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1、Contents lists available at ScienceDirectToxicology and Applied Pharmacologyjournal homepage: use of vitamin E and nimodipine ameliorates dibutyl phthalate-induced memory deficit and apoptosis in mice by inhibiting the ERK 1/2pathwayBiao Yan,Yanling Sun,Jie Zeng,Yingying Chen,Chongyao Li,Peng Song,L
2、in Zhang,Xu Yang,Yang Wu,Ping MaLaboratory of Environment-Immunological and Neurological Diseases,School of Basic Medical Sciences,Hubei University of Science and Technology,Xianning 437100,ChinaA R T I C L E I N F OKeywords:Learning disabilitiesDibutyl phthalateHippocampal neuronsERK 1/2 pathwayVit
3、amin ENimodipineA B S T R A C TLearning disabilities(LDs)in children are a serious global problem.Dibutyl phthalate(DBP),a plasticizer widelyused in daily life,has been linked to triggering childhood LDs,however the mechanism underlying this remainsunclear.Studies have shown that the ERK 1/2 pathway
4、 is closely related to apoptosis of hippocampal neurons.On the basis of these links between LDs,DBP and the ERK 1/2 pathway,we explore whether DBP induceshippocampal neuron apoptosis and increases behavioral disorders in mice via the ERK 1/2 pathway.We lookedat oxidative stress,examined the calcium
5、signal,detected the ERK 1/2 pathway and evaluated apoptosis as wellas using histological observations,and found that DBP significantly increased oxidative damage and apoptosis inhippocampal neurons via the ERK 1/2 pathway in mice.We also found that pretreatment with the dihy-dropyridines(DHPs)Ca2+an
6、tagonist,nimodipine(NMDP),combined with the antioxidant Vitamin E(VE),attenuated ERK 1/2 phosphorylation and DBP-mediated disorders,suggesting that a combined use of VE andNMDP can ameliorate DBP-induced memory deficit and apoptosis via inhibiting the ERK 1/2 pathway.Theseresults indicate that DBP p
7、redisposes oxidative damage and apoptosis in hippocampal neurons by activation ofthe ERK 1/2 pathway,and may be proposed as a possible mechanism underlying LDs in children.Moreover,VEand NMDP may play a certain protective role in the targeted treatment of childhood LDs.1.IntroductionLearning disabil
8、ities(LDs)often occur in childhood.Also known aschildrens LDs,these children exhibit a variety of learning-relatedsymptoms,such as difficulty in learning and remembering.The 5thedition of the Diagnostic and Statistical Manual of Mental Disorders(DSM-5)defines LDs to be neurodevelopmental disorders.T
9、hese aresome of the most important disorders for children globally,and causewidespread concern(Miodovnik et al.,2014).Studies show that onaverage,one in six children in the United States have LDs,and about20%to 25%of these children exhibit moderate to severe LDs(Boyleet al.,2011;Landrigan et al.,201
10、2;Iuculano et al.,2015).The globalproblem of environment-related LDs in children is so severe,that acommittee of experts convened by the National Academy of Sciencesconcluded that 3%of neurodevelopmental disorders are directly causedby exposure to environmental toxins,and that a further 25%of neu-ro
11、developmental disorders are due to the widely understood interactionbetween environmental factors and genetic susceptibility(Landriganet al.,2012).Dibutyl phthalate(DBP)is a typical persistent organic neurotoxicpollutant(Kobrosly et al.,2014).It is a widely used plasticizer thatresults in high expos
12、ure levels in normal daily life(Guo et al.,2011).Studies have reported the occurrence of phthalate metabolites in urinespecimens from the U.S.,Germany,Sweden,Mexico,Japan and Korea,https:/doi.org/10.1016/j.taap.2019.02.008Received 11 September 2018;Received in revised form 12 February 2019;Accepted
