(8.6)--Role of TRPV1 on ozone-exacerbat环境与健康环境与健康.pdf

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1、Role of transient receptor potential cation channel subfamily Vmember 1(TRPV1)on ozone-exacerbated allergic asthma in mice*Jinquan Lia,b,c,1,Yushan Chena,1,Qiao Yi Chend,Dan Liua,Lang Xua,Guirong Chenga,Xu Yange,Zhenzhong Guob,*,Yan Zenga,c,*aBrain and Cognitive Dysfunction Research Center,School of

2、 Medicine,Wuhan University of Science and Technology,Wuhan,430065,ChinabHubei Province Key Laboratory of Occupational Hazard Identification and Control,Medical College,Wuhan University of Science and Technology,Wuhan,430065,ChinacBig Data Science and Engineering Research Institute,Wuhan University o

3、f Science and Technology,Wuhan,430065,ChinadDepartment of Environmental Medicine,New York University School of Medicine,57 Old Forge,Tuxedo,NY 10987,United StateseSection of Environmental Biomedicine,Hubei Key Laboratory of Genetic Regulation and Integrative Biology,College of Life Sciences,Central

4、China NormalUniversity,Wuhan 430079,Chinaa r t i c l ei n f oArticle history:Received 21 August 2018Received in revised form18 January 2019Accepted 18 January 2019Available online 24 January 2019Keywords:OzoneAllergic asthmaTransient receptor potential cation channelsubfamily V member1Calcitonin gen

5、e-related peptideThymic stromal lymphopoietina b s t r a c tAround the globe,worsening air pollution is spawning major public health and environmental concerns,especially in the poorest and most populous cities.As a major secondary air pollutant,ozone is a po-tential risk factor for exacerbated asth

6、ma,although the underlying mechanisms remain uncertain.In thisstudy,we aim to investigate the role of ozone on asthma exacerbation using a classic asthmatic modelwith allergic airway inflammation by treating Balb/c mice with ovalbumin(OVA).Our study shows ozoneexposure significantly exacerbated OVA-

7、induced asthmatic phenotypes,including serum immunoglob-ulin,Th cytokines,inflammatory cell counts,mucus production,airway remodeling,and airway hyper-responsiveness(AHR).Interestingly,expression of transient receptor potential cation channel subfam-ily V member1(TRPV1)was also significantly elevate

8、d in ozone-exacerbated asthmatic mice and thattreatment with TRPV1 antagonist effectively suppressed AHR,airway inflammation and remodeling.Theunderlying mechanisms of these effects may be associated with suppression of neuropeptide calcitoningene-related peptide(CGRP)and thymic stromal lymphopoieti

9、n(TSLP),an epithelial cell-derived cyto-kine.Base on the role of TRPV1 in allergic asthma,this study further revealed that inhibition of TRPV1 byTRPV1 antagonist has significant anti-inflammatory effects on ozone-induced asthma exacerbation inthis study.Induction of TRPV1 expression may be an import

10、ant mechanism underlying the increasedrisks for asthma after exposure to environmental pollutants.2019 Elsevier Ltd.All rights reserved.1.IntroductionAllergic asthma characterized by airway inflammation,airwayremodeling,and airway hyperresponsiveness(AHR),is a highlyprevalent chronic airway disease

11、affecting nearly 300 millionpeople worldwide.Acute exacerbation of asthma can be caused byenhancedairwayinflammation,hypercontractilityofairwaysmooth muscle and airway wall edema(Wang et al.,2018a).Envi-ronmental allergens such as air pollutants and viruses can triggerasthma exacerbations(Bowatte et

12、 al.,2017).As a major airpollutant,ozone has high oxidation potential and can have adverseimpacts on human health,especially the respiratory system(Duanet al.,2017b).Previous studies have reported that increasedground-level ozone is positively associated with the averagenumber of emergency roomvisit

13、s byasthmatic patients,suggestinga possible role of ozone in the exacerbation of asthma(Tian et al.,2018b).In fact,previous studies have shown that continuousexposure to ozone is particularly harmful for asthma patients(Goodman et al.,2018).In our studies,1.0 ppm acute ozone expo-sureinducedlunginfl

14、ammationandexacerbatedasthmatic*This paper has been recommended for acceptance by Dr.David Carpenter.*Corresponding author.*Corresponding author.Brain and Cognitive Dysfunction Research Center,Schoolof Medicine,Wuhan University of Science and Technology,Wuhan,430065,China.E-mail addresses:(Z.Guo),(Y

15、.Zeng).1Jinquan Li and Yushan Chen contributed equally to the work.Contents lists available at ScienceDirectEnvironmental Pollutionjournal homepage: Elsevier Ltd.All rights reserved.Environmental Pollution 247(2019)586e594symptoms in asthmatic mice model(Duan et al.,2017b;Zhu et al.,2016).Understand

16、ing the mechanisms of ozone-induced asthmaexacerbation may lead to potential therapeutic solutions forasthma patients.Transient receptor potential cation channel subfamily V mem-ber1(TRPV1)is a non-selective cation channel,which can be acti-vated by a wide variety of endogenous and exogenous factors

17、 aswell as physical and chemical ligands,including noxious chemicals,low pH,and high temperatures(Vandewauw et al.,2018).Inaddition to playing a critical part in nociceptive neuron sensorytransmission,increasing attention has been drawn towards the roleof TRPV1 in the pathophysiology of asthma(Baker

18、 et al.,2016;Choiet al.,2018).Not only is TRPV1 associated with childhood onset ofasthma,patients with severe asthma also exhibit significantlyincreased TRPV1 expression in the airway epithelia(McGarveyet al.,2014).It is believed that TRPV1 is gated by electrophilic orlipophilic ligands,both of whic

19、h can be derived from exogenousand endogenous sources.Reactive oxygen species(ROS),forexample,are endogenous electrophilic compounds capable ofinducingoverexpression of TRPV1 invitro(Dinget al.,2016a).Otherstudies have indicated that TRPV1 may serve as an integrator of“noxious”stimuli in activating

20、the immune-inflammatory cascade(Wang et al.,2018b;Wang et al.,2018c).For instance,activation ofTRPV1 can lead to an intracellular Ca2rise in airway cells,whichcan result in the induction of NF-kB and NFAT with subsequent pro-inflammatory mediator production and release(Jia et al.,2014).Moreover,envi

21、ronmental risk factors such as particulate matter 2.5(PM2.5)and diesel exhaust particles(DEP)can worsen symptomsof asthma and have been shown to elevate levels of TRPV1 in ani-mal models.Inparticular,electrophilic components of conventionalDEP can stimulate ROS production and promote inward currents

22、 inairway epithelial cell lines via TRPV1 activation(Akopian et al.,2016).Specifically,environmental PM generated from coal,oil flyash,and ash from Mount St.Helens have all been shown to activateTRPV1 expression(Lvet al.,2016).These studies suggest that TRPV1channel may play a critical role in pollu

23、tant-induced asthmaexacerbation.Although it is known that inhaled ozone can stimu-late the production of ROS and other oxidation products in thepulmonary system,it is still unclear whether ozone exposure candirectly influence TRPV1 channel activation.In this study,we aim to identify the exacerbating

24、 effects ofozone by examining serum immunoglobulin concentrations,cyto-kine contents,airway leukocyte infiltration,histopathologicalchanges in lung tissue,and airway hyper-responsiveness(AHR)inan allergic asthma mouse model.Furthermore,we will determinethe amount of TRPV1 expression in the lung and

25、examine theantagonistic effects of capsazepine(CPZ)on ozone-exacerbatedasthma features,including allergic inflammation,airway remod-eling and AHR.Finally,we aim to elucidate the potential molecularmechanisms of TRPV1 on ozone-exacerbated allergic asthma.2.Materials and methods2.1.AnimalsAll experime

26、nts were performed according to relevant guidelinesandregulations.HubeiProvinceExperimentalAnimalCenter(Wuhan,China)provided 4e5 weeks,201.5g,male Balb/c mice and com-mercial diet for this study.The Office of Scientific Research Manage-ment of Central China Normal University approved this experiment

27、alprocedure and the approval ID is CCNU-IACUC-2016-003.2.2.General procedures for ozone exposureThere are 5 groups in this experiment and 6 mice in each group.The first group is control group which is exposed to filtered air(FA).The second to fourth group are 1.0 ppm ozone alone exposuregroup,OVA gr

28、oup and OVA1.0 ppm ozone exposure group,respectively.The last group is OVA 1.0 ppm ozone and CPZ(2 mgCPZ/kg via intratracheal instillation)group.On days 1,8 and 15,mice were treated with saline or same volume OVA solution(83.33mg OVA and 2.92 mg Al(OH)3in 500mL saline)per day byintraperitoneal injec

29、tion.Then,saline or 1%OVA aerosol was usedto challenge(30 min/d)on days 19 through 25.Mice were exposedto ozone(1 ppm)for 3h/day from days 19e25.Ozone was pro-duced using a KTB portable ozone generator(Guangzhou,China).Ozone control mice were exposed to filtered air for 3 h/day fromdays 19e25 in an

30、adjacent chamber.The apparatus delivers aregulated air flow with controlled temperature(24C)and relativehumidity(50%).In this study,the concentration of ozone andquantity of CPZ were determined according to our previous studies(Duan et al.,2017a;Wu et al.,2013).CPZ was administered as anantagonist 3

31、 h after exposure to 1.0 ppm ozone.Detailed protocol isshown in Fig.1A.In order to avoid the interaction between various tests,threesets of experiments were conducted in this study.In the first round,after administration,the four groups mice(6 mice/group)wereused for testing serum Ig production,Th c

32、ytokine concentrations,and the levels of TRPV1,CGRP and TSLP in lung via commercialELISA kits.Moreover,inflammatory cell counting was also finishedin this round.In the second experiment,a separate set of 20 mice(5mice/group,4 groups)were treated under the same conditions andthen used for lung histol

33、ogical evaluation,immunofluorescentstaining for TRPV1 and immunohistochemical staining of CGRP andTSLP.In the last round,after same treatment,another 20 mice(5mice/group,4 group)were used for AHR test.2.3.Immunofluorescent staining for TRPV1Immunofluorescence staining of mouse lung tissue for TRPV1w

34、as carried out according to a previously described procedure(Duan et al.,2017b).The slides were incubated with anti-TRPV1(1:50,Abcam,MA,USA)as primary antibodies.DAPI was used tovisualize cell nuclei.All immunofluorescence staining was per-formed under a confocal microscope.The intensity of TRPV1 si

35、gnalswas quantified using ImageJ.The mean intensity of five non-overlap fields was averaged for each stained slide and the surfacearea examined held constant across all samples.Five mice lungtissues were analyzed per group.2.4.Assessment of AHROn day 26,all mice were tested for AHR by Lung Function

36、Sys-tem(AniRes,2005;Beijing,China)according to a previouslydescribed procedure(Li et al.,2014).Briefly,24h after the finalchallenge,mice were anesthetized with pentobarbital sodium(100mg/kg)via intraperitoneal injection.When the mouse entereda deeply anesthetized state,the trachea was exposed by car

37、efullycutting the skin of the neck.A cannula was then inserted into thetrachea and quickly connected to a small computer-controlledventilator.The ratio of breath/inspiration is preset to 1.5:1 andthe actual respiration rate is preset to 90 breaths per minute.During the AniRes 2005 software running,5

38、0ml of methacholinewas injected into the jugular vein at 5min intervals using a syringeneedle through a fixed tracheal tube.Due to the MHC was directlyinjected into the jugular vein instead of inhaling,the concentra-tions of MHC were 0.025,0.05,0.1 and 0.2 mg/kg,respectively.Within 250s after MCH en

39、try into the vein,R-area of inspiratoryresistance(RL)and expiratory resistance(Re)were recorded.Thelowest pulmonary dynamic compliance(Cldyn)(the trough of theJ.Li et al./Environmental Pollution 247(2019)586e594587compliance curve versus the baseline Distance)was marked toquantitatively assess lung

40、compliance.2.5.Lung histological assayLung tissue was fixed in 4%buffered formaldehyde and paraffinembedded under standard conditions.Tissue sections(5mm)werestained with haematoxylin&eosin(H&E)and periodic acid-schiff(PAS)for lung histological assay following the manufacturersstandard protocol(Sigm

41、a-Aldrich).All sections of the lung tissuewere assessed by pathologists in a blinded fashionwith a DM4000Bmicroscope.Inflammation score of H&E-stained lung sections wasperformed as previously described(Duan et al.,2017b).Briefly,fiverandom fields for each stained slide were determined by using asemi

42、-quantitative severity score(0e3)for inflammatory cell infil-tration and alveolar epithelial injury(0e3).The average opticaldensity(AOD)of the PAS stained sections were performed byImage-Pro Plus 6.0.The optical density of five random fields wasaveraged for each stained slide and the surface area ex

43、amined heldconstant across all samples.Five mice lung tissues were analyzedper group.2.6.Cytokine measurements and inflammatory cell countingBronchoalveolar lavage fluid(BALF)was obtained by instillingwithatrachealcannula.TheBALFisthencentrifugedat1500 rpm at 4C for 10min.The supernatant was collect

44、ed fordetecting levels of IL-4,IL-5,IL-13,and IFN-gusing commercialELISA kits(ebioscience,CA,USA).After the BALF is centrifuged,cellsediment suspended in saline is used to facilitate cell counting.Total cells,neutrophils,eosinophils,and lymphocytes in suspensionwere counted using a Blood Cell Analys

45、is system(MTN-21,Matee3nu Technology Corp.,Jinan,China).Lung tissue homogenatewas prepared according to a previously described procedure(Duanet al.,2017b)and the supernatant was collected for TRPV1,CGRPand TSLP testing.The concentrations of TRPV1,CGRP and TSLP inlung were measured using commercial E

46、LISA kits(elabscience)according to manufacturers protocols.2.7.Quantitative analyses of total serum IgE,OVA-IgE,and OVA-IgG1All mice were sacrificed 24h after the final OVA challenge.Bloodsamples were collected via cardiac puncture and kept at roomtemperature for 30 min.Then,all samples centrifuged

47、at 3000rpmfor 10 minat room temperature for serum preparation.Next,total-IgE(eBioscience,San Diego,USA),OVA-IgE(BlueGene,Shanghai,China),and OVA-IgG1(BlueGene,Shanghai,China)commercialELISA kits were used to measure the serum samples at 450 nm by amicroplate reader according tothe manufacturers inst

48、ructions.Thesensitivities of the ELISA kits were15 pg/mL for total-IgE and 0.1ng/mL for both OVA-IgE and OVA-IgG1.2.8.Immunohistochemical staining of CGRP and TSLPImmunohistochemical staining of CGRP and TSLP was carriedout according to a previously described procedure.Sections of thelung tissue wer

49、e incubated with primary antibodies anti-CGRP(1:50,Abcam,MA,USA)and anti-TSLP(1:100,Abcam,MA,USA).The negative control was obtained by omitting the primaryFig.1.TRPV1 production is induced in the lung after ozone exposure in OVA-induced mouse models of asthma.(A)Experimental protocol.(B)Immunofluore

50、scence stainingfor TRPV1 in lung tissue(pan marker,red),nuclei were stained with DAPI(blue)(n 5).(C)Fluorescence Intensity analysis of immunofluorescent staining(n 5).*:p 0.05,*:p 0.01,compared with the Control group;#:p 0.01,compared with the OVA group.(D)TRPV1 concentrations in lung by ELISA(n 6).