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1、Vitamin E antagonizes ozone-induced asthma exacerbation in Balb/cmice through the Nrf2 pathwayLiju Duana,d,Jinquan Lib,Ping Mac,Xu Yangb,Shunqing Xua,*aKey Laboratory of Environment and Health(Huazhong University of Science and Technology),Ministry of Education&Ministry of EnvironmentalProtection,an
2、d State Key Laboratory of Environmental Health(Incubation),School of Public Health,Tongji Medical College,Huazhong University of Scienceand Technology,Wuhan 430030,ChinabSection of Environmental Biomedicine,Hubei Key Laboratory of Genetic Regulation and Integrative Biology,College of Life Sciences,C
3、entral China NormalUniversity,Wuhan 430079,ChinacResearch Center of Basic Medical Sciences,School of Basic Medical Sciences,Hubei University of Science and Technology,Xianning 437100,ChinadCollege of Public Health,Zhengzhou University,Zhengzhou 450001,Chinaa r t i c l e i n f oArticle history:Receiv
4、ed 21 August 2016Received in revised form10 June 2017Accepted 13 June 2017Available online 15 June 2017Keywords:OzoneVitamin EOxidative stressAllergic asthmaNrf2 pathwaya b s t r a c tMillions of people are regularly exposed to ozone,a gas known to contribute significantly to worseningthe symptoms o
5、f patients with asthma.However,the mechanisms underlying these ozone exacerbationeffects are not fully understood.In this study,we examined the exacerbation effect of ozone in OVA-induced asthma mice and tried to demonstrate the protective mechanism of vitamin E(VE).Anasthma mouse model was establis
6、hed,and used to identify the exacerbating effects of ozone by assessingcytokine and serum immunoglobulin concentrations,airway leukocyte infiltration,histopathologicalchanges in lung tissues,and airway hyper-responsiveness.We then determined the amount of reactiveoxygen species(ROS)accumulated,the e
7、xtent to which VE induced ROS elimination,and examined theantagonistic effects of VE on the ozone-induced exacerbating effects.This study showed that 1-ppmozone exposure could exacerbate OVA-induced asthma in mice.More importantly we found thatozone induced oxidative stress in asthmatic airways may
8、lead to the inhibition of Nuclear factor-erythroid 2-related factor 2(Nrf2),and may subsequently induce even more exaggerated oxidativestress associated with asthma exacerbation.Through VE induced Nrf2 activation and the subsequentincrease in Nrf2 target protein expression,this study suggests a nove
9、l mechanism for alleviating ozoneexacerbated asthma symptoms.2017 Elsevier Ltd.All rights reserved.1.IntroductionAsthma is a widespread global health issue characterized byairwayinflammationmediatedthroughhyperresponsiveness(AHR),remodeling,and infiltration of eosinophils,neutrophils andmast cells i
10、nto the airway wall.(Halwani et al.,2010;Kim et al.,2010).The morbidity of asthma has progressively increased overthe past several decades(Barnes,2010).Both epidemiological evi-dence and experimental evidence have demonstrated an associa-tion between exacerbation of asthma and levels of atmosphericp
11、ollutants(Alexis and Carlsten,2014;Kelly and Fussell,2011;Nastos et al.,2010).Ozone(O3)is a robust oxidizing agentroutinely affects millions of people worldwide,and significantlycontributes to worsened symptoms in patients with asthma andchronic obstructive pulmonary disease(COPD)(Saingam et al.,201
12、6;Tetreault et al.,2016).Studies in animal models haveconfirmed this finding,and demonstrated that ozone exposure canpromote allergic sensitization and exacerbate allergic responses(Bao et al.,2014;Liang et al.,2013).However,the mechanismsunderlying these ozone induced exacerbated effects in asthmap