13、14 February 2019Abbreviations:LDs,Learning disabilities;DBP,Dibutyl phthalate;ERK 1/2,Extracellular signal-regulated kinases 1/2;VE,Vitamin E;NMDP,Nimodipine;DHPs,Dihydropyridines;ROS,Reactive oxygen species;GSH,Glutathione;MDA,Malondialdehyde;CaM,Calmodulin;CaMK II,Ca2+/calmodulin-dependent protein
14、 kinaseII;PKC,Protein kinase C;BDNF,Brain-derived neurotrophic factor;CREB,cAMP-response element-binding protein;cyt C,Cytochrome C;TNF-,Tumour necrosisfactor-Corresponding authors at:Laboratory of Environment-Immunological and Neurological Diseases,School of Basic Medical Sciences,Hubei University
15、of Scienceand Technology,Xianning 437100,China.E-mail addresses:(Y.Wu),(P.Ma).Toxicology and Applied Pharmacology 368(2019)117Available online 15 February 20190041-008X/2019 Published by Elsevier Inc.Tindicating the widespread exposure of humans to phthalates.Based onthe urinary concentrations of ph
16、thalate metabolites,Guo et al.(2011)estimated that the daily intake of DBP in China was 12.2g/kg bw/day,exceeded the tolerable daily intake of 10g/kg bw/day,proposed forDBP,by the European Food Safety Authority.Infants and children ex-posed to DBP through diet,inhalation and skin contact show advers
17、ecognitive and behavioral outcomes,with DBP particularly affectingtheir neurodevelopment(Ejaredar et al.,2015).Epidemiological studiesby Cho et al.(2010)suggest that exposure to DBP can significantlyreduce students academic achievement and IQ.Other studies confirmthat prolonged exposure to DBP durin
18、g pregnancy can induce LD inchildren(Lien et al.,2015;Olesen et al.,2017).Lien et al.(2015)foundpositive associations between maternal DBP exposure and externalizingdomain behavior problems in 8-year-old children.Olesen et al.(2017)reported that boys who experienced increased prenatal DBP exposuresc
19、ored lower in language development assessments.The brain is an important target organ for exogenous stimulation,including DBP(Wjtowicz et al.,2017),while the hippocampus is a keyfunctional region for learning and memory(Whitlock et al.,2006).There is emerging evidence that phthalate including DBP ma
20、y havedeleterious effects on the developing brain in 012-year-old childrenwith behavioral problems(Ejaredar et al.,2015).The supporting la-boratory studies and proposed mechanisms underlying the adverseimpacts of DBP in relation to hippocampal toxicity have also been re-ported(Holahan and Smith,2015
21、).Moreover,DBP-induced apoptosisin the hippocampal neurons of DBP-exposed immature rats are alsodemonstrated(Li et al.,2013;Wjtowicz et al.,2017).Although theobserved associations are based on limited studies with a broad range ofendpoints,the implications of such outcomes are of concern from apubli
22、c health standpoint and merit further investigation given the non-negligible influence of the DBP exposure to those LDs(Miodovnik et al.,2014).Recent studies have shown that the extracellular regulated proteinkinase 1 and 2(ERK1 and ERK2),commonly referred to collectively asERK 1/2,are members of th
23、e mitogen-activated protein kinase(MAPK)family.This family of kinases transport signals from the surface of thecell to the nucleus,thereby mediating the activation of nuclear tran-scription factors that participate in cell apoptosis and other biologicalfunctions(Cargnello and Roux,2011;Ciccarelli an
24、d Giustetto,2014).ERK 1/2,and their phosphorylations(p-ERK 1/2)play a notable role inneurobehavioralresponsesandcognitiveprocessesthatincludelearning and memory(Johnson and Lapadat,2002).In addition,di-(2-ethylhexyl)phthalate,a homologue of DBP,can induce excessive re-active oxygen species(ROS)and c
25、ause calcium aggravation in hepa-tocytes,which were isolated from the liver of male albino rats of theWistar strain weighing approximately 120130g(4weeks of age),ex-posed to DEHP for 24h at doses of 5,10,25,50,100,and 200M(Ghosh et al.,2010).But even more importantly,intracellular ROSaccumulation an
