The Lung Diseases And Molecular Mechanism In Response To Maternal NO₂ Exposure

Air pollution has always been a major threat to human health.In the early 20th century,developed countries in Europe and America experienced a series of air pollution events,such as Meuse Valley fog in Belgium?1930?,photochemical smog in Los Angeles?1943?and Great Smog in London?1952?,which led to dozens,even thousands of death.China also suffered frequent air pollution attack in the 21st century.In 2013,China experienced widespread haze,which was the worst air pollution in 52 years and affected more than 100 large or medium-sized cities.Importantly,nitrogen dioxide?NO₂?,as a traffic related air pollutant,is also a key factor in the occurrence of haze.As lung is the target organ of NO₂ exposure,many researches indicate NO₂ is related to respiratory health problems,such as asthma.Further,the theory of fetal origins of adult disease?FOAD?pointed out the developmental deficiency of fetus in utero could lead to increased diseases susceptibility in adult.Air pollutants can affect the uterine environment and thus lead to fetal hypoplasia through penetrating the placenta barrier into the uterus or causing toxic effects on the mother.Therefore,fetal stress was proposed as a crucial factor for allergic airway response occurred in offspring.However,existing evidences about the toxic characteristics of NO₂ exposure and its health effects on lung mainly focused on child and/or adult,rarely touching whether maternal exposure could affect the asthma susceptibility in the offspring.For this reason,this study will focus on toxicity effects and genetic/epigenetic mechanisms of direct or maternal NO₂ exposure induced lung injuries based on lung disease model.1.Direct NO₂ exposure could reduce lung function,however,the exact mechanism is not clear.In directly acute NO₂ exposure study,Wistar rats were exposed to either NO₂?2.5 ppm,5 h/day?or air daily for seven days,then the lung inflammation and possible mechanisms were investigated.Lung histopathological changes were observed by Htoxylin eosin?H&E?staining.Ultrastructural changes of lung tissues were examined by transmission electron microscopy?TEM?.The mRNA expression levels of interleukin-1??IL-1??,IL-6,intercellular cell adhesion molecule-1?ICAM-1?,janus kinases?JAK?1,JAK3 and signal transducer and activator of transcription?STAT?6were detected by real-time quantitative RT-PCR.The results showed that NO₂exposure could alter the pathological and ultrastructure of lung tissue and increase the infiltration of inflammatory cells in the lungs when compared with the control group.Further,proinflammatory cytokines IL-6 and ICAM-1were 1.36-and 2.01-fold of control,respectively.Finally,NO₂ exposure also promoted the expression of JAK3 and STAT6?2.19-and 1.30-fold of control,respectively?,suggesting that the JAK-STAT6 signaling pathway may be involved in the pulmonary inflammatory response induced by NO₂.This study provides experimental evidence for exploring the correlation between NO₂ exposure and lung injury.2.Researches about NO₂ induced lung injuries mainly focused on direct NO₂ exposure,however many recent studies investigated the relationship between maternal NO₂ exposure and lung injuries in the offspring,and the specific mechanism is not clear.In maternal NO₂ exposure study,pregnant BALB/c mice were exposed to either NO₂?2.5 ppm,5 h/day?or air daily throughout the gestation period.Offspring were sacrificed on postnatal days?PNDs?1,7,14,21 and 42.Lung histopathological changes were observed by H&E staining and Masson staining.Total number of cells and differential of pulmonary inflammatory cells?macrophages?Mac?,eosinophil?Eos?,neutrophils?Neu?and lymphocytes?Lym??were detected by blood cell counts and fluorescence-activated cell sorting?FACS?,respectively.Type II cytokine IL-4 and IL-13 and type I cytokine interferon-gamma?IFN-??were detected by enzyme-linked immunosorbent assay?ELISA?.Finally,the methylation levels in promoter of IL4 and IL13 in the lungs of the mice were investigated by bisulfite sequencing PCR?BSP?.The results showed that maternal NO₂ exposure can induce asthma related symptoms in the offspring,including histopathological changes,significant increase in the numbers of total cells,macrophages,eosinophils and lymphocytes on PND1.Also,increased IL-4 and IL-13 and decreased IFN-?level were observed in maternal NO₂ exposed group on PND1.We also found that the methylation level of IL4 and IL13 promoter region decreased 20%and 10%in maternal NO₂ exposed offspring on PND1.More importantly,these changes did not show significant sex differences and gradually restored to the normal level with the development of offspring.The results above indicated that maternal exposure to indoor environmental NO₂ could cause allergic asthma-related consequences in offspring absent any subsequent lung provocation.Present study provides experimental evidences for the maternal NO₂ exposure induced lung injuries and the recovery effect during postnatal development.3.The research above indicated that maternal NO₂ exposure could lead to lung inflammation which could restore to normal level with development process absent any subsequent lung provocation.Do these results suggest that the toxicity on offspring is terminated,or will it enhance the susceptibility of related diseases in later life processes?What mechanism participates in the offspring damage?In this section,pregnant BALB/c mice were daily exposed to 2.5 ppm NO₂ throughout gestation period,then the offspring were challenged to allergen?ovalbumin,OVA;30 min/day,14 days?.OVA specific immunoglobulin?Ig?E in the serum was detected by ELISA,airway hyperresponsiveness?AHR??Resistance of expiration?R_e?,Resistance of lung?R_L?,Respiratory dynamic compliance?Cdyn??of the mice were detected by Anires2005 system.Lung histopathological changes were observed by H&E staining.Proinflammatory cytokines?Tumor necrosis factor-alpha,TNF-??and IL-1?,key cytokine IFN-?,IL-4,IL-13 and IL-17 in the lung of the mice were determined by ELISA.As for signaling pathway,the protein expression of T-bet?Th1 cell key transcription factor?,GATA3?Th2 cell key transcription factor?,JAK1,STAT6 and p-STAT6 in lung tissues were detected by Western blot?WB?.The results showed that maternal NO₂ exposure enhanced OVA induced elevation of OVA-IgE level in serum,and caused AHR and pathological changes in offspring.Furthermore,maternal NO₂ exposure promoted OVA induced increased expression of proinflammation factors and impaired the Th2/Th1 balance.In addition,JAK-STAT6 pathway participated in these processes.Our study implied maternal NO₂ exposure could increase the potential risk of airway sensitivity to allergen.4.Epidemiological studies suggest that maternal exposure to NO₂contributes to the increased morbidity and mortality of allergic asthma later in life.In this section,pregnant BALB/c mice were exposed to either NO₂?2.5 ppm,5 h/day?or air daily throughout the gestation period.The adult offspring were sensitized by OVA injection followed by an OVA aerosol challenge during postnatal weeks 7-9.Total and OVA-IgE in the serum were detected by ELISA,AHR?R_e,R_L and Cdyn?of the mice was detected by Anires2005 system and lung histopathological changes were observed by H&E staining,Masson staining and AB/PAS staining.Our results showed that following OVA sensitization/challenge,maternal NO₂ exposure enhanced the levels of allergic asthma-characterized OVA-IgE,AHR,airway inflammation,fibrosis and mucous cell metaplasia in adult offspring.Further,total number of cells and differential of pulmonary inflammatory cells?Mac,Eos,Neu and Th2?were detected by blood cell counts and FACS,respectively.The results indicated that OVA sensitization and challenge increased the number of Mac,Eos and Th2 cells and maternal NO₂ exposure enhanced these effects.In order to investigate the specific makers,key cytokine IFN-?,IL-4 and IL-13 in the lung of the mice were detected by ELISA.The results showed that maternal NO₂ exposure could enhance alternation of these cytokines in lungs of asthma mice.Finally,the methylation levels in promoter of IL4 and IL13 in the lung tissues of the mice were investigated by BSP,and demethylation of IL4 gene occurred during the process?9%decrease,NOO vs.AOO?.This study provides experimental evidences for clarifying maternal NO₂ exposure induced adverse effects on offspring and revealing early lung injury and enhanced susceptibility to adverse environmental stimuli in adulthood.In this study,we first discussed the effects of acute NO₂ exposure on inflammatory and histopathological changes in lungs of rats.Then,we determined the effects of maternal NO₂ exposure on the lung injuries of the offspring mice,and proposed that the changes were reversible with the development of the offspring.Further,we evaluated whether maternal NO₂exposure could enhance offspring sensitivity to allergen and its mechanism.Finally,we analysed the relationship between maternal NO₂ exposure and asthma susceptibility in offspring mice based on DNA methylation.In the present study,we focused on direct and maternal NO₂ exposure,revealed its direct toxicity on animal and indirect effect on early life injuries and disease risks in offspring,and provided new ideas for health risk management.