Effects Of Fine Particulate Matter On Lung Function In Patients With Bronchial Asthma And Its Effect And Mechanism On Airway Hyperresponsiveness In Asthmatic Mice

The Particulate Matter（PM）in the air,especially the Fine Particulate Matter（PM2.5）,has become one of the most serious environmental and public health problems in many countries,especially in northern China.The sources of PM are complex,is also a complex mixture.PM2.5 is also considered to be the most harmful particulate matter to the lungs due to its small particle size and large specific surface area,containing a large number of toxic and harmful substances that can pass through the bronchioles,reach the alveoli and even enter the blood.As one of the major cities in the beijing-tianjin-hebei region,Affected by multiple pollution factors such as traffic related pollution,steel,cement and winter heating,shijiazhuang’s air pollution level ranks among the top in China.Shijiazhuang is the fifth contributor to pollution-related mortality in 190 cities in China,accounting for 2.02%of the total deaths caused by air pollution.Bronchial asthma（asthma）is a heterogeneous disease,is usually charact-erized by chronic airway inflammation,with airway hyperrespons-iveness（AHR）as its pathological and physiological characteristics.Its occurrence and development are associated with exposure to external agents（e.g,upper respiratory viruses,dust mites,and air pollutants）.Previous epidemiological investigations have shown that PM2.5 exposure is associated with increased incidence,hospitalization rate and mortality of asthma.At present,the clinical studies on the effect of PM2.5 on asthma mainly focus on epidemiological investigation,and there are less studies on the direct correlation between PM2.5 and lung function of asthma patients.Previous studies on the effect of PM2.5 on lung function in asthma patients mainly focused on the effect of the average concentration of PM2.5 on asthma symptoms and lung function during the period,lacking point-to-point studies on the correlation between PM2.5 and daily lung function changes in asthma patients.In the basic experimental research,there is a lack of animal models simulating the real world chronic exposure of PM2.5.Whether chronic exposure to PM2.5 aggravates airway hyperresponsiveness and its pathoge-nesis have not been determined.Our study is divided into two parts:Part one,the clinical part,to evaluate daily changes of lung function in patients with asthma responses to short-term ambient air pollutants（including fine particle matter and poisonous gases）levels.These data include lung function data continuously monitored by different individuals and continuous air pollutant data,so a multi-level mixed effect model was used for analysis,and the lag effect of PM2.5 was analyzed,which was more accurate and comprehensive than previous studies.The second part:animal experiment,we treated the different groups of BALB/c mice to exposed to high concentrations of PM2.5 for 8 consecutive weeks using a nose-only“PM 2.5 online enrichment system”,and observe the effects of PM 2.5 on the pathology and AHR of BALB/c mice undergoing sensitization and challenge with ovalbumin（OVA）and to observe the effects of apoptosis and T-cell immunoglobulin and mucin domain 1（TIM-1）in this process.Part Ⅰ Effects of Fine Particulate Matter of Lung Function in Patients with Asthma:Short-term Exposure StudyObjective:To evaluate daily changes of lung function in patients with asthma responses to short-term ambient air pollutants（including fine particle matter,SO₂,NO₂ and O₃）levels.Mehtods:We used observational methods.From February 2018 to March2019,we recruited patients with bronchial asthma living in the urban area of shijiazhuang city,whose condition is under control,and confirmed them into the group after excluding other diseases.During the baseline enrollment,the general condition was recorded and the clinical diagnostic basis was verified.We gave each patient a spirometer to record the daily peak expiratory flow（PEF）,forced vital capacity（FVC）,FVC%,forced expiratory volume in one second（FEV1）,FEV1%,FEV1/FVC,Maximal expiratory flow at 75%（MEF75）,MEF50,MEF25.Each patient was monitored for 10 consecutive days,and their test results were stored online.We obtained the atmospheric fine particulate matter and toxic gases（including SO₂,NO₂ and O₃）of the corresponding date from shijiazhuang environmental protection bureau.After summarizing the obtained data,we used the multilevel mixed effect model to analyze the data.Univariate analysis was performed on the relationship between PM2.5 and lung function in asthma patients,and the relationship between SO₂,NO₂,O₃ and lung function.We analyzed the lagged effect of PM2.5.We conducted multivariate analysis according to the results of univariate analysis.Results:1.We recruited a total of 42 patients,including 14 males and 28 females.There was no significant difference in BMI and treatment regimen between the two groups.2.The fixed effect analysis results of the mixed effect model with PM2.5univariate analysis（including the lagged effect）show that:The 0-order lag value of PM2.5 was negatively correlated with the variation of pulmonary function parameters PEF（z=-2.28,P=0.023）,FVC（z=-2.51,P=0.012）、FEV1%（z=-2.18,P=0.029）、FEV1（z=-2.21,P=0.027）.There was no correlation between the 1-order lag value of PM2.5 and the variation of lung function parameters.3.The fixed effect analysis results of the mixed effect model with SO₂,NO₂ and O₃ univariate analysis show that:There was a negative correlation between O₃ and PEF（z=-2.16,P=0.030）.4.The mixed effects of PM2.5 and O₃ were analyzed by multivariate analysis:Both PM2.5（z=-2.54,P=0.011）and O₃（z=-2.37,P=0.018）showed significant differences in the variance of PEF variation,with negative correlation between them.Meanwhile,in the analysis of the interaction effect between PM2.5 and O₃,PM2.5 still showed the negative correlation with the variation in FEV1（z=-2.17,P=0.030）and FEV1%（z=-2.30,P=0.028）,which once again confirmed that PM2.5 could affect FEV1 and FEV1%.Summary:42 patients with asthma were observed for 10 consecutive days in different time periods.The obtained data show that:1.PM2.5 has negative effects on PEF,FEV1,FEV1%,and FVC of asthma patients,but has no significant effects on MEF25,MEF50,and MEF75.The 1-day lag effect of PM2.5 was statistically insignificant.2.The maximum 8 hours of O₃ per day showed a negative correlation with PEF value,while no significant effect was found between SO₂,NO₂ and lung function.3.Combined analysis of PM2.5 and O₃ showed that both had an negative correlation with PEF,while PM2.5 still had a negative correlation with FEV1and FEV1%.Part Ⅱ Effects of Fine Particulate Matter on Airway Hyperresponsi-veness in Asthmatic Mice and its Mechanism ResearchObjective:The aim of this study was to observe the effects of exposure to high concentrations of PM 2.5 for 8 weeks on the pathology and airway hyperresponsiveness（AHR）of BALB/c mice undergoing sensitization and challenge with ovalbumin（OVA）and to observe the effects of apoptosis and T-cell immunoglobulin and mucin domain 1（TIM-1）in this process.Methods:1.Animals and exposure regimen:Forty female BALB/c mice were divided into four groups:a control group,an OVA group,an OVA/PM group,and a PM group（n=10）.All mouse cage in individually ventilated cages（IVCs）.Mice in the control group bound for 4 h per day and 5 days per week for a total of 8 weeks.Mice in the OVA group were sensitized and challenged with OVA.Mice in the OVA/PM group were sensitized and challenged as in the OVA group and then exposed to PM 2.5 for 4 h per day and 5 days per week for a total of 8 weeks using a nose-only“PM 2.5 online enrichment system”in the Second Hospital of Hebei Medical University at Shijiazhuang City,Hebei Province,China.2.Measurement indexes:During the experiment,quartz membrane was used to collect the particles in the pipeline,and the mass concentration of PM2.5 particles was measured by the gravimetric method with a lab analytical balance.AHR was detected.Bronchoalveolar lavage fluid（BALF）was collected for cell classification.The levels of interleukin-4（IL-4）,IL-5,and IL-33 in BALF were measured using enzyme-linked immunosorbent assay.Changes in histological structures were examined by light microscopy,and changes in ultramicrostructures were detected by electron microscopy.Apoptosis was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling（TUNEL）assay in the lung tissues.Western blotting and immunohistochemistry were utilized to analyze the expression of Bcl-2,Bax,and TIM-1 in the lungs.3.Statistical analysis:Data are expressed as the mean±standard deviation（SD）and analyzed using SPSS software version 19.0（SPSS Inc.,Chicago,IL,USA）.Differences among treatment groups were evaluated by one-way analysis of variance（ANOVA）.A value of P<0.05 was considered statistically significant.Results:1.Particle concentration and composition analysis:BALB/c mice were exposed for 8 consecutive weeks with an average daily exposure concentration of 703μg/m³.The composition analysis of exposed particles showed that the top five concentrations were NO₃^-,Organic carbon（OC）,SO₄^2-,NH₄⁺and elemental carbon（EC）.2.Observation of lung histopathology and microstructure under electron microscope:the OVA/PM group had mild loss of tracheal epithelial cells,widened alveolar septum,inflammatory cell infiltration,and bronchiolar smooth muscle hyperplasia.The PM group showed peribronchiolar inflamm-atory cells and alveolar epithelial hyperplasia.Mitochondrial abnormalities,mild fusion degeneration of nuclear membrane and mitochondrial ridge were observed in OVA/PM group and PM group under electron microscope.3.Lung mechanics and airway hyperresponsiveness in different groups:The results showed that AHR in the OVA/PM group was significantly more severe than that in the OVA and PM groups（P<0.05）.AHR in the PM group was also considerably more severe than that in the control group（P<0.05）.4.Cell counts and cytokine determination in BALF:The BALF of OVA/PM group（28.00±6.08 vs.12.33±4.51,t=4.631,P=0.002）and PM group（29.00±3.00 vs.12.33±4.51,t=4.927,P=0.001）had more lymphocytes than the BALF of the control group.The number of neutrophils in the BALF of the OVA/PM group（6.67±1.53 vs.3.33±1.53,t=2.886,P=0.020）and PM group（6.67±1.53 vs.3.33±1.53,t=2.886,P=0.020）was much higher than those in the BALF of OVA group.The concentration of IL-4（77.44±11.19 vs.48.02±10.02 pg/ml,t=4.595,P=0.002）and IL-5（15.65±1.19 vs.12.35±0.95pg/ml,t=3.806,P=0.005）in the OVA/PM group were increased compared to those in the OVA group.The concentration of IL-4（77.44±11.19 vs.41.47±3.40 pg/ml,t=5.617,P=0.001）and IL-5（15.65±1.19 vs.10.99±1.40pg/ml,t=5.374,P=0.001）in the OVA/PM group were increased compared to those in the PM group.The concentration of IL-4（41.47±3.40 vs.25.46±2.98 pg/ml,t=2.501,P=0.037）in the PM group were also higher than those in the control group.5.Expression of apoptotic cells:TUNEL assays showed that the number of apoptotic cells in the OVA/PM group was significantly higher than that in the OVA group（Tunel immunohistochemical scores[IHS%],1.20±0.18 vs.0.51±0.03,t=8.094,P<0.001）and PM group（Tunel IHS%,1.2±0.18 vs.0.51±0.09,t=8.094,P<0.001）,and that the number of apoptotic cells in the PM group was significantly higher than that in the control group（Tunel IHS%,0.51±0.09 vs.0.26±0.03,t=2.894,P=0.020）.6.Expressions of Bax/Bcl-2 and TIM-1:The Bax/Bcl-2 ratio（1.51±0.18 vs.0.4±0.10,t=9.654,P<0.001）and TIM-1/β-actin ratio（0.78±0.11vs.0.40±0.06,t=6.818,P<0.001）in the OVA/PM group were increased compared to those in the OVA group.The Bax/Bcl-2 ratio（1.51±0.18 vs.0.97±0.16,t=5.000,P=0.001）and TIM-1/β-actin ratio（0.78±0.11 vs.0.31±0.06,t=8.545,P<0.001）in the OVA/PM group were increased compared to those in the PM group.The Bax/Bcl-2 ratio（0.97±0.16 vs.0.18±0.03,t=7.439,P<0.001）and TIM-1/β-actin ratio（0.31±0.06 vs.0.02±0.01,t=5.109,P=0.001）in the PM group were also higher than those in the control group.Summary:PM 2.5 may induce increased airway hyperresponsiveness and pathologic pulmonary inflammation in mice sensitized with OVA.This process may be associated with apoptosis and TIM-1 activation.This study will provide a new target for the prevention and treatment of asthma caused by PM2.5.Conclusions:In clinically,it can be observed that the increase of PM2.5can reduce the PEF,FEV1,FEV1%and FVC of asthma patients,and PM2.5has no significant effect on lung function with a 1-day lag.In animal experiments,inhalation exposure of high concentration PM 2.5 for 8 weeks resulted in increased airway hyperresponsiveness and increased pathologic pulmonary inflammation in mice with OVA sensitization.This process that may be associated with apoptosis and TIM-1 activation.