26、d a Ca2+imbalance can activate the ERK 1/2 pathwaysin hippocampal neurons(Mccubrey et al.,2006;Kemmerling et al.,2007).On the basis of there being a possible relationship between DBP andthe ERK 1/2 pathway,we conducted this research to determine whetherDBP induces hippocampal neuron apoptosis and wh
27、ether it increasesthe observed behavioral disorders via the ERK 1/2 pathway.We ex-amined the ROS,GSH and MDA content to evaluate oxidative stress.The levels of CaM,CaMK II and PKC were examined to assess the Ca2+signaling pathway.We determined ERK 1/2 and ERK 1/2 pathway-re-lated proteins(sensitive
28、biomarkers)including BDNF and p-CREB levelsafter DBP treatment to study the role of ERK 1/2 in DBP-mediated ef-fects.The levels of cyto C,caspase-3 and TNF-were also analysised toevaluate apoptosis in hippocampal neurons.We also looked for changesin mouse behavior,and observed histopathological chan
29、ges and im-munohistochemistry in the hippocampus.In addtion,we used the an-tioxidant VE and the DHP Ca2+antagonist,NMDP,to demonstrate thatthe ERK 1/2 pathway mediates oxidative stress and the Ca2+signalingpathway induced by DBP.Although this study is an animal experiment,it still provides a better
30、understanding of the possible mechanism bywhich neurotoxic environmental pollutants induce LDs in children.2.Materials and methods2.1.Ethics statementAll experimental procedures were approved by the Office ofScientific Research Management at Hubei University of Science andTechnology(Xianning,China),
31、with a certificate of Application for theUse of Animals(approval ID:HBUST-IACUC-2018-001).2.2.Animal preparationMale Kunming mice(3-week-old male,18 1g)were obtainedfrom the Hubei Province Experimental Animal Centre(Wuhan,China).All mice were housed under SPF conditions at 2025 C with 5070%humidity
32、and a 12h light/dark cycle.A commercial diet and filteredwater were provided ad libitum.Mice were quarantined for 3daysprior to the study.Nine mice were used in each group to minimize thenumber of experimental animals needed while simultaneously ensuringthe statistical validity of the results.2.3.Re
33、agents and kitsDBP(99%,CAS:84-74-2),VE(CAS:59-02-9),NMDP(98%,CAS:66085-59-4),2,7-dichlorodihydrofluorescein(DCFH-DA),2-thiobarbituric acid(TBA)and Hoechst 33258 were purchased fromSigma-Aldrich(St.Louis,MO,USA).Tween 80(CAS:9005-65-6)wasobtained from Amresco(Solon,OH,USA).Mouse ELISA kits for GSHwer
34、e obtained from(Nanjing Jiancheng Bioengineering Institute,Nanjing,China).Mouse kits for CaM,CaMK II,PKC,ERK 1/2,BDNF,p-CREB,cyt C,caspase-3 and TNF-were purchased from eBioscience(San Diego,CA,USA).Rabbit anti-p-ERK 1/2-antibody,goat-anti-rabbitIgG-antibody,rabbit IgG peroxidase conjugated streptav
35、idin-biotincomplex(SABC-POD)kit and a diaminobenzidine(DAB)kit were ob-tained from Boster Bio-engineering(Boster Bio-engineering,Wuhan,China).All other chemicals were of the highest grade available com-mercially,or as indicated.2.4.Experimental protocolsThe KM mice were divided randomly into eight g
36、roups of nine miceeach.The groups were treated as follows:(A)control(saline,oral ex-posure),(B)50mg/kg/d DBP(oral exposure),(C)50mg/kg/d VE(oralexposure),(D)VE(50mg/kg/d)2h after the administration of DBP(50mg/kg/d)(DBP+VE),(E)2mg/kg/d NMDP(intraperitoneal in-jection),(F)NMDP(2mg/kg/d)30min before t
37、he administration ofDBP(50mg/kg/d)(DBP+NMDP),(G)50mg/kg/d VE oral exposurecombined with 2mg/kg/d NMDP(VE+NMDP),(H)DBP and VE wereadministered via gavage at a regular time every day,before which allthe mice were treated with NMDP(DBP+VE+NMDP).Based onprevious repeated studies in our laboratory,the do
38、sage of DBP(50mg/kg/d)was selected according to Wu et al.(2017),and is similar to thecontent of DBP in daily foods ranged from 0 to 46.50mg/kg.VE acts asan antioxidant was administrated 2h after DBP treatment according toMurugesan et al.(2005).NMDP acts as a calcium antagonist was in-jected 30min be
39、fore DBP treatment(Karande and Bende,2010).Ex-perimental mice received daily doses according to group protocol for aperiod of 4weeks.The hidden-platform acquisition test was undertakenfrom day 22 to day 26.Day 27 was the forgetting-period,and on day 28the probe trial test was performed(Fig.1).After
40、the 28days,the micewere sacrificed,the brains of the mice were collected and processedaccording to the requirements of the various tests described below.B.Yan,et al.Toxicology and Applied Pharmacology 368(2019)11722.5.Morris water maze(MWM)The MWM test was conducted to evaluate spatial learning abil
41、itiesand memory of the experimental mice(Vorhees and Williams,2005).The maze consists of a black circular pool(diameter 2.14m,height80 cm),filled with water at 23 1C to a height of 50cm.Acquisitionand maintenance of spatial memory by the mice was assessed by usingthe MWM as previously described(Ma e
42、t al.,2015).The Ethevision XTVersion 12.0(Noldus Ltd.,Netherlands)monitoring and analysis soft-ware recorded the escape latency of the mice from the different treat-ment groups.2.6.Brain coefficientsThe brain of each mouse was trimmed of extraneous tissue andweighed.The brain coefficient(calculated
43、as 100%brain weight(g)/body weight(g)was used as an indicator of organ-specific,as well asgeneral toxicity in the mice.2.7.Immunohistochemical assayCoronal sections were cut at bregma 3.8m.These sections of braintissue(3 sections were quantified per mouse)were quenched of en-dogenous peroxides using
44、 3%H2O2.These sections were then boiled insodium citrate(0.01 mol/L,pH6.0)to retrieve antigens so as to unmaskthe antigen epitopes,following which the sections were permeabilizedwith 0.2%Triton X-100 for 10min,and blocked with 10%goat serumfor a further 20min at room temperature.Sections were then i
45、ncubatedwith diluted primary antibodies(rabbit anti phospho-44/42 ERK 1/2antibody,1:200 dilution)overnight at 4C.Slides were washed withPBS and incubated with a secondary antibody(goat anti-rabbit IgG;1:200 dilutions)for 30min at 37C.p-ERK 1/2 was detected with arabbit IgG peroxidase conjugated SABC
46、-POD kit,followed by incuba-tionwithaDABkit.p-ERK1/2weredeterminedbyim-munohistochemical staining(brown color stain).Immunostained sec-tions were viewed under a DM 4000B microscope(Leica,Berlin,Germany)(3 images were quantified from each section).Using Image-Pro Plus 6.0 software(Media Cybernetics,B
47、ethesda,MD,USA),the p-ERK 1/2 staining intensity was taken to be the average optical density.A non-stained region was selected and used as the background.Thestatistical area was 0.134mm2.2.8.Histopathological examinationBrain tissues of the mice were isolated for the preparation of his-topathologica
48、l slides.All of the samples were incubated in a fixative(4%paraformaldehyde)solution for 24 h at room temperature,fol-lowing which the distal pieces were embedded in paraffin,sectionedinto 10m slices,stained with Haematoxylin&Eosin(H&E)and Nisslaccording to standard protocols(Ma et al.,2015),and obs
49、erved using aBX53 microscope(Olympus,Tokyo,Japan).The tissue sections wereexamined qualitatively by two experienced pathologists in a blindedmanner.After routine embedding and slicing,the dewaxed and transparentslices were washed twice with saline(0.9%NaCl)for 3min per wash,and were then added to a
50、0.5mL Hoechst 33258 dye solution(workingfluid concentration is 5mg/L)for 5min.After this procedure,the bluenuclei(Hoechst-DNA)in the tissue sections were detected using afluorescent microscope.2.9.Detection of oxidative stressTen percent hippocampal tissue homogenates were collected for usein the de