-Hydroxy-2-nonenal

Cyclooxygenase-2

Abstract

Background: Chorioamnionitis is one of the important

causes of preterm labor. Preterm labor with chorioamnio-

nitis is associated with oxidative stress. We reported that

4-hydroxy-2-nonenal (4-HNE), a major end product of oxida-

tive fatty acid metabolism, is accumulated in the placenta

with chorioamnionitis. The aim of this study was to confirm

the effect of 4-HNE on cyclooxygenase-2 (COX-2) and pros-

taglandin (PG) induction in the uterine myometrial tissues.

We also examined the effect of N-acetylcysteine (NAC) on

4-HNE-induced COX-2 expression. Methods: Uterine myo-metrial tissues were obtained from 5 patients. One of them

underwent elective cesarean section without labor, and 4 of

them underwent hysterectomy because of placental previa

or atonic bleeding. We stimulated the uterine myometrial

tissues with 4-HNE. In addition, the tissues were pretreated

with NAC before 4-HNE treatment. The expression of COX-2

mRNA was observed by real-time PCR. PGE2 and prosta-

cyclin release into the supernatants of the tissue cultures

Received: April 19, 2010

Accepted after revision: October 29, 2010

Published online: January 6, 2011

Koichiro Shimoya, MD, PhDDepartment of Obstetrics and GynecologyKawasaki Medical School577 Matsushima, Kurashiki City 701-0192 (Japan)Tel. +81 86 462 1111, Fax +81 86 462 1199, E-Mail shimoya @ med.kawasaki-m.ac.jp

© 2011 S. Karger AG, Basel0378–7346/11/0721–0037$38.00/0

Accessible online at:www.karger.com/goi



Temma-Asano et al.Gynecol Obstet Invest 2011;72:37–4238

cytokine concentrations [6, 7]. Activated fetal immuno-competent cells also contribute to elevation of the cyto-kine concentrations in the cord serum [8]. These process-es participate in the fetomaternal defense mechanism.On the other hand, the cytokines and endotoxins stimu-late prostaglandin (PG) synthesis causing uterine myo-

metrial contractions. This is believed to be one of the po-tential pathways from bacterial invasion to preterm de-livery.

In infectious diseases such as chorioamnionitis(CAM), activated phagocytes migrate from the maternalcirculation to the fetal membranes, where they encircleand digest bacteria in order to protect the mother [9]. Inthe next step, powerful reactive oxygen species are pro-duced by phagocyte NADPH oxidase [10]. During theoxidative stress, reactive aldehydes such as 4-hydroxy-2-nonenal (4-HNE) are generated by a free radical chainreaction mechanism [11]. We have already reported that

4-HNE, a major end product of oxidative fatty acid me-tabolism, is accumulated in the placenta with CAM [12].We also reported that oxidative stress affects sEndoglinand S100B protein expression from villous and amniotictissues [13]. Oxidative stress has been postulated to be in-

volved in the pathophysiology of numerous diseases [14,15]. In some of these diseases, cyclooxygenase-2 (COX-2)or PG overexpression has been detected [16–18]. Pretermlabor in CAM may be associated with oxidative stress.The fetal membrane mainly produces uterotonic PGs. Wehave already reported that the fetal tissues produced PGE2 (PGE2) in the human. The predominant myometrialPG produced just prior to labor is prostacyclin (PGI2), asmooth muscle relaxant. Activation of PGI2 receptor up-regulated the expression of several contractile proteinsand the gap junction protein connexin 43 through cAMP/PKA signaling in human myometrial tissue in organ andcell culture [19]. We have hypothesized that the lipid per-oxidation product 4-HNE produced at CAM may con-tribute to preterm labor, along with the upregulation ofCOX-2. In the present study, we confirmed the effect of4-HNE in uterine myometrium on COX-2 expressionand PG synthesis, such as PGE2 and PGI2. The regula-

tion of uterotonic PGs and uterorelaxant PGs in the myo-metrium is important for understanding the regulationmechanism of preterm labor. N-Acetylcysteine (NAC) isa thiol-containing antioxidant that increases intracellu-lar glutathione (GSH) concentrations or acts as a free rad-ical scavenger. Oral NAC was found to reduce the recur-rence of preterm birth in patients with bacterial vaginosis[20]. We also examined the effect of NAC on 4-HNE-in-duced COX-2 expression in human uterine myometrium.

