Introduction. Shuko Tokuriki 1 Aiko Igarashi. Hironobu Naiki 2 Yusei Ohshima

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1 Lung (2017) 195: DOI /s ACUTE LUNG INJURY Tretment with Gernylgernylcetone Induces Het Shock Protein 70 nd Attenutes Neontl Hyperoxic Lung Injury in Model of Bronchopulmonry Dysplsi Shuko Tokuriki 1 Aiko Igrshi 1 Tkshi Okuno 1 Genrei Oht 1 Hironou Niki 2 Yusei Ohshim 1 Received: 13 Ferury 2017 / Accepted: 17 April 2017 / Pulished online: 26 April 2017 Ó The Author(s) This rticle is n open ccess puliction Astrct Purpose Bronchopulmonry dysplsi (BPD) is respirtory compliction chrcterized y norml lveolr development in premture infnts. Gernylgernylcetone (GGA) cn induce het shock protein (HSP) 70, which hs cytoprotective effects ginst vrious stressors. Here, we investigted whether GGA protected neontl lungs from hyperoxic stress in murine BPD model, nd mesured the serum HSP70 levels in preterm humns treted with oxygen. Methods Neworn mice were exposed to [90% oxygen nd dministered GGA or vehicle lone orlly on dys 1, 2, nd 3 of life. At 2 dys of ge, HSP70 expression in the lung ws determined y western lotting. At 8 dys of ge, the lungs were processed for histologicl nlysis. Rdil lveolr count (RAC) nd men liner intercept (MLI) were mesured s prmeters of lveolriztion. Apoptosis ws evluted y the terminl deoxynucleotidyl trnsferse-medited dutp nick end leling (TUNEL) method nd cleved cspse-3 immunohistochemistry. Serum HSP70 levels in preterm humns treted with oxygen were mesured y enzyme-linked immunosorent ssy. Results GGA dministrtion enhnced the HSP70 expression to two-fold compred with normoxi-exposed nd vehicle-treted mice. Hyperoxi reduced HSP70 & Shuko Tokuriki shu@u-fukui.c.jp 1 2 Deprtment of Peditrics, Fculty of Medicl Sciences, University of Fukui, 23-3 Shimoizuki, Mtsuok, Eiheijicho, Yoshid-gun, Fukui , Jpn Deprtment of Pthologicl Sciences, Fculty of Medicl Sciences, University of Fukui, 23-3 Shimoizuki, Mtsuok, Eiheiji-cho, Yoshid-gun, Fukui , Jpn expression, wheres GGA rogted the effects. Hyperoxi-exposed mice exhiited more poptotic cells in lung prenchym nd more simplified lveolr structure with less RAC nd lrger MLI thn normoxi-exposed mice. GGA suppressed the increse in poptotic cells nd the structurl chnges of the lungs induced y hyperoxi. Serum HSP70 levels of preterm humn infnts grdully decresed with ge. Conclusions GGA my ttenute hyperoxic injury in neontl lungs nd therey my prevent the development of BPD. Keywords Bronchopulmonry dysplsi Gernylgernylcetone Hyperoxi Alveolriztion Introduction Bronchopulmonry dysplsi (BPD) is mjor respirtory compliction in premture infnts [1]. The etiology of BPD is multifctoril, nd includes oxygen toxicity, ventiltorinduced pulmonry injury, nd ntentl nd/or postntl inflmmtion [2, 3]. BPD is chrcterized y poor lveolriztion nd vsculriztion in the developing lungs of immture infnts [4]. Currently, effective postntl therpies re limited nd new therpeutic interventions to prevent or tret BPD re required. Het shock proteins (HSPs) constitute fmily of intrcellulr proteins ctivted y vriety of stressors, including ischemi nd inflmmtion. HSPs ssist in delivering trget proteins to the uiquitin protesome system for degrdtion together with cochperone molecules [5, 6]. Among them, HSP70 hs cytoprotective function vi nti-poptotic nd nti-inflmmtory effects in vitro nd in vivo. Incresed expression of HSP70