13、atients are not fully understood.Elucidating the underlyingmechanisms is pivotal in the prevention and treatment of ozone-induced asthma exacerbation.Inhaled ozone interacts with airway lining fluid in the pulmo-nary system to produce reactive oxygen species(ROS)and otheroxidation products,which may
14、 result in oxidative injury(Leroyet al.,2015).Oxidative damage is an important etiology in thepathophysiology of respiratory diseases,especially in pollution-*Corresponding author.School of Public Health,Tongji Medical College,Huaz-hong University of Science and Technology,Wuhan 430030,China.E-mail
15、address:(S.Xu).Contents lists available at ScienceDirectFood and Chemical Toxicologyjournal homepage: Elsevier Ltd.All rights reserved.Food and Chemical Toxicology 107(2017)47e56induced exacerbations(Ciencewicki et al.,2008b).Nuclear factorkappa binding(NF-kB)is a key transcription factor that coord
16、inatesthe expression of various inflammatory genes(Lawrence,2009).ROS,created by a variety of pollutants,have various inhibitory orstimulatory roles in the NF-kB signaling system(Morgan and Liu,2011).Various studies have reported the importance of the NF-kBpathway in ozone-induced lung inflammation
17、and damage(Choet al.,2007;Connor et al.,2012).Phosphorylation of NF-kB p65(serine 536)plays an important role in activating NF-kB followingozone exposure and promoting inflammatory stimuli(Wu et al.,2010).Nuclear factor-erythroid 2-related factor 2(Nrf2)is a redox-sensitive transcription factor that
18、 plays an important role in thepromoter region of genes,encoding antioxidant and/or detoxifyingenzymes and other related stress-responsive proteins(Jang et al.,2016;Kim and Keum,2016).Evidence suggests that there isfunctional cross talk between NF-kB and Nrf2 pathways.Theabsence of Nrf2 is associate
19、d with increased oxidative stress,exacerbated NF-kB activity and increased cytokine production,whereas NF-kB can modulate Nrf2 transcription and activity(Ganesh Yerra et al.,2013).Interventions focusing on restoring thebalance between Nrf2 and NF-kB pathways in persons at risk forpollutant-induced d
20、iseases,represent a novel approach to pre-venting asthma exacerbation.Given the vital role of oxidative stress in the pathophysiology ofasthma exacerbation,it seems likely that antioxidants could beused as agents for the treatment of asthma caused by environ-mental factors such as ozone.Deficiencies
21、 in antioxidants likevitamin E have been linked to asthma exacerbation and severity(Romieu et al.,2002).Peh et al.(2015)have shown that Vitamin Eacts not only as a direct free-radical scavenger,but also as an anti-inflammatory and antioxidative agent,by inhibiting NF-kB nucleartranslocation,and prom
22、oting nuclear Nrf2 levels.In a previousstudy,we also showed that VE enhanced the expression of Nrf2,up-regulated the antioxidant genes heme oxygenase-1(HO-1)andquinone oxidoreductase 1(NQO1),and could very possibly decreasethe levels of oxidative stress and alleviate ozone-induced lunginjury(Zhu et
23、al.,2016).In this study we established an allergic asthma mouse model,and used it to identify the exacerbating effects of ozone byassessing cytokine and serum immunoglobulin concentrations,airway leukocyte infiltration,histopathological changes in lungtissue,and AHR.We then determined the amount of