Materials and Methods

SamplesUterine myometrial tissues were obtained from 5 postpartum

women. One of them underwent elective cesarean section withoutlabor, and 4 of them underwent hysterectomy because of placentalprevia or atonic bleeding. This study was approved by the localethics committee of the Department of Obstetrics and Gynecol-ogy, Osaka University Graduate School of Medicine. Informedconsent was obtained from each patient.

Tissue Culture and Effect of HNE in Myometrial TissueThe myometrial tissues were thoroughly washed and separat-

ed from the serosa and the endometrium to produce tissue blocks3 mm in diameter (about 100 mg). The samples were incubatedwith 1 ml of medium (RPMI 1640 containing 10% heat-inactivat-ed fetal bovine serum and 1% penicillin) at 37° C in an atmosphereof 95% air and 5% CO2 in 24-well flat-bottomed microplates. Af-ter 12 h of culture, culture medium was removed and the tissueswere treated with various concentrations of 4-HNE. After the tis-sues were cultured for a specific period, the supernatants and tis-sues were collected and frozen in liquid nitrogen and stored at–80 ° C until use.

Effect of NAC in Myometrial Tissue on 4-HNE-Induced COX-2 ExpressionThree samples of tissue cultures were pretreated with various

concentrations of NAC (0, 5, 10, 20, and 30 mM ), 1 h before 4-HNEtreatment (25 M ). After the medium was removed, the tissueswere cultured for 18 h and the supernatants and tissues were col-lected and frozen in liquid nitrogen and stored at –80 ° C until use.

RNA ExtractionTotal RNA was extracted from frozen myometrial samples

with Trizol regent according to the manufacturer’s recommen-dations (Invitrogen, Carlsbad, Calif., USA). RNA samples weretreated with RNase-free DNase I to preclude genomic DNA con-tamination. RNA integrity was confirmed by agarose gel electro-phoresis, and quantified by spectrophotometric analysis.

Real-Time PCROne microgram of total RNA was reverse transcribed using

the Superscript II for RT-PCR according to the manufacturer’srecommendations (Invitrogen). Real-time quantif ication of COX-2 mRNA was performed with a TaqMan real-time PCR assay, cal-culating relative gene expression with normalization of results forGAPDH mRNA.

Protein AssayTo determine protein concentration of the supernatants of

tissue culture, Pierce BCA protein assay kit (Thermo scientific,Rockford, Ill., USA) was used.

Enzyme-Linked Immunosorbent Assay of PGE2To measure the level of PGE2 in the culture medium of tissues

treated with 4-HNE (25 M ), a PGE2 enzyme-linked immunosor-bent assay (ELISA) kit-monoclonal (Cayman Chemical, Ann Ar-bor, Mich., USA) was used. The intra- and interassay CVs were! 10%.



Effect of 4-HNE and NAC on COX-2 in

the Uterus

Gynecol Obstet Invest 2011;72:37–42 39

Enzyme-Linked Immunosorbent Assay of PGI2To measure the level of PGI2 in the culture medium of tissues

treated with 4-HNE (25 M ), a PGI2 EIA kit (Usen Life Science,Inc., Wuhan, China) was used. The intra- and interassay CVs were! 10%.

Statistical AnalysisThe data were subjected to one-way analysis of variance using

the Statview statistics package (Abacus Concepts, Inc., Berkeley,Calif., USA). p ! 0.05 was considered significant.

Results

We stimulated myometrial tissues with 4-HNE at theconcentration of 0, 10, 25, 50 M and analyzed the dose-dependent effect of 4-HNE on COX-2 expression by real-time PCR. As shown in figure 1, COX-2 mRNA expres-sion was significantly increased compared with controlwithout treatment of 4-HNE, and the maximal expres-sion was observed at 25 M (1.62-fold of the control;

p = 0.04). Figure 2 demonstrates the kinetics of COX-2mRNA expression. COX-2 mRNA expression was signif-icantly increased in a time-dependent manner. The max-imal expression was observed during 12 h of incubation.We also measured the amount of PGE2 released into thesupernatant of medium by ELISA. PGE2 concentrationin the supernatant increased in a time-dependent fashion(fig. 3). There was no significant difference in the re-sponse of PGE2 release by 4-HNE treatment between

each tissue. The PGI2 concentrations in the supernatantstimulated with 4-HNE at 0, 2, 4, 8, and 18 h after treat-ment were 11.68 5.2, 13.08 5.2, 13.98 7.2, 20.38 18.7,and 21.18 19.1 pg/mg protein, respectively. There wasno significant effect on PGI2 release of myometrial cul-ture stimulated by 4-HNE. Preterm labor is thought to be

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Fig. 1. Dose-dependent induction of COX-2 mRNA in the uterinetissues treated with 4-HNE for 6 h. Real-time quantification ofCOX-2 mRNA was performed with TaqMan real-time PCR assay.Gene expression level was normalized to GAPDH and expressedrelative to control without 4-HNE (five tissue samples in each

group, error bar represents SE, * p = 0.04).

Fig. 2. Time-dependent induction of COX-2 mRNA in the uterinetissues treated with 4-HNE. Real-time quantification of COX-2mRNA was performed with TaqMan real-time PCR assay. Geneexpression level was normalized to GAPDH and expressed rela-tive to the COX-2 expression before 25 M 4-HNE treatment (five

tissue samples in each group, error bar represents SE, * p ! 0.05).

Fig. 3. Time-dependent production of PGE2 by 4-HNE. Uterinetissues were incubated with 25 M 4-HNE, and the amount ofPGE2 released into the medium was measured by ELISA. The datawere adjusted by the protein concentrations in the supernatants(five tissue samples in each group, error bar represents SE, * p ! 0.01).




associated with the upregulation of PGE2, which is syn-thesized by COX-2. Our findings therefore suggest thatuterine contraction and cervical ripening may be inducedunder oxidative stress, resulting in preterm labor.

To determine whether NAC inhibits 4-HNE-inducedCOX-2 expression, we examined the effect of pretreat-ment with NAC. As shown in figure 4, COX-2 mRNAexpression was significantly inhibited by NAC pretreat-ment. PGE2 released into the supernatant was also de-creased by NAC pretreatment (fig. 5). In figure 4, we dem-onstrate mRNA levels of the tissues treated with 4-HNEfor 6 h, and in figure 5 we show PGE2 release in the su-pernatants stimulated by 4-HNE for 18 h. The discrep-ancy of mRNA and PGE2 protein might be dependent onthe difference in kinetics.

Discussion

4-HNE is a toxic aldehyde generated by lipid peroxida-tion contributing to the deleterious effects of oxidativestress [21, 22]. It has been shown to be involved in thepathogenesis of various diseases such as Alzheimer’s dis-ease, atherosclerosis, alcoholic hepatitis and cancer [23–26]. Some evidence suggested that 4-HNE can affect in-tracellular signaling cascades in a concentration-depen-dent manner [27]. Low levels of 4-HNE have been shownto promote proliferation of cells [28] and induce various

enzymes including protein kinase C, phospholipase Cand adenylate cyclase [27]. High concentrations of 4-HNEcan cause cell cycle arrest, differentiation and apoptosis[29]. We previously reported that 4-HNE was accumu-lated in the placenta with CAM and had the effect of COX-2 induction in chorion tissues [12]. In this study, we firstconfirmed that 4-HNE induced the COX-2 expressionand PGE2 synthesis in uterine myometrial tissues as wellas chorion. The myometrium produces various PGs, suchas PGI2 [19]. PGI2 is a strong relaxant of myometrium.We measured PGI2 in the supernatants treated with 4-HNE, but PGI2 production was not affected by 4-HNEtreatment. These results suggest that 4-HNE has an effecton PGE2 production, but not on PGI2 production. Themaximum response of COX-2 mRNA induction was ob-served at the concentrations of 25 M 4-HNE. Ruef et al.[29] reported that 100 M 4-HNE induced the apoptosisof vascular smooth muscle cell. Higher concentrations of4-HNE might have the toxicity of cells and tissues. Oxida-

tive stress induces COX-2 expression through the produc-tion of 4-HNE which activates p38MAPKinase, suggest-ing that 4-HNE links oxidative stress and chronic inflam-mation through the activation of cyclooxygenase [30].

To determine the role of induced COX-2 mRNA, weinvestigated the release of PGE2 in the supernatants ofculture medium. Time-dependent increase in PGE2 inthe supernatant was also observed. These results suggest-ed that 4-HNE induced COX-2 expression and PGE pro-

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Fig. 4. Effect of NAC pretreatment on HNE-induced COX-2 in-duction. Uterine tissues were incubated with 0–20 mM NAC for1 h, and then treated with 25 M 4-HNE for 6 h. COX-2 inductionwas examined by real-time PCR analysis. Gene expression levelwas normalized to GAPDH and expressed relative to control

without 4-HNE treatment (three tissue samples in each group, er-ror bar represents SE, * p ! 0.01).

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Fig. 5. Effect of NAC pretreatment on 4-HNE-induced PGE2 re-lease. Uterine tissues were incubated with 0–30 mM NAC for 1 h,and then treated with 25 M 4-HNE for 18 h. The amount of PGE2released into the medium was measured by ELISA (three tissuesamples in each group, error bar represents SE, * p ! 0.05, * * p !

0.01).



Effect of 4-HNE and NAC on COX-2 in

the Uterus

Gynecol Obstet Invest 2011;72:37–42 41

duction in the uterine myometrium. PGs such as PGE2act to mediate cervical ripening and stimulate uterinecontraction [31]. Our studies suggest that 4-HNE gener-ated in CAM may increase PGs within maternal myome-trium leading to infection-induced preterm labor.

NAC is a membrane-permeable cysteine precursor

that does not require active transport and delivers cyste-ine to the cell [32]. NAC is an antioxidant and a free rad-ical scavenger that increases intracellular GSH concen-trations [33]. The efficacy of NAC as an antioxidant hasgenerally been reported. NAC has been shown to have thebeneficial effect on nervous system ischemia and isch-emia/reperfusion, and the preventive effects on trauma-induced oxidative brain tissue damage [34]. Arakawa etal. [35] have reported that NAC exerts significant protec-tive effects against 4-HNE-induced neuronal cell death.Tam et al. [36] have shown that oxidative injuries in pros-tatic tumorigenesis can be modulated by antioxidants

such as NAC, which is used as dietary supplements. In thefield of obstetrics, it was reported that maternal admin-istration of NAC reduced the rate of preterm birth in miceassociated with inflammation [37]. The effect of NAC onlipopolysaccharide (LPS)-induced preterm labor and fe-tal demise in murine model was investigated. NAC pre-treatment abolished these effects of LPS in the placenta[38]. We then investigated the effect of NAC on 4-HNE-induced COX-2 expression. 4-HNE-induced COX-2 ex-pression and PG production were inhibited by NAC pre-treatment in a concentration-dependent manner. Most ofcellular 4-HNE is metabolized via its conjugation to GSHthrough reactions catalyzed by glutathione S-transferas-es. The resulting conjugate of GSH and 4-HNE can be

further metabolized by aldose reductase or RLIP76 [39].NAC, an antioxidant and GSH precursor, could inhibitthe effect of 4-HNE on signaling.

Our findings suggest that uterine contraction may beinduced under oxidative stress, resulting in preterm la-bor. In preterm labor, CAM induces oxidative stress, and

oxidative stress produces 4-HNE in the chorion and fetalmembranes. 4-HNE might play a role in the preterm la-bor in CAM via COX-2 induction. NAC might have a pro-tective effect against preterm labor. Ongoing trials areassessing whether antioxidant supplementation is bene-ficial to pregnant women. A recent article reported thatsupplementation with vitamins C and E during pregnan-cy does not reduce the risk of outcomes [40]. Oral NACwas found to reduce the recurrence of preterm birth inpatients with bacterial vaginosis [20]. However, the sam-ple size was too small to detect whether NAC is beneficialduring pregnancy. NAC has low toxicity and is approved

by the Food and Drug Administration. NAC might pre- vent preterm birth and protect preterm labor in the high-risk patients. Further investigations will be necessary toexamine the ability of antioxidants such as NAC to pre-

vent preterm birth in CAM.

Acknowledgements

This work was supported, in part, by Grant-in-Aid for Scien-tif ic Research (No. 21592118) from the Ministry of Education, Sci-ence, and Culture of Japan (Tokyo, Japan), Health Labor Sciences

Research Grant of Research on Child and Families (Tokyo, Ja-pan), and the grants of Kawasaki Medical School Project 21-411(Kurashiki, Japan).

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