2 470 Lung (2017) 195: ttenutes hyperoxi-induced lipid peroxidtion nd ATP depletion in humn respirtory epithelil cells in vitro [7]. HSP70-deficient mice exhiit incresed mortlity under hyperoxi, nd denovirl delivery of HSP70 to the lungs effectively rescues oth HSP70-deficient nd wild-type mice [8]. The induction of HSP70 is potentil therpeutic trget ginst severl stressor-induced lung injuries. Gernylgernylcetone (GGA), n cyclic polyisoprenoid widely used for ulcer therpy, induces HSP70 expression [9, 10]. GGA exhiits cytoprotective nd ntiinflmmtory effects in leomycin-induced lung firosis mouse models [11, 12] nd reduces hydrogen peroxideinduced poptotic cell deth y enhncing HSP production [13]. GGA prevents oxidtive stress in liver, rin, kidney, nd retin. However, whether GGA dministrtion cn prevent hyperoxi-induced neontl lung injury remins uncler. Thus, we exmined the effects of GGA on lveolriztion of neontl lungs exposed to hyperoxi in murine BPD model. Methods Animls nd Study Design C57BL/6J mice were otined from Chrles River Jpn (Shizuok, Jpn). Neontl pups were rndomly divided into four groups sed on hyperoxi exposure nd GGA tretment. The pups were kept in room ir (normoxi) unless exposed to hyperoxi with n oxygen concentrtion of [90% in 46.7-liter Plexigls exposure chmer (Fukui, Jpn) from 1 to 3 dys of ge. Mternl mice were swpped every 24 h etween normoxi nd hyperoxi groups. Ech mother ws given four to eight pups. The pups were treted with 500 mg/kg/dy of GGA (Tokyo Chemicl Industry Co., Tokyo, Jpn) or 10 ml/kg of vehicle lone (5% ric gum nd 0.6% Tween 80) during the 3 dys of hyperoxi exposure. As the ody weight of 1 3-dy-old pups ws only g, we dministered 10 ll of 5% GGA solution (50 mg/ml) per 1 g of ody weight. All pups were nesthetized y isoflurne inhltion nd orlly dministered GGA or vehicle using the outer cylinders of 24-guge indwelling needles. In order to confirm relile dministrtion of GGA or vehicle into their stomch, orlly dministered solution ws stined lue using food-coloring gent. GGA emulsified in vehicle ws freshly prepred nd then dministered to the mice once dy. At 2 nd 8 dys of ge, pups were scrificed to otin lung tissue. The study protocol ws pproved y the Experimentl Animl Ethics Committee t Fukui University (Permit No ). Study Popultion of Humn Neontes nd Mesurement of Serum HSP70 Between Mrch 2012 nd April 2015, 347 infnts were dmitted to the Neontl Intensive Cre Unit, Growing Cre Unit, or Nursery t the University of Fukui Hospitl. Twenty-six infnts were excluded due to deth, chromosoml normlity, metolic diseses, severe intrventriculr hemorrhge, nd surgicl diseses or hospitl trnsfer efore 1 month of ge. Fifty-seven of the remining 321 infnts, of which serum smples hd een otined etween postntl dys 0 nd 3, were enrolled in this study. Of these, serum smples of 10 infnts orn t \32 weeks of gesttionl ge hd lso een otined etween postntl dys 28 nd 30. Demogrphic nd clinicl dt regrding the infnts perintl nd postntl courses were extrcted from the medicl records. The independent vriles were gesttionl ge, irth weight, sex, prentl steroid use, Apgr scores t 1 nd 5 min, surfctnt use, the presence of histologiclly determined choriomnionitis (Stge 2 or 3) [14], nd BPD, defined s oxygen dependence t 36 weeks of postmenstrul ge. The smples were frozen t -20 C until they were nlyzed. HSP70 levels were mesured in duplicte using enzyme-linked immunosorent ssy kits (Enzo Life Sciences Inc., NY, USA). The study ws pproved y the Institutionl Ethics Committee t the University of Fukui. Western Blotting Anlysis of HSP70 Expression Murine lung tissues resected t 2 dys of ge were homogenized in ice-cold tissue lysis uffer consisting of 10 mm Tris se-ph 7.4, 0.1% sodium dodecyl sulfte (SDS), 150 mm NCl, 1 mm ethylenediminetetrcetic cid (EDTA), 1% octylphenoxypolyethoxyethnol, 0.1% sodium deoxycholte, nd protese inhiitor. The totl extrcted cell proteins were electrophoresed y SDS-PAGE (20 lg protein/lne). Proteins were trnsferred to polyvinylidene difluoride memrne (Bio-Rd Lortories Inc., CA) nd immunolotted with polyclonl rit nti-hsp70 ntiody (Enzo Life Sciences, Inc., NY, USA) or nti--ctin ntiody (Cell Signling Technology, Dnvers, USA). Blots were then incuted with pproprite HRP-conjugted secondry ntiodies nd nds were visulized using ImmunoStr Ò (Wko Pure Chemicl Industries, Ltd., Osk, Jpn). The mount of protein on the memrne ws quntified y chemiluminescence imging nlyzer (ImgeQunt LAS 4000mini, GE Helthcre, Tokyo, Jpn) nd the expression levels were recorded s rtios to the level of -ctin.

