| With the development of urban industrialization,air pollution has become a serious threat to human health,especially PM2.5.PM2.5 can induce the exacerbation of respiratory diseases,reflected by an increase in hospitalization rates and mortality of patients with asthma and COPD.Airway hyperresponsiveness(AHR)refers to an increase in sensitivity of respiratory tract in response to non-antigenic stimulation,as evidenced by the excessive contraction of bronchial smooth muscle,thus causing airway stenosis and increased airway resistance.AHR as a classicalcharacteristic of asthma is positively correlated with severity of asthma to a certain degree.Currently,the underlying mechanisms by which PM2.5-induced AHR including inflammation,oxidative stress,the imbalance of immune system,cell apoptosis,excessive autophagy,epigenetics alterations and so on.However,the exact molecular mechanisms remain unclear.Bromodomain protein 4(BRD4)as a "adapter" of chromatin can bind to chromosomes during the whole mitotic cycle,and recruit different transcriptional regulators(i.e.,Mediator,P-TEFb)to modulate numerous genes expression.In addition,BRD4 participates in genes transcription mediated by RNA Polymerase II,and plays a key role in regulating the cell cycle,inflammation,oxidative stress and so on.Currently,the role played by BRD4 in asthma mainly focuses on promoting the release of inflammatory cytokines and inducing airway remodeling.This study aims to explore the role played by BRD4 in PM2.5-induced AHR and uncover the exact molecular mechanisms.Our study is divided into three parts : Part one:animal experiment,the mice model with AHR was established with nose-only PM2.5 exposure system(Huironghe Technology Co.,Beijing,China)for 12 weeks,in Shijiazhuang,Hebei Province.Then we evaluatedAHR of mice with PM2.5 exposure,and explore the role of BRD4 in PM2.5-induced AHR.Part two: PM2.5 powder was dissolved in DMSO,then filtered by a 0.22 micron filterto obtain PM2.5 mother liquor,and then PM2.5mother liquor was diluted in standard culture mediumto form 12.5μg/ml concentrations.Then h ASMCs were treated with 12.5μg/ml PM2.5 suspension for 24 h to observe the role of BRD4 in the function of h ASMCs contraction,and explore the exact molecular mechanisms.Part three: PM2.5 powder was dissolved in DMSO,then filtered by a 0.22 micron filter to obtain PM2.5mother liquor,and then PM2.5 mother liquor was diluted in standard culture mediumto form different PM2.5 concentrations.Thenh ASMCs were treated with different concentration of PM2.5 suspension(0,3.125,6.25,12.5,25μg/ml)for 24 h toevaluate the function of h ASMCs migration,and uncover the role of BRD4 in the above process.PART One PM2.5 increases airway hyperresponsiveness by promoting BRD4 expressionObjective: To explore the role of BRD4 in PM2.5-induced AHR in mice.Mehtods:1.In total,40 male C57BL/6J mice(Wild-type,6-7-week-old,20 ± 2g)were obtained from Beijing Sibeifu biotechnology Co.,Ltd.Animals were housed in a specific-pathogen-free(SPF)room and provided with water and standard mouse chow.No animals became ill or died before the experimental endpoint.The experimental procedures of all animals were approved by the Ethical Committee of the Second Hospital of Hebei Medical University,China(approval number:2020-AE011).As our previous work described,the mice model with AHR was established with nose-only PM2.5 exposure system(Huironghe Technology Co.,Beijing,China)for 12 weeks in Shijiazhuang,Hebei Province.After acclimatizing to the laboratory environment for one week,all animals were randomly assigned to four groups(n = 10/group): FA(filtered air): The mice received filtered air for 4 h per day,5 days per week for 12 weeks.PM2.5: The mice received PM2.5 exposure for 4 h per day,5days per week for 12 weeks.PM2.5+ZL0420: One hour before PM2.5exposure,mice were treated with 10mg/kg BRD4 inhibitor(ZL0420)by intraperitoneal injection,once a day,5 days per week,for12 weeks.PM2.5+Vehicle: One hour before PM2.5 exposure,mice were treated with vehicle by intraperitoneal injection,once a day,5 days per week,for12 weeks.2.Mice were challenged by different concentrations of methacholine(Mch:(mg/ml)0,3.125,6.25,12.5,25 and 50)24 h after the last exposure to PM2.5,andthe values of airway resistance [RN],respiratory system resistance[Rrs],andtissue damping(G)were measured and recorded;Broncho alveolar lavage fluid(BALF)of mice were obtained 24 h after the last PM2.5exposure.Thesupernatants wereobtained for themeasurement of cytokine levels(IL-1β,IL-6,IL-8 and BK);The left lung tissuewas fixed using 4%neutral formaldehyde solution for 72 h.Tissue sections were stained with hematoxylin-eosin(HE)to visualize the histopathological abnormalities of lung tissue;Immumohistochemical assay was used to evaluate the expression of BRD4 in lung tissue of mice;Western blotting was selected to further assess related-protein levels(i.e.,BRD4、KLK14、B2R、MMP2、MMP9 and Vimentin).3.Statistical analysis : Results are presented as the means?±?SEM and analyzed with SPSS version 19.0(IBM,Armonk,NY).Comparisons between two groups were evaluated with two sample Student’s t-test.P < 0.05 was considered significantstatistically.`Results: 1.During the PM2.5 exposure period,the average concentration of daily PM 2.5 was 90μg/m3.The data was provided by the Shijiazhuang Environmental Protection Bureau.The average concentration of daily PM2.5online exposure was 820μg/m3.The maximum concentration of PM2.5exposure was 2000μg/m3,and theminimum concentration of PM2.5 exposure was 500μg/m3.2.BRD4 inhibition contributed to alleviating PM2.5-induced AHR.The role of BRD4 in AHR induced by PM2.5 was elaborated next.PM2.5had an adverse effect on lung function.The values of Rrs,RNand G in PM2.5group were much higher than that in FA groupunder the stimulation of Mchata certain concentration.Compared with PM2.5+Vehicle group,PM2.5+ZL0420 group showed an apparent decrease in lung function(i.e.,Rrs,RN,and G)under the stimulation of Mch at a certain concentration.Moreover,under the stimulation of 12.5 mg/ml Mch,the Rrs,RN,and G of PM2.5 group were more than 2 folds as high as baseline value.However,the concentration of Mch that caused a 2 folds increase of airway resistance(G,RN,Rrs)in FA and PM2.5+ZL0420 group was much higher.In brief,PM2.5 could enhance AHR,and BRD4 inhibition couldimprove PM2.5-induced AHR.3.BRD4 inhibition could alleviate lung histopathology induced by PM2.5.PM2.5 was indicated to lead to alveolar space widening,airwaywall thickening and increased inflammatory cytokines.Compared with FA group,PM2.5 group could increase IL-1β,IL-6,IL-8 and BK levels in BALF.Here,we found that BRD4 played a critical part in lung damage induced by PM2.5.Compared with PM2.5+Vehicle group,BRD4 inhibition could ameliorate lung pathology,as evidenced by improvement of alveolar structure,amelioration of airwaywall thickening,and reduction of inflammatory cytokines.4.Confirmation of BRD4 localization lung tissueand assessment of BRD4 expression level with immunohistochemistry.Immunohistochemistry was selected to assess BRD4 protein expression in lung tissue cells.Positive signs of BRD4 antibody are visualized as brownish yellow granules.5.Verification of BRD4,KLK14,B2 R,MMP2,MMP9,and Vimentin levelsin lung tissue by Westernblotting.The protein levels of BRD4,KLK14,B2 R,MMP2,MMP9,and Vimentin in lung tissue were further measured by Western blotting.BRD4,KLK14,B2 R,MMP2,MMP9,and Vimentin levels of PM2.5 group were upregulated in comparison with that of the FA group.The expression of BRD4 was downregulated in PM2.5+ZL0420 group in comparison with that in PM2.5+Vehicle group,and then the reduction of BRD4 was accompanied by a drastic decrease in KLK14,B2 R,Vimentin,MMP2 and MMP9 levels.In other words,BRD4 participated in regulating the above protein expression in lung tissue.Conclusion:BRD4 participates PM2.5-induced AHR by modulating the expression of KLK14、B2R、MMP2、MMP9 and Vimentin in lung tissue of mice.PART Two PM2.5 promotes the contraction of h ASMCs by increasing BRD4 protein expressionObjective: To observe the role of BRD4 in PM2.5-induced airway smooth muscle cell constraction.Method:1.PM2.5 powder was dissolved in DMSO,then filtered by a 0.22 micron filterto obtain PM2.5 mother liquor,and then PM2.5 mother liquor was diluted in standard culture mediumto form different concentrations.Then h ASMCs were treated with different concentration of PM2.5 suspension(0,3.125,6.25,12.5,25μg/ml)for 24 h.Moreover,three separate BRD4-specific si RNA sequences as well as a negative control si RNA sequence(Ribo Bio;China)wastransfected using the Lipofectamine?RNAi MAX Reagent(Thermo-Fisher Scientific,Waltham,MA,USA).Then experiment was randomly assigned to four groups:NCsi RNA+DMSO group,BRD4 si RNA+DMSO group,NCsi RNA+ PM2.5 group,BRD4 si RNA+PM2.5 group.2.On the other hand,one hour before exposure to PM2.5 DMSO extracts,h ASMCs were treated with BRD4 inhibitor(ZL0420),followed by exposure to PM2.5 DMSO extracts for 24 h.The expression of associated proteins was assessed by western blotting 24 h after exposure to PM2.5 DMSO extracts.Then experiment was assigned to four groups:Control+DMSOgroup,Control+PM2.5group,ZL0420+ DMSO group,ZL0420+ PM2.5 group.3.Immunofluorescence was performed to examine the localization of BRD4 in h ASMCs;The levels of BRD4,KLK14 and B2 R in h ASMCs were assessed by western blotting;The level of BK in thesupernatant of h ASMCs was measured with ELISA kit;Cell Contraction Assay Kit was selected to assess the contractile function of HASMCs.4.Statistical analysis : Results are presented as the means?±?SEM and analyzed with SPSS version 19.0(IBM,Armonk,NY).Comparisons between two groups were evaluated with two sample Student’s t-test.P < 0.05 was considered statistically significant.`Results: 1.BRD4 expression in hASMCs was increased 24 hours after treatment with different concentrations of PM2.5(3.125,6.25,12.5,25μg/ml).Following treatment with 12.5μg/ml PM2.5,the expression level of BRD4 in hASMCs was the highest.The results of immunofluorescence showed that the expression of BRD4 was increased after exposure to 12.5μg/ml PM2.5 for24 h,which was consistent with the result of western blotting.Moreover,the result of immunofluorescence revealed that BRD4 was located in the nucleus rather than cytoplasm of h ASMCs.2.Compared with NCsi RNA+DMSO group,NCsi RNA+PM2.5 group could promote contraction of h ASMCs,as evidenced by the reduction of gel diameter.However,BRD4 gene knockdown could amelioratethe above response before and after PM2.5 exposure,reflected by inhibition of the reduction of gel diameter.ZL0420,a BRD4 inhibitor,inhibited h ASMC contraction,which was consistent with the results of BRD4 gene silencing,thus further supporting the view that BRD4 participates in PM2.5 DMSO extracts-induced h ASMC contraction.3.BRD4 knockdown and ZL0420 could lead to the reduction of KLK14 and B2 R expression,thereby inhibiting BK secretion in the supernatant of cells.Conclusion:PM2.5 could promote the contraction of h ASMCs,and that was associated with increased BRD4 expression.Notably,BRD4 could regulate KLK14 and B2 R expression,thereby mediating PM2.5-induced hASMCs contraction.PART Three PM2.5 promotes the migration of h ASMCs byincreasing BRD4 protein expressionObjective: To observe the role of BRD4 in PM2.5-induced airway smooth muscle cell migration.Method1.PM2.5 powder was dissolved in DMSO,then filtered by a 0.22 micron filter to obtain PM2.5 mother liquor,and then PM2.5 mother liquor was diluted in standard culture mediumto form different concentrations.Then hASMCs were treated with different concentration of PM2.5 suspension(0,3.125,6.25,12.5,25μg/ml)for 24 h.Moreover,the experiment was assigned to four groups: NCsi RNA+DMSO group,BRD4 si RNA+DMSO group,NCsi RNA+ PM2.5 group,BRD4 si RNA+ PM2.5 group.2.On the other hand,one hour before exposure to PM2.5 DMSO extracts,h ASMCs were treated with 40μmol/L BRD4 inhibitor(ZL0420),followed by exposure to PM2.5 DMSO extracts for 24 h.The expression of associated proteins was assessed by western blotting 24 h after exposure to PM2.5DMSO extracts.Then experiment was assigned to four groups:Control+DMSO group,Control+PM2.5group,ZL0420+ DMSO group,ZL0420+ PM2.5 group.3.The levels of MMP2,MMP9 and Vimentin in h ASMCs were assessed by western blotting;Transwell chamber method was used to evaluate the migration function of h ASMCs.4.Statistical analysis : Results are shown as the means ± SEM and analyzed with SPSS version 19.0(IBM,Armonk,NY).Comparisons between two groups were evaluated with two sample Student’s t-test.P < 0.05 was considered statistically significant.Results1.PM2.5 could promote h ASMCs migration,which was assessed by calculating the average number of cells in five random fields under a microscope equipped with a×10 objective.Following treatment with6.25μg/ml PM2.5,the amount of h ASMCs migration was the highest.Meanwhile,this alteration was often accompanied by increased MMP2,MMP9,and Vimentin expression in h ASMCs.2.BRD4 gene knockdown and ZL0420 could inhibit h ASMCs migration,as demonstrated by an apparent decrease in the amount of h ASMCs migration.Meanwhile,BRD4 gene knockdown and ZL0420 could inhibit the expression of MMP2,MMP9,and Vimentin in h ASMCs,thus further supporting the view that BRD4 participated in PM2.5-induced h ASMCs migration.Conclusion PM2.5 could promote the migration of h ASMCs,and that was associated with increased BRD4 expression.Notably,BRD4 could regulate MMP2,MMP9 and vimentin protein expression to modulate the migration of h ASMCs. |
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