Effects Of PM2.5 On Acute Exacerbation Of Chronic Obstructive Pulmonary Disease:An Experimental Study And Epidemiological Analysis

In recent years,air pollution,especially the fine particulate matters（aerodynamic diameter<2.5μm,PM2.5）,pose a huge threat to public health,especially the impact on respiratory system.PM2.5 could lead to lung injury or chronic inflammation.For the impact on chronic airway inflammatory diseases such as chronic obstructive pulmonary disease（COPD）,epidemiological data show that the short-term increase in PM2.5 exposure concentration can lead to increased hospitalization risk for acute exacerbation of COPD.However,the exact damage effect and mechanisms of PM2.5 need further study..In the field of environmental and health epidemiology,some recent reports analyzed the relationship between PM2.5 and respiratory diseases,such as COPD,which showed that PM2.5 has an important role in affecting the rate of COPD hospitalization,and air pollution was indeed paid more attention than ever before.In 2013,the Chinese government launched the National Air Pollution Prevention and Control Action Plan（2013-2017）,with strict legislation.In 2017,the air quality began to be improved significantly.To observe the health benefits of air quality improvement,whether the hospitalization risk of acute exacerbation of COPD caused by PM2.5 has changed,is what we want to study further..PM2.5 contains a variety of polycyclic aromatic hydrocarbons（PAHs）,which are environmental organic pollutants.As the starting point of detoxification of smoke and environmental pollutants（such as PAHs）,Aryl hydrocarbon receptor（AhR）is densely distributed in the lung.AhR plays an important role in the pathogenesis of COPD.AhR is related to the molecular mechanisms of prooxidative and pro-inflammatory effect of PM2.5.The role of AhR pathway in the mechanism of acute exacerbation of COPD caused by PM2.5 is not clear.Clara cells（CC）in airway epithelium have anti-inflammatory and immunomodulatory effects.CC are highly responsive to AhR ligands,and are main target cells of PAHs cytotoxicity and Cytochrome P450 1A1（CYP1A1）expression stimulated by AhR activation.Clara cell specific protein（CCSP）is a sensitive marker of lung injury,and an index to evaluate the severity of COPD.PM2.5 can induce CC damage,but the mechanism is not clear.We hypothesized that the pathophysiology of acute exacerbation of COPD caused by PM2.5 may be manifested by CC damage,and the mechanism of which may be related to AhR pathway.The concentrated ambient particles（CAPS）is an useful tool for us to study PM2.5 inhalation exposure.The mass concentration,particle sizes and composition of PM2.5 can be monitored,which can more accurately reflect the characteristics of inhaled particulate aerosol.In addition,the inhalation exposure of PM2.5 was more closer to the real effect of PM2.5 on respiratory system than PM2.5 extract dropped in airway before.We are also exploring some protective treatments to reduce PM2.5-induced lung injury.It has been proved in our previous studies that hydrogen（H₂）inhalation can reduce PM2.5-related lung injury in rats,which is manifested in improving lung function,reducing airway mucus（MUC5AC）hypersecretion,reducing lung inflammation and oxidative stress.But the mechanism of hydrogen protection is not clear.Some studies have shown that hydrogen molecules can alleviate hyperoxia induced lung injury through Nrf2pathway,and the activation of Nrf2 pathway is mostly mediated by AhR.Whether the protective mechanism of hydrogen is related to AhR needs further research.Our research consists of three parts.The first part is the epidemiological study.Based on the data of 2015-2019 in Shijiazhuang area,we applied six factors of environmental pollutants（PM2.5,PM10,SO₂,NO₂,CO and O₃）and temperature and humidity as the ambient air quality indicators to evaluate the impact of these indicators on the daily number of COPD hospitalization,and to evaluate the change in the trend of daily COPD admission after the improvement of air quality.The evaluation methods of the health benefits for pollutants reduction were also explored.The second part is the basic experiment.We used PM2.5 exposure system to observe the effect of inhalation concentrated ambient PM2.5 on lung injury in healthy rats and COPD rats,and the relationship between the expression of AhR and CYP1A1protein and lung injury.We explored the mechanism of PM2.5-induced airway CC injury in AhR-CYP1A1 signal pathway.The third part is the basic experiment to explore the protective treatment.CCSP is the lung injury index,we further evaluated the lung protective effect of hydrogen on CC injury induced by PM2.5,and preliminarily explored the mechanism of inhaling high concentration hydrogen（66.7%H₂）to reduce PM2.5-related lung injury..Part One The influence of air quality in Shijiazhuang area on the daily number of COPD hospitalization in 2015-2019Objective:To evaluate the impact of ambient air quality indicators on the daily number of COPD hospitalization,and to estimate the change of which after air pollution reduced.Methods:The data of medical insurance patients hospitalized for acute exacerbation of COPD in Shijiazhuang city from January 1,2015 to December31,2019 were collected,and the daily average concentrations of six factors（PM2.5,PM10,SO₂,NO₂,CO,O₃）for air quality in Shijiazhuang City during the period were obtained,and temperature and humidity indexes added.Data were collected and analyzed in two periods:the first stage the trend of the data in 2015-2018 were described,and the short-term impacts of air pollutants on the number of COPD inpatients were analyzed by using the distributed lag nonlinear models（DLNM）;the second stage the data in 2015-2019 were analyzed by using the VAR model to evaluate the trend of the daily number of COPD hospitalization,and the related air pollutants were analyzed.Results:1. Periodic fluctuation of each index（descriptive analysis）:PM2.5,PM10,SO₂,NO₂,CO and the daily number of COPD hospitalization are all at the peak in winter（around January）,while O₃ and temperature are on the contrary,falling to the minimum in winter,and at the peak in summer（around July）every year.2.Year by year changes in trend of each index（descriptive analysis）:from January 2015 to January 2017,PM2.5,PM10,SO₂,NO₂,CO and other pollutants showed an annual upward trend in the same season.In the winter of2017（around January 2018）,the indexes began to show a downward trend compared with the same period before,air quality was improved compared with the previous two years,while O₃ always showed an upward trend year by year.However,the number of COPD inpatients increased year by year from January 2015 to January 2018,but after January 2018（the end of 2017）,the growth trend was to be flat.3.DLNM model results:PM2.5 has a certain lag effect and cumulative effect on the daily number of COPD inpatients;temperature has a immediate effect and cumulative lag effect when dropped below-8℃.4.VAR model results:the daily number of COPD inpatients with increasing trend year by year was mainly affected by their own cunmulative factors（its own sequence）,but it may also be affected by SO₂ and PM2.5partly.Summary:1.From the above linear and non-linear models,it can be seen that the daily number of COPD inpatients in Shijiazhuang during 2015-2019 may be affected by the short-term lag effect of PM2.5,SO₂ and temperature drop.2.The increasing trend of the number of COPD inpatients in Shijiazhuang area tends to be gentle after the end of 2017.The trend of the number of COPD inpatients per day is mainly affected by its own cumulative factors（its own sequence）year by year,and may also be affected by the changes of atmospheric PM2.5 and SO₂ concentration partly.Part Two Effects of concentrated ambient PM2.5 on lung injury in hydrocarbon receptor（AhR）Objective:To observe the impacts of PM2.5 on lung injury and the expression of AhR signal pathway protein in normal and COPD rats,and to explore the role of AhR in the mechanism of PM2.5-induced CC injury.Methods:1. Experimental animal grouping and PM2.5 exposure protocols:48healthy and male SD rats were divided into 8 groups（6 rats in each group）:Normal+FA 2w,Normal+PM 2w,Normal+FA 4w,Normal+PM 4w,COPD+FA 2w,COPD+PM 2w,COPD+FA 4w and COPD+PM 4w.All the control groups inhaled clean filtered air（FA）in the exposure tower,all the exposure groups inhaled PM2.5（PM）in the exposure tower,the exposure time was 2 weeks（2w）or 4 weeks（4w）,5 days/week,5 consecutive hours/day.In addition,9 healthy male SD rats were divided into three groups:normal control group（FA group）,PM exposure group（PM group）and PM exposure antagonist group（PM+TMF group）.The exposure time of inhaled FA or PM2.5（PM）was 2 weeks,5 days/week,5 consecutive hours/day.On the first day of each week,1 ml of DMSO or AHR antagonist（TMF）solution was injected intraperitoneally for 2 weeks.2.Detection indexes and methods:during the exposure period,the concentration of PM2.5（filter membrane weighing method）,particle size distribution and PAHs composition in PM2.5 samples were measured;after the exposure,the invasive lung function（Rrs and PEF）of rats were measured,Balf and serum were collected,lung tissues were stored or paraffin sections were made.Enzyme linked immunosorbent assay（ELISA）was used to detect the CCSP levels in Balf and serum.Immunohistochemistry（IHC）was used to detect the expression of CCSP in airway.Western blot was used to detect the expression of AhR,CYP1A1 and CCSP in lung tissue.Results:1. During the exposure period,the average concentration of PM2.5 is 750[585,1310]μg/m³,and the size distribution of PM2.5 meets the size range of PM2.5.Concentrated ambient PM2.5 play a role in concentrating PAHs in the atmosphere（concentration times were 3-12 times）.2. Lung function:the lung function of normal rats did not change significantly after 2 weeks of PM exposure,but decreased significantly after 4weeks of PM exposure;the lung function of COPD rats was lower than that of normal rats,and further decreased after 2 and 4 weeks of PM exposure.3. Balf and serum CCSP:PM2.5 can lead to the decrease of CCSP in BALF of normal rats,and the CCSP in serum can also be reduced after 4weeks of exposure;COPD rats innately have the decrease of CCSP in Balf,which was further reduced after 4 weeks of PM2.5 exposure;while CCSP in serum was increased after 2 and 4 weeks of PM2.5 exposure.4. Immunohistochemistry for airway CCSP:after exposure to PM2.5 for2w or 4w,the CC of normal rats was reduced.After exposure to PM2.5 for 4w,the morphology of CC was changed obviously.However,there was an innate decrease in the number of CC in COPD rats.After exposure to PM2.5 for 2w,the number of CC was not changed obviously,the morphology of CC was thinned.After exposure to PM2.5 for 4w,the number of CC was decreased obviously,the morphology of CC was changed,CC was damaged,airway structure was disordered,and epithelial continuity was damaged.5. The expression of AhR and CYP1A1 protein in lung tissues（Western blot）:after 2 weeks for PM2.5 exposure,AhR of normal rats can be activated,that is to say,AhR is down regulated and CYP1A1 is up regulated,while AhR of COPD rats is lower than that of normal rats and CYP1A1 is higher than that of normal rats,indicating that there is innately AhR activation in COPD.After further PM2.5 exposure for 4 weeks,AhR was still down regulated,but CYP1A1 is up regulated less.AhR was down regulated in normal and COPD rats after 4-week PM2.5 exposure,and the down-regulation was more significant in COPD rats,while CYP1A1 was not significantly increased both in normal and COPD rats than controls,indicating that although AhR was activated and down regulated in 4-week PM2.5 exposure,up-regulation of CYP1A1 may be inhibited.6. The effect of AhR antagonist（TMF）on the expression of CCSP:TMF reversed the decrease of CCSP in BALF（ELISA method）and airway epithelium（IHC method）and the down-regulation of CCSP protein expression（WB method）induced by PM2.5 exposure.Summary:1.Exposure to concentrated ambient PM2.5 for 2 and 4 weeks can result in different lung injury manifestations in normal rats and COPD rats;the injury in COPD rats may be more severe than in normal rats.2.PM2.5 exposure can activate the AhR-CYP1A1 pathway,while the protein expression of the AhR-CYP1A1 pathway induced by PM2.5 is different due to duration of PM2.5 exposure;3.PM2.5 can lead to damage to CC,but it is more serious in COPD rats,which may cause damage to the barrier of airway epithelium of COPD;4.PM2.5 could induce damage to CC,which may be mediated by AhR.Part Three Hydrogen could reduce PM2.5-induced lung injury throughObjective:To evaluate the protective effect of hydrogen against lung injury with CC damage induced by PM2.5,and to explore the protective mechanism of inhaling high concentration hydrogen（67%H₂）.Methods:1.Experimental animal grouping and PM2.5 exposure protocols:35healthy male Wistar rats were divided into 5 groups（7 rats in each group）:Filtered air control group（FA）,PM2.5 exposure group（PM）,PM2.5 exposure+nitrogen oxygen group（PM+N）,PM2.5 exposure+H₂ group（PM+H）,H₂control group（FA+H）.Clean filtered air FA or PM2.5（PM）were inhaled for5h/day,5 days per week,for 4 consecutive weeks;"N"refers to the mixture of 67%N₂ and 33%O₂ for 2h after PM2.5 exposure;"H"refers to the mixture of 67%H₂ and 33%O₂ for 2h after PM2.5 exposure.2.Detection indexes and methods:dynamic concentration（mass concentration detector）and particle size distribution of PM2.5 during exposure;Then lung tissues of rats were stored and pathological paraffin section were made.The expression of CCSP and AhR were determined by IHC and Western blot.Results:1.The average daily concentration of PM2.5 in the ambient air during PM2.5 exposure was 122.4±96.9μg/m³.The dynamic particle mass concen-tration in PM2.5 exposure tower measured by aerosol mass concentration monitor fluctuating between 452μg/m³（minimum）and 2786μg/m³（maximum）,with an average mass concentration of 1328±730μg/m³.The size distribution of PM2.5 meets the size range of PM2.5.2.Immunohistochemistry of airway CCSP:the inhalation of PM2.5resulted in a significant decrease of CCSP-positive CC and a slight disorder of airway epithelial structure.The CC was damaged by PM2.5,but the damage was reversed when PM2.5 exposure was combined with H₂ inhalation.3.PM2.5 can reduce the level of AhR,and H₂ inhalation can inhibit the decrease of AhR induced by PM2.5.Summary:1.Subacute（4 weeks）exposure of PM2.5 could cause damage of airway Clara cells（CC）in rats,while high concentration of H₂ inhalation could reverse the damage of CC;2.H₂ could ameliorate lung injury induced by PM2.5 possibly through AhR-dependent mechanisms.Conclusion:In terms of epidemiological research,the daily number of COPD hospitalization in Shijiazhuang from 2015 to 2019 may be affected by PM2.5 and SO₂,while the increasing trend of the number of COPD inpatients tends to be flat after the end of 2017.In terms of animal experiment,concentrated ambient PM2.5 exposure could induce different degrees of lung injury demonstrated by lung function and CC damage in normal rats and COPD rats.The damage in lungs may be more serious in COPD rats than in normal rats.The mechanism of PM2.5-induced lung injury and the protective effect of high concentration of H₂may be related to AhR.