24、reactiveoxygen species(ROS)accumulated,the extent to which vitamin E(VE)induced ROS elimination,and examined the antagonisticeffectsofVEontheozone-inducedexacerbatingeffects.Furthermore,by detecting the expression of Nrf2 and NF-kB inlung tissues,we tried to elucidate the potential molecularmechanis
25、ms of VE in attenuating the effect on ozone exacerbatedallergic asthma.2.Materials and methods2.1.Animals5-6 weeks SPF male Balb/c mice were purchased from the HubeiProvince Experimental Animal Center(Wuhan,China).All micewere housed in pathogen-free cages at(24e26)!C and 12 h light-dark cycle with
26、55%e75%humidity.A commercial diet(HubeiProvince Experimental Animal Center)and filtered water wasprovided ad libitum.All experiments were performed according torelevant guidelines and regulations.The experimental procedureswere approved by the Office of Scientific Research Management ofCentral China
27、 Normal University(ID:CCNU-SKY-2011-008).2.2.Ozone exposure and experimental designO3was produced using a KTB portable ozone generator(Guangzhou,China)from ambient air.An ozone dosimeter insidethe chamber was used to monitor the different concentrations ofozone.The experiment comprised 4 groups:(A)C
28、ontrol group,(B)Ovalbumin(OVA)group,(C)OVA1.0 ppm ozone exposure group,(D)OVA 1.0 ppm ozone and VE(a-tocopherol,100 mg VE/kg viaintraperitoneal injection)group.The mice in the control groupwere exposed to filtered air(FA).In this study,the concentration ofozone and quantity of vitamin E were determi
29、ned according to ourprevious studies(Li et al.,2014a;Zhu et al.,2016).The mice were sensitized with OVA Al(OH)3(83.33mg OVAand 2.92 mg Al(OH)3in 500mL saline)or saline on days 1,8 and 15by intraperitoneal injection.Then the mice were exposed to anaerosol challenge of 1%OVA or saline(30 min/d)using a
30、n ultrasonicnebulizer(Yuyue 402A type I,China)on days 19 through 25.At thesame time,mice were exposed to clean air or 1.0-ppm ozone for3 h/day from days 19e25.VE was administered as an antioxidant3 h after exposure to 1.0-ppm ozone.The detailed protocols areshown in Fig.1.2.3.Quantitative analyses o
31、f total serum IgEOn day 26,all mice were anaesthetized with 100 mg/(kg bw)pentobarbital.Heart blood was centrifuged at 3000 rpm for10 min at room temperature,after which the serum was collected.The concentration of total-IgE in the serum was detected usingeBioscience(San Diego,CA,USA)ELISA kits acco
32、rding to the man-ufacturers instructions.2.4.Examination of bronchoalveolar lavage fluid(BALF)Mice were sacrificed and subjected to BAL after serum collec-tion.BALF was obtained by lavaging three times with saline via atracheal cannulawhile the mouses chest was gently massaged.Thevolume of total lav
33、age fluid was about 1.5 mL.The collected BALFwas centrifuged at 1500 rpm at 4!C for 10 min,and the cell sedi-ment was then resuspended in 0.5 mL saline.Lymphocytes,eo-sinophils and neutrophils were counted using an automatichematology analyzer(Matenu,MTN-21,China).2.5.Tissue homogenate preparation a
34、nd detection of cytokinesAftercollecting bronchoalveolar lavage fluid,the lung tissue wasexcised and washed in ice-cold phosphate-buffered saline(PBS).10%lung tissue homogenate samples were prepared by homoge-nizing on ice using 10 mL/g of ice-cold PBS(pH 7.5).Afterwards,tissue samples were centrifu
35、ged at 12,000 rpm for 10 min at 4!C.Finally,the supernatant was collected for evaluating the concen-tration of lung cytokines(IL-4,IL-5,IL-13 and IFN-g)and TSLP usingeBioscience ELISA kits.A modified BCA protein assay kit was pur-chased from Sangon Biotech(Shanghai,China)to determine totalprotein.2.
36、6.Analysis the level of glutathione(GSH)and malonaldehyde(MDA)In accordance with the manufacturers instructions,GSH con-tent was measured by assay kit(Nanjing Jiancheng BioengineeringInstitute,China).Malondialdehyde(MDA)concentrationwasdetermined using the thiobarbituric acid(TBA)method(Li et al.,20
37、14a).The protein concentration was determined using themodified BCA protein assay kit(Sangon Biotech,China).L.Duan et al./Food and Chemical Toxicology 107(2017)47e56482.7.Lung histological assayThe left lung was removed for preparation of histopathologyslices.Samples were fixed in 10%formalin soluti
38、on at 4!C overnightand cut into 5mm slices for haematoxylin and eosin(H&E),massontrichrome(MT)and periodic acid-schiff(PAS)staining,as previ-ously described(Wu et al.,2013).A DM4000B microscope(LeicaMicrosystems GmbH,Wetzlar,Germany)was used to examine thestained section.Scoring of cell infiltration
39、 in H&E-stained lungsections was performed as previously described(Goh et al.,2013).Pictures were analyzed with Image-Pro Plus 6.0 to determine theaverage optical density(AOI)of the MT and PAS stained sections(You et al.,2016).All lung tissue sections were examined by pa-thologists in a blinded fash
40、ion.2.8.Immunohistochemical staining of Nrf2,HO-1 and NF-kB(phospho S536)Proteinexpressiondeterminedbyimmunohistochemicalstaining was described previously(Li et al.,2014b).Briefly,Immu-nohistochemical staining of Nrf2,HO-1 and NF-kB were assessedusing the primary antibodies anti-Nrf2(1:100,Proteinte
41、ch,Chi-cago,USA),anti-HO-1(1:50,Proteintech,Chicago,USA)and anti-phospho-p65(S536)(1:100,Abcam,MA,USA),respectively.Thesections were then incubated in an appropriate biotinylatedimmunoglobulin and avidin-biotin peroxidase complex.The IHCstaining control was obtained by omitting the primary antibody.
42、ADM4000B Microscope was used to view the stained sections.Theintensity of protein Nrf2,HO-1 and NF-kB staining was estimated asaverage optical density(AOD)using Image Pro Plus 6.0 software.2.9.Immunofluorescent staining for mast cell tryptaseThe slides of mouse lung tissue were incubated for 2 h s i
43、nblocking solution containing 5%(v/v)of appropriate serum with0.25%Triton X-100.The slides were then rinsed three times withPBS and incubated with anti-mast cell tryptase(1:50,Abcam,MA,USA)overnight at 4!C.After three further washes,the sectionswere incubated with specific secondary antibodies for 1
44、 h at roomtemperature.4,6-diamidino-2-phenylindole(DAPI)was used tovisualize cell nuclei.All immunofluorescence staining was per-formed under a confocal microscope.The intensity of mast celltryptase signals was quantified using ImageJ(Manetti et al.,2014).2.10.Measurement of AHROn day26,the remainin
45、g 20 mice weretested for AHR accordingto a previously described method(Guo et al.,2012).The R-areas ofRi and Re,and the valley value of Cldyn were recorded using theAniRes2005 lung function system(Bestlab,version 2.0,China).2.11.Statistical analysesThe data are presented as the mean standard deviati
46、on.Thestatistical graphs were made using GraphPad Prism 5.0(San Diego,CA,USA).Results were evaluated statistically using a one-wayanalysis of variance(ANOVA).p 0.05 was considered to be asignificant difference and p 0.01 to be an extremely significantdifference.Data analyses were carried out using S
47、PSS ver13(SPSS,Chicago,IL,USA).3.Results3.1.Effects of ozone on serum total IgE levels and cytokineconcentrationsAll OVA-sensitized groups revealed an up-regulation of IgE,IL-4,IL-5,IL-13 and TSLP concentrations compared with the salinegroup.The 1.0 ppm O3exposure group showed significantlyelevated
48、levels(Fig.2A,B,C,D and F),while the IFN-gconcentra-tions(Fig.3C)decreased in the OVA 1.0 ppm O3group comparedwith the OVA group(Fig.2E).In agreement with the results for thecytokines,there is a significant increase in the concentration of IgE(Fig.2A).Interestingly,treatment with vitamin E dramatica
49、llyFig.1.Experimental design and animal exposure.L.Duan et al./Food and Chemical Toxicology 107(2017)47e5649reduced the levels of IL-4,IL-5,IL-13 and TSLP as well as the con-centration of total serum IgE,compared with the OVA 1.0 ppm O3group.3.2.Effect of ozone exposure on inflammatory cell recruitm
50、ent andthe elimination effects of vitamin EAs shown in Fig.3,the examination of the cellular profile of thebronchoalveolar lavage fluid showed that the number of total cells(macrophages,neutrophils and eosinophils)in the OVA 1.0 ppmO3treatment groupwas greaterthan that for the OVAonlygroup.Inadditio