3 Lung (2017) 195: Tissue Preprtion nd Morphologicl Assessment of the Lung After euthnsi, the pulmonry rtery ws perfused with sline. The right lung ws emedded in optiml cutting temperture (OCT) compound (Tissue-Tek Ò ; Skur, Jpn) nd frozen for immunohistochemistry. The left lung ws inflted with 10% formldehyde t 20 cm H 2 O pressure y clmping the trche. Sections from the formlinfixed, prffin-emedded locks were stined with hemtoxylin nd eosin for morphologicl evlution. At lest 10 imges of the lung tissue were rndomly recorded in linded fshion with chrge-coupled device cmer system (OLYMPUS FX380; Olympus Co., Tokyo, Jpn). Alveolriztion ws ssessed y mesuring rdicl lveolr counts (RAC) nd men liner intercept (MLI), s descried previously [15, 16]. Evlution of Apoptosis in Murine Lung Tissue A terminl deoxynucleotidyl trnsferse-medited dutp nick end leling (TUNEL) ssy ws performed using the In Situ Cell Deth Detection Kit (Roche Dignostics GmH, Mnnheim, Germny) ccording to the mnufcturer s instructions. Fluorescence microscope imges were used to clculte the re stined with TUNEL using Metmorph softwre (Moleculr Devices, Sunnyvle, CA, USA). To evlute cleved cspse-3, 10-lm frozen lung tissue sections were fixed in 4% prformldehyde solution for 20 min t room temperture, permeilized with 0.2% Triton Ò X-100 in phosphte-uffered sline (PBS) for 2 min on ice, nd incuted with locking uffer (Blocking One Histo Ò ; Ncli Tesque, Inc., Kyoto, Jpn) for 10 min t room temperture, followed y incution with nti-ctive cspse-3 pa (Promeg Co., Mdison, WI, USA) (1:100) overnight t 4 C, nd then Alex Fluor 488 got nti-rit immunogloulin G (1:5000) for 60 min t room temperture. Smples were mounted with Prolong Ò (Thermo Fisher Scientific, Wlthm, MA, USA) nd inspected under fluorescence microscope. The numer of cleved cspse-3-positive cells in the whole re of section ws counted nd expressed s per the totl re clculted y Metmorph softwre. Sttisticl Anlysis Murine dt re presented s men ± stndrd error of the men (SEM), nd were nlyzed using one-wy nlysis of vrince (ANOVA) or Student s t test. Humn dt re presented s medins nd rnges, nd were nlyzed using the Mnn Whitney U nd Wilcoxon signed-rnk tests. Correltions were evluted using the non-prmetric Spermn s rho test. A significnt difference ws considered t p \ Results Hyperoxi Suppressed Lung HSP70 Expression, Wheres GGA Enhnced it We first exmined the effects of GGA dministrtion nd hyperoxi exposure on HSP70 expression in the lung. HSP70 protein expression ws enhnced 24 h fter in vivo GGA dministrtion (Fig. 1). HSP70 expression levels in the lungs treted with 500 mg/kg GGA were more thn two-fold higher thn those treted with vehicle lone (2.4 ± 0.4 vs. 1.1 ± 0.3, p \ 0.05). In contrst, 24 h of hyperoxi exposure suppressed HSP70 protein expression compred with normoxi (0.3 ± 0.0 vs. 1.1 ± 0.3, p \ 0.05). GGA dministrtion rogted hyperoxi-induced suppression of HSP70 protein expression compred with vehicle dministrtion (0.9 ± 0.1 vs. 0.3 ± 0.0, p \ 0.01). β-cn HSP70/β-cn Fig. 1 Gernylgernylcetone (GGA)-induced expression of het shock protein 70 (HSP70) in the lungs. Representtive immunolotting dt for HSP70 nd -ctin expressions 24 h fter the dministrtion of GGA or vehicle lone (vehi) under normoxi or hyperoxi re shown. Expression levels of HSP70 normlized y -ctin signl were clculted y densitometry nd compred etween the vehicle-treted group (open rs) nd the 500 mg/kg GGA-treted group (closed rs). vehi vehicle dministrtion. Dt shown represent mens ± the stndrd error of the men (SEM) (normoxi/vehicle group: n = 8; normoxi/gga group: n = 7; hyperoxi/ vehicle group: n = 6; hyperoxi/gga group: n = 7). Comprison etween the indicted groups (*p \ 0.05, **p \ 0.01)

