Effects And Mechanism Of Kallikrein-bradykinin System On Acute Attack Of Asthma And Airway Hyperresponsiveness Induced By Fine Particulate Matter

Part one Effects of Fine Particulate Matter(PM2.5)on ospitalization Risk of Acute Attack of Asthmatic PatientsObjective: To explore the effects of PM2.5 on hospitalization risk of acute attack of asthma patients.Methods: This study included all adult inpatients with acute asthma attack in the database of Shijiazhuang Medical Insurance Center from January1,2013 to December 31,2016.It described the average daily number of hospitalized patients and time series trend of air pollutant concentrations;To explore the relationship between the number of hospitalized patients with acute asthma attack and the concentration of air pollutants by using generalized addictive model(GAM);To establish a distributed lag nonlinear model(Distributed lag non-linear models,DLNM)through building a PM2.5cross-basis function matrix thereby exploring the exposure effects and lag effects of PM2.5 concentration on hospitalized patients with acute asthma attack;Refering to air pollution classification standards of World Health Organization and China,to study the effects of PM2.5 concentrations under different standards on hospitalized patients with acute asthma attack;According to gender and age grouping,to further analyze impacts of PM2.5on the hospitalization risk of patients with acute asthma attack.Results: In the database of Shijiazhuang Medical Insurance Center from2013 to 2016,there were 4274 inpatients(over 18-year-old)with acute asthma attack.The average daily hospitalization was 2.90±2.21.The average daily PM2.5 concentration was 117.1±98.8?g/m3.The average daily temperature was 14.6±10.8°C and the average relative humidity was 57.6%.Results of the study showed that the median PM2.5 concentration of 88?g/m3 was used as a reference.When the PM2.5 reached the maximum after 0 days,risk of acute asthma attack was the highest.For every 10 ?g/m3 increase in PM2.5concentration,the risk of hospitalization of acute attack among asthma patients increased by 2.88 times(RR=2.88(95%CI=1.37-6.07))and the cumulative risk of 7 days increased by 2.55 times(RR=2.55(95%CI=1.04-8.36)).According to the PM2.5 concentration standard recom-mended by World Health Organization,7-day cumulative acute attack hospitalization risk for asthma patients in Shijiazhuang City would be reduced by 36%(RR=0.64(95%CI=0.45-0.92)).According to air quality standards of China,when the air quality was rated excellent,good,lightly polluted,moderately polluted,and heavily polluted,the cumulative risk of hospit-alization for asthma in 7 days was 0.86(95%CI=0.78-0.95),1.02(95%CI= 0.99-1.04),1.07(95%CI=0.94-1.21),1.12(95%CI=1.01-1.37)and 1.26(95%CI=1.14-1.68)respectively.The risk of acute attack in male patients was greater than women;the risk of acute attack in patients with asthma under the age of 60 was greater than that of patients over the age of 60.Summary: High concentration of PM2.5 significantly increases the risk of hospitalization for acute attacks of asthma patients.Reducing the concentration of PM2.5 in the air can effectively reduce the risk of hospitalization for acute attack of asthma patients.Part two Effects of Fine Particulate Matter on Airway Responsiveness,Bronchial-pulmonary Deposition and Lung-related Inflamm-atory Cytokines in MiceObjective: To observe the effects of PM2.5 on airway responsiveness,bronchial-pulmonary pathology and related inflammatory factors in the lungs of mice through animal experiments and to observe the role of kallikreinbradykinin system during this process.Mehtods:1.Experimental animal grouping and experimental conditions: 40wild-type C57BL/6 healthy male mice were randomly divided into clean air(FA)group,PM2.5 group,PM2.5+vehicle group,PM2.5+SBTI Group(10 in each group).From November 26,2016 to January 6,2017,there was a continuous exposure lasting for 6 weeks(5 days a week & 4 hours a day).In FA group,clean air was filtered by the filter membrane.In the other three groups,PM2.5 online enrichment oral and nose exposure system were utilized.Apart from this,12 hours before daily PM2.5 exposure,mice in PM2.5+SBTI group and PM2.5+vehicle group were intraperitoneally injected with SBTI and comfort agent(phosphate buffer saline,PBS).2.Observation inflammatory factors and methods: After the above experiment,the small animal pulmonary function instrument was used to compare the changes of airway resistance in each group.The enzyme-linked immunoassay was used to detect the inflammatory factors(interleukin-4,interleukin-10,interleukin-13,interleukin-33)and bradykinin(BK)level changes in bronchoalveolar lavage fluid(BALF).Meanwhile,light microscope and electron microscope were used to observe the pathological changes and ultrastructural changes of the bronchi-pulmonary tissues of mice in each group.Western Blotting and Real-time PCR(RT-PCR)were used to detect the expression of kallikrein in bronchial-lung tissues of each group.3.Statistical analysis: One-Way analysis of variance was used to evaluate the differences between different groups.P<0.05 indicated that the difference was statistically significant.Results:1.During the whole experiment,the average daily PM2.5 concentration was 306?g/m3,and the average concentration of PM2.5 inhalation online exposure was 1513?g/m3(720?g/m3~2514?g/m3).In PM2.5 group,the concentration of acetylcholine required for doubling the initial airway resistance was 12.5 mg/ml which was much lower than FA group(>25mg/ml)(P<0.05);compared with PM2.5+vehicle group,the administration of kallikrein inhibitor before exposure could significantly reduce the airway responsiveness of mice(P<0.05).2.Bronchial-pulmonary histopathology and microstructure: Compared with FA group,the other three groups had different degrees of airway wall thickening,inflammatory cells infiltration and alveolar structure damaged.In PM2.5 + SBTI group,SBTI could reduce the infiltration of inflammatory cells and improved the structure of bronchi-pulmonary.Observed under electron microscope,ultrastructure of the lungs in FA group was basically normal.In PM2.5 group,type II alveolar epithelial cells swelled in cytoplasm and nucleus.Some mitochondria were abnormal.Nuclear membrane and mitochondrial crest were slightly fused and blurred.Rough endoplasmic reticulum was slightly expanded and the particles were fused and degranulated.Compared with PM2.5+vehicle group,intervention of kallikrein inhibitors could improve the ultrastructure of the lungs microscopically showing mild swelling of terminal bronchioles and of mitochondrial cristae and membranes.3.Determination of inflammatory factors in BALF: The concentrations of pro-inflammatory factors IL-4,IL-13,and IL-33 in PM2.5 group were significantly higher than those in FA group,while the anti-inflammatory factor IL-10 had no statistical difference in both PM2.5 group and FA group.IL-4,IL-13,IL-33,IL-10 had no statistical difference in PM2.5+SBTI group and PM2.5+vehicle group.4.Detection of the expression level of kallikrein: The expression of kallikrein in bronchial-lung tissues of PM2.5 group was significantly higher than that of FA group.Compared with PM2.5+vehicle group,administration of kallikrein inhibitors before exposure significantly reduced the expression of bronchial-pulmonary kallikrein and the BK level in BALF.Summary: PM2.5 inhalation exposure increases airway hyperresponsiveness in mice and aggravates pathological changes of bronchialpulmonary inflammation.Kallikrein inhibitors can alleviate the above changes.It is suggested that the increase of airway hyperresponsiveness by PM2.5 is relevant to the activation of the kallikrein-bradykinin system signaling pathway.This study will provide a new target for the prevention and treatment of asthma caused by PM2.5.Part three Research on Effects of Fine Particles on the Contractile Function of Human Airway Smooth Muscle Cell(hASMC) and Its MechanismObjective: To observe effects of different concentrations of PM2.5collected by the enrichment system on cell contraction function of human airway smooth muscle cells and to explore the role of the kallikreinbradykinin system during this process.Methods:1.Cellular immunofluorