PM_2.5 Exposure Induced Lung Injuries And Related Molecular Mechanisms

Due to the rapid development of economy,the adverse environment and health problem induced by air pollution has emerged in China in a compressed manner,which is experienced by developed countries during the different industrial development stages in the past 100 years.The most prominent event is the frequent atmospheric haze in recent years,which involves a wide range of areas with high intensity and long duration.Considering that the sources and causes of atmospheric fine particulate matter(PM_2.5)in China are complex,and that its toxic components and mechanism are not clear,we could not copy foreign research patterns and analysis results.Therefore,it is of great significance to study the healthy effects and toxicological mechanism of PM_2.5 in the atmosphere combining with the characteristics of haze,which will help us analysis the harmful effects of haze and promote the development of environmental pollution and health research in China.In our study,we propose to investigate the combinded effects of PM_2.5 and gaseous pollutants including SO₂ and NO₂ on lung injuries.Then,we study the susceptibility of mice to PM_2.5 at different life stages,and the recovery effects of PM_2.5 exposure on mice with the strongest susceptibility.Finally,we analyze the effects of maternal PM_2.5 exposure on lung development of offspring.1.The role of ambient air pollution is considered to be important in the development of chronic obstructive pulmonary disease?COPD?,and pulmonary hypertension?PH?is a common clinical manifestation of COPD.However,many studies have mainly focused on the adverse health effects of a single air pollutant,ignoring the combined toxicities of multiple pollutants.In the present study,C57BL/6 mice were randomly divided into three groups: control group?aspiration of saline every other day and exposure to clean air every day?;lower concentration group(1 mg/kg bw PM_2.5 every other day by oropharyngeal aspiration and 0.5 mg/m³ SO₂ and 0.2 mg/m³ NO₂ by dynamic inhalation simultaneously?6 h/d?);higher concentration group(3 mg/kg bw PM_2.5,3.5 mg/m³ SO₂ and 2 mg/m³ NO₂).After exposure ended,lung function?forced expiratory volume in 0.1 s?FEV0.1?/forced vital capacity?FVC??was determined by Anires2005 system,histopathological abnormalities were determined via hematoxylin-eosin?HE?staining and ultrastructural changes was observed by transmission electron microscopy?TEM?.Then,western blot?WB?assays were used to determine the expression of the marker of PH?endothelin-1?ET-1?and endothelial nitric oxide synthase?e NOS??.In addtion,micro RNAs?mi RNAs?microarray combined with real-time quantitative reverse transcription PCR?RT-PCR?were used for determining mi RNA expression profiles,and dual-luciferase reporter gene was used for analysis of mi RNA expression and the target gene expression of mi RNA.The results indicated that PM_2.5,SO₂ and NO₂ co-exposure induce airflow limitation,histopathological and ultrastructural alteration and abnormal ET-1 and e NOS expression in mice lung.Further,mi RNA arrays identified three significantly changed mi RNAs homologous with humans?mi R-338-5p,mi R-450b-3p and mi R-142-5p?,and we targeted mi R-338-5p based on RT-PCR validation.Furthermore,bioinformatic and dual-luciferase reporter gene analysis suggested that mi R-338-5p bound to 3'-untranslated region?UTR?of hypoxia-inducible factor 1??HIF-1??m RNA and down-regulation of mi R-338-5p which led to the increased expression of HIF-1? and its related gene four-and-a-half LIM?Lin-11,Isl-1 and Mec-3?domain 1?Fhl-1?and contributed to PH.These findings provide a basis for the role of mi RNAs in lung injuries induced by air pollutants co-exposure.2.PM_2.5 itself is a complex and is associated with many respiratory diseases.However,experimental studies about the correlation between PM_2.5 and lung injuries mainly focus on the same age group and lack comparison among different age groups.Therefore,C57BL/6 mice at different ages?4 weeks?4W?,4 months?4M?,10 months old?10M??received oropharyngeal aspiration of PM_2.5?3 mg/kg bw?every other day for 4 weeks.Then,lung function was determined by Anires2005 system and histopathological abnormalities were observed by HE staining.Next,the level of ROS and activity of antioxidant enzymes?superoxide dismutase?SOD?and glutathione peroxidase?GSH-Px??were determined.Finally,RT-PCR was used to determine inflammatory cytokines such as Tnf-? and Il-1?.The results showed that PM_2.5 exposure altered lung function in 4W and 10 M mice,and could cause airway hyperresponsiveness in mice at different ages.HE staining results suggested that PM_2.5 exposure can cause changes in airway structure in 4W and 10 M mice.In addition,PM_2.5 exposure elevated levels of inflammatory factors and ROS in 4W and 10 M mice and altered antioxidant enzyme activities in 10 M mice.These results compare the susceptibility of mice at different stages to PM_2.5 exposure induced lung injury,and provide the theoretical basis for seeking the best time for the prevention and mitigation effects of air pollution.3.Epidemiological and experimental studies have progressively provided a better knowledge of the underlying mechanisms by which fine particulate matter(PM_2.5)exerts its harmful health effects.However,limited studies focused on the effect and following recovery after the particulate exposure ended.Therefore,this study focus on elderly mice which is most suspective to PM_2.5 exposure,and then establish PM_2.5 exposure and recovery model,aim to determine recovery process and mechanism of lung injury induced by PM_2.5 exposure.10-month C57BL/6 mice were treated by oropharyngeal aspirations of 3 mg/kg bw PM_2.5 every other day for 2 weeks or 4 weeks,respectively;another mice in were treated by oropharyngeal aspirations of 3 mg/kg bw PM_2.5 for 4 weeks following 1 week or 2 weeks recovery,respectively.Then,lung function was determined by Anires2005 system.Wright's-Giemsa staining was used to determine cell differential counts and particulate depositionin bronchoalveolar lavage fluid?BALF?.Further,western blot and enzyme-linked immunosorbent assay?ELISA?was used to determine the H3K27 ac expression and level of histone acetyltransferase?HAT?and histone deacetylase?HDAC?.Chromatin immunoprecipitation-sequence?Ch IP-seq?analysis was used to determine the genomic distribution of H3K27 ac peaks.Finally,inflammtory cytokines and chemokine levels were determined by ELISA.The results revealed that PM_2.5 exposure for 4 weeks significantly decreased the lung function,and the changes returned to normal levels after 1-week recovery.However,we observed persistent particle alveolar load following 2-week recovery.Interestingly,the alterations of H3K27 ac expression and related enzyme activities mimicked the changes of respiratory function during the process,and Ch IP-seq suggested that these PM_2.5-associated differentials H3K27 ac markers participated in immune responses and chemokine signaling pathway with signal transducer and activator of transcription 2?stat2?and breast cancer anti-estrogen resistance 1?bcar1?being two important genes.Consistently,the expression of pro-inflammatory cytokines and chemokines elevated after PM_2.5 exposure for 4-week,and reversed to normal levels following 2-week recovery.The study highlighted that PM_2.5 aspiration caused histone modification associated lung dysfunction and inflammation,and the action restored after exposure ending and 2-week recovery.Also,persistent particle alveolar load might be a long-term potential risk for lung diseases.4.A variety of particulate exposure models established in our previous study have confirmed the correlation between particulate matter exposure and respiratory damage.However,the adverse effect of particulate air pollution on respiratory health starts in utero.Epidemiological evidence indicates that maternal exposure to PM_2.5 is related to increased risk of bronchopulmonary dysplasia?BPD?.Further,the researches about maternal PM_2.5 exposure and lung development of offspring were rarely reported.Therefore,we aim to test whether maternal PM_2.5 exposure causes BPD in male offspring,and,if so,to investigate whether these adverse effects could be restored with the development.The plug-positive mice received 3 mg/kg bw PM_2.5 by oropharyngeal aspiration every other day throughout the gestation period,starting on gestation day?GD?0.At embryonic?E?18.5,maternal placenta and lung were harvested for analysis of metal content in placenta and inflammation reaction in maternal lung and placenta.Offspring at postnatal days?PND?1,7,14 and 21 were sacrificed for analysis.HE staining was used to determine alveolar development,immunofluorescence,western blot and RT-PCR were used to test angiogenesis,immunohistochemistry,TEM and RT-PCR were used to analysis secretory function.Then,lung inflammation were determined by RT-PCR.Finally,lung function was determined by Anires2005 system.The results showed that maternal PM_2.5 exposure increased the levels of Pb and Mn and altered the presence of inflammatory factors in the placenta.During the postnatal development stage,maternal exposure to PM_2.5 caused low birth weight and typical BPD-like symptoms,including hypoalveolarization,decreased angiogenesis,suppressed production of secretory and surfactant proteins,and increased inflammation,in the lungs of male offspring.However,maternal PM_2.5 exposure induced only hypoalveolarization and inflammation in the lungs of female offspring.Furthermore,these alterations were reversed during postnatal development.In the present study,we first investigated the underlying molecular mechanism of lung injuries induced by co-exposure of PM_2.5 and gaseous pollutants based on the epigenetic regulation of mi RNAs.Then,we established PM_2.5 exposure model of mice at different age stages to clarify the susceptibility of lung injuries in these mice.Third,we further established PM_2.5 exposure and recovery model of the most sensitive aged mice to discuss the recovery effect and mechanism of PM_2.5 exposure-induced lung injuries in aged mice from the perspective of histone modification.Finally,we determined the adverse effects of maternal PM_2.5 exposure on the lung development of the offspring based on the theory of fetal origin of adult diseases?FOAD?,and identified the root causes of increased susceptibility of diseases in later adulthood.Our present study evaluated the adverse effects and molecular mechanism of air pollution on respiratory system by a variety of exposure models and various epigenetic mechanisms.This study also provided new experimental evidences for the development of the research on environmental pollution and health effect in China.