4 472 Lung (2017) 195: GGA Improved Hyperoxi-Induced Filure to Thrive The totl numer of pups treted postntlly ws 15 in the normoxi/vehicle-treted group, 22 in the normoxi/ggatreted group, 13 in the hyperoxi/vehicle-treted group, nd 15 in the hyperoxi/gga-treted group, nd the survivl rte in ech group ws 93.3, 81.8, 92.3, nd 100%, respectively. Pup survivl rte ws not significntly Fig. 2 Pup survivl rte. The totl numer of pups treted postntlly ws 15 in the normoxi/vehicle group, 22 in the normoxi/gga group, 13 in the hyperoxi/vehicle group, nd 15 in the hyperoxi/gga group, respectively. The Kpln Meier plot shows tht the survivl rte of pups ws not significntly different mong groups. GGA tretment prevented hyperoxi-induced filure to thrive. Dt re expressed s rtes of increse from ody weight t 1 dy of ge. Dt shown represent men ± SEM (normoxi/vehicle group: n = 6; normoxi/gga group: n = 6; hyperoxi/vehicle group: n = 7; hyperoxi/gga group: n = 6). ***Comprison etween the indicted group nd normoxi/vehicle, normoxi/gga, nd hyperoxi/gga groups (***p \ 0.001) different mong groups (Fig. 2). None of the totl 12 pregnnt mice swpped every 24 h etween normoxi nd hyperoxi groups died during the experimentl period. Hyperoxi exposure significntly reduced ody weight gin rte (Fig. 2). At 8 dys of ge, the ody weight of hyperoxi/vehicle-treted neontes ws 1.7 ± 0.1 times the ody weight t dy 1, wheres in normoxi/vehicle-treted neontes it ws 2.9 ± 0.2 times the ody weight t dy 1 (p \ 0.001). Tretment with 500 mg/kg GGA did not ffect the ody weight gin of neontes kept in room ir. Of note, GGA tretment improved the hyperoxi-induced reduction in ody weight gin. GGA Restored Hyperoxi-Induced Hypo- Alveolriztion As just 3 dys of hyperoxi exposure led to the susequent filure of neontes to thrive, we investigted whether the hyperoxi resulted in ny oservle sequele in neontl lungs. The lungs of neontes exposed to hyperoxi showed smller RAC nd lrger MLI thn those of neontes kept in normoxic conditions (RAC: 4.2 ± 0.4 vs. 7.2 ± 0.5, p \ 0.001; MLI: 68.4 ± 1.9 vs ± 1.4 lm, p \ 0.001) (Fig. 3). Thus, 3 dys of hyperoxi exposure resulted in hypo-lveolriztion or emphysemtous chnges with significntly decresed RAC nd incresed MLI. Tretment with 500 mg/kg GGA incresed RAC nd decresed MLI in the lungs of hyperoxi-exposed neontes, suggesting tht GGA suppressed hyperoxi-induced lung injuries (RAC: 5.4 ± 0.2 vs. 4.2 ± 0.4, p \ 0.05; MLI: 59.9 ± 1.6 vs ± 1.9 lm, p \ 0.01). GGA Exhiited Cytoprotective Effects Aginst Hyperoxi-Induced Lung Injury To clrify the mechnisms involved in the structurl chnges oserved in hyperoxi-exposed neontl lungs, we exmined poptosis of pulmonry prenchyml cells vi the TUNEL ssy. Hyperoxi exposure for 3 dys pprently incresed the numer of TUNEL-positive cells in neontl lungs t 8 dys of ge (Fig. 4, c). In conjunction with structurl chnges, GGA tretment drmticlly inhiited the increse in TUNEL-positive cells ssocited with hyperoxi exposure, suggesting tht GGA tretment prevented hyperoxi-induced poptosis. We lso ssessed cleved cspse-3, mrker of poptosis. Hyperoxi-exposed mice exhiited more cspse- 3-positive cells in lungs thn did normoxi-exposed mice. GGA significntly suppressed the hyperoxi-induced increse in cspse-3-positive cells in lungs (Fig. 4, d).

5 Lung (2017) 195: significntly decresed s the infnts ged (medin 0.33 ng/ml, rnge ng/ml vs. medin 0.20 ng/ml, rnge ng/ml; p \ 0.05). There were no sttisticlly significnt differences in the serum HSP70 levels t postntl dys etween the infnts with nd without BPD (Fig. 5). Discussion Serum HSP70 Levels in Humn Infnts Decresed with Age Fifty-seven infnts were enrolled in this study; medin gesttionl ge ws 35 weeks (rnge weeks), medin irth weight ws 1951 g (rnge g), medin Apgr score t 1 min ws 8 (rnge 0 9), nd medin Apgr score t 5 min ws 9 (rnge 4 10). Twenty-eight infnts (49.1%) were mle, 25 (43.9%) received prentl steroids, 17 (29.8%) received surfctnt, 6 (10.5%) hd choriomnionitis, nd 7 (12.3%) hd BPD. Serum HSP70 levels etween postntl dys 0 nd 3 were not significntly correlted with gesttionl ge, irth weight, or Apgr score. While serum HSP70 levels in infnts dministered surfctnt t irth were significntly higher thn those of non-dministered infnts (medin 0.34 ng/ml, rnge ng/ml vs. medin 0.26 ng/ml, rnge ng/ml; p \ 0.05), there were no significnt effects of sex, ntentl steroid use, choriomnionitis, or the development of BPD on serum HSP70 levels t postntl dys 0 3. Notly, serum levels μ Fig. 3 GGA tretment reverted histologicl chnges induced y hyperoxi. Histology of neontl lungs t 8 dys of ge. All imges t 9400 mgnifiction. Scle r: 100 lm. Rdil lveolr count (RAC) nd men liner intercept (MLI) were compred etween the vehicle-treted group (open rs) nd the 500 mg/kg GGA-treted group (closed rs). Dt re the men ± SEM (normoxi/vehicle group: n = 6; normoxi/gga group: n = 6; hyperoxi/vehicle group: n = 7; hyperoxi/gga group: n = 6). Comprison etween the indicted groups (*p \ 0.05, **p \ 0.01) We demonstrted tht GGA dministrtion induced HSP70, which my hve exerted nti-poptotic nd cytoprotective effects ginst hyperoxi insults, resulting in improved hypo-lveolriztion of neontl lungs in mouse BPD model. Although oxygen therpy is essentil for sving the lives of immture infnts, prolonged hyperoxi exposure my cuse lung injury through the production of highly rective nd destructive oxygen rdicls, resulting in BPD. In mouse models, hyperoxi-exposed neontl lungs exhiit severely simplified nd enlrged lveolr structure, resemling the pthologicl chnges oserved in BPD [17]. Severl experimentl pproches using nti-oxidnts, growth fctors, or stem cells hve een shown to prevent the development of BPD or restore norml lveolr structure. However, most of these pproches re not redily pplicle to humn neontes due to sfety considertions. GGA, n nti-ulcer drug, is non-toxic HSP inducer [9]. Orlly dministered GGA t 600 mg/kg induces HSP70 expression in mouse lungs, strting t 8 h nd reching mximum t 24 h [11]. In our pilot study, the expression levels of HSP70 in the lungs treted with single dose of 250 mg/kg of GGA only tended to e higher thn those treted with vehicle. Although 24 h of hyperoxi exposure suppressed HSP70 expression in neontl lungs, Zhng et l. [8] reported tht HSP70 mrna expression is induced in dult mouse lung fter C48 h of hyperoxi exposure. For these resons, in the present study, we dministered 500 mg/kg of GGA dily during 3 dys of hyperoxi exposure. Zhng et l. [8] demonstrted tht extrcellulr HSP70 trnsduces nti-poptotic nd cytoprotective signls through toll-like receptor 4 nd Trif-nucler fctor kpp B pthwy, which induces Bcl-2 expression nd inhiits cspse-3 ctivtion. HSP70 expression in dult lungs fter prolonged hyperoxi exposure my e due to n utoregultory protective mechnism ginst hyperoxi insults. Although poptosis occurs in ll stges of lung development, dysfunctionl poptotic ctivity my reduce lveolr numer. In fct, in one study, prolonged ventiltion-induced lung epithelil poptosis in neontl mice coincided with significnt reduction in lveolriztion [18]. Thus, rpid induction of HSP70 y GGA could protect neontl immture lung tissues from insults cused y rective nd

6 474 QRUPR[LD GGA YHKL GGA YHKL '$3, 781(/ '$3, FDVSDVH 781(/ SRVLWLYH DUHDV c QRUPR[LD destructive oxygen rdicls. GGA dministrtion my e useful tretment for BPD. HSP70 levels in the mternl nd umilicl cord ser re up-regulted upon preterm delivery [19]. Gesttionl ge, irth weight, nd choriomnionitis re known risk fctors for the development of BPD. In the present study, none of these fctors were directly ssocited with the neontl K\SHUR[LD K\SHUR[LD d &OHDYHG FDVSDVH 3RVLWLYH FHOOV WRWDO DUHD [ Fig. 4 GGA tretment inhiited hyperoxi-induced poptosis in lungs. Nucleus nd poptotic cells were identified vi DAPI stining nd the terminl deoxynucleotidyl trnsferse-medited dutp nick end leling (TUNEL) method (), nd cleved cspse-3 immunohistochemistry () in the hyperoxi-exposed neontl lungs treted with 500 mg/kg GGA or vehicle lone. TUNELpositive res (c) nd cleved cspse-3-positive cells (d) expressed per the totl re of the lung section of ech slide were compred etween the vehicle-treted group (open rs) nd the GGA-treted group (closed rs) under normoxi or hyperoxi. Arrows indicte TUNEL-positive cells () nd cleved cspse-3positive cells (). All imges t 9200 mgnifiction. Scle r: 100 lm. Dt re the men ± SEM (normoxi/ vehicle group: n = 6; normoxi/gga group: n = 6; hyperoxi/vehicle group: n = 7; hyperoxi/gga group: n = 6). Comprison etween the indicted groups (*p \ 0.05) Lung (2017) 195: QRUPR[LD K\SHUR[LD HSP70 serum levels etween postntl dys 0 nd 3. HSP70 serum levels in humn neontes re higher thn those of norml dults, ut grdully decrese from irth to dys of ge. This trnsient increse in HSP70 my fford protection ginst excessive stress ssocited with irth. In the present study, orl GGA dministrtion during hyperoxi exposure mintined the HSP70 levels nd

7 Lung (2017) 195: Fig. 5 Correltions etween serum HSP70 levels nd gesttionl ge () nd irth weight (). Chronologicl chnges in serum HSP70 levels in preterm humn infnts with or without BPD (open circle nd dshed line, BPD, n = 4; cross nd solid line, no-bpd, n = 8) (c). Dt re the medin nd rnge. *p \ 0.05 c ρ ρ restored hypo-lveolriztion of neontl mice lungs. Fullterm mice re orn in the scculr stge of lung development, which corresponds to humn lung development t weeks of gesttion. Mice lung development progresses to the lveolr stge t postntl dy 5, which corresponds to humn lung development t weeks of gesttion [20]. Thus, our murine BPD model ppers to e useful model of therpeutic intervention with GGA in preterm humn infnts. If HSP70 expression ws kept t high levels under mechnicl ventiltion nd oxygen dministrtion, the immture lung tissues my e protected from insults ssocited with BPD risk fctors. Conclusion Orl dministrtion of GGA might e useful nd sfe pproch for the prevention of BPD cused y hyperoxi exposure. Further studies re required to elucidte the mechnisms underlying the development of lveolr structure in immture lungs, nd the potentil side effects of GGA. Funding This study ws supported y the Jpn Society for the Promotion of Science (Numer: ). Complince with Ethicl Stndrds Conflict of interest The uthors declre tht they hve no conflict of interest. Ethicl Approvl All procedures performed in studies involving nimls were in ccordnce with the ethicl stndrds of the institution or prctice t which the studies were conducted. Ethicl Stndrds All procedures performed in studies involving humn prticipnts were in ccordnce with the ethicl stndrds of the institutionl reserch committee nd with the 1964 Helsinki declrtion nd its lter mendments or comprle ethicl stndrds. Informed Consent The prents of the infnts provided written consent. Open Access This rticle is distriuted under the terms of the Cretive Commons Attriution 4.0 Interntionl License ( tivecommons.org/licenses/y/4.0/), which permits unrestricted use, distriution, nd reproduction in ny medium, provided you give pproprite credit to the originl uthor(s) nd the source, provide link to the Cretive Commons license, nd indicte if chnges were mde.

8 476 Lung (2017) 195: References 1. Northwy WH Jr, Rosn RC, Porter DY (1967) Pulmonry disese following respirtor therpy of hyline-memrne disese. Bronchopulmonry dysplsi. N Engl J Med 276: Ozdemir R, Erdeve O, Dizdr EA et l (2011) Clrithromycin in preventing ronchopulmonry dysplsi in Ureplsm urelyticum-positive preterm infnts. Peditrics 128:e1496 e Reyurn B, Mrtin RJ, Prksh YS et l (2012) Mechnisms of injury to the preterm lung nd irwy implictions for long-term pulmonry outcome. Neontology 101: Sliv DM, Nrdiello C, Pozrsk A et l (2015) Recent dvnces in the mechnisms of lung lveolriztion nd the pthogenesis of ronchopulmonry dysplsi. Am J Physiol Lung Cell Mol Physiol 309:L9 L Soti C, Pl C, Ppp B et l (2005) Moleculr chperones s regultory elements of cellulr networks. Curr Opin Cell Biol 17: Soti C, Ngy E, Guricz Z et l (2005) Het shock proteins s emerging therpeutic trgets. Br J Phrmcol 146: Wong HR, Menendez IY, Ryn MA et l (1998) Incresed expression of het shock protein-70 protects A549 cells ginst hyperoxi. Am J Physiol 275:L836 L Yi Zhng, Zng X, Shn P et l (2013) A protective HSP70-TLR4 pthwy in lethl oxidnt lung injury. J Immunol 191: Hirkw T, Rokutn K, Nikw T et l (1996) Gernylgernylcetone induces het shock proteins in cultured guine pig gstric mucosl cells nd rt gstric mucos. Gstroenterology 111: Otk M, Ymmoto S, Ogswr K et l (2007) The induction mechnism of the moleculr chperone HSP70 in the gstric mucos y gernylgernylcetone (HSP-inducer). Biochem Biophys Res Commun 353: Fujiyshi T, Hshimoto N, Jijiw M et l (2009) Protective effect of gernylgernylcetone, n inducer of het shock protein 70, ginst drug-induced lung injury/firosis in n niml model. BMC Pulm Med. doi: / Tnk K, Tnk Y, Nm T et l (2010) Het shock protein 70 protects ginst leomycin-induced pulmonry firosis in mice. Biolchem Phrmcol 80: Kim YJ, Kim JY, Kng SW et l (2015) Protective effect of gernylgernylcetone ginst hydrogen peroxide-induced oxidtive stress in humn neurolstom cells. Life Sci 131: Redline RW, Fye-Petersen O, Heller D et l (2003) Amniotic infection syndrome: nosology nd reproduciility of plcentl rection ptterns. Peditr Dev Pthol 6: Thoms PC, Willim MT (1982) The rdil lveolr count method of Emery nd Mithl: repprisl 1- postntl lung growth. Thorx 37: Vivek B, Jen-Ruey T, Anne M et l (2003) Mild hypoxi impirs lveolriztion in the endothelil nitric oxide synthse-deficient mouse. Am J Physiol Lung Cell Mol Physiol 284:L964 L Perrone S, Ttrnno ML, Buonocore G (2012) Oxidtive stress nd ronchopulmonry dysplsi. J Clin Neontol 1: Mokres LM, Pri K, Hilgenodorff A et l (2010) Prolonged mechnicl ventiltion with ir induces poptosis nd cuses filure of lveolr septtion nd ngiogenesis in lungs of neworn mice. Am J Physiol Lung Cell Mol Physiol 298:L23 L Chng A, Zhng Z, Ji L et l (2013) Altertion of het shock protein 70 expression levels in term nd preterm delivery. J Mtern Fetl Neontl Med 26: Nrdiello C, Mižíková I, Morty RE (2017) Looking hed: where to next for niml models of ronchopulmonry dysplsi? Cell Tissue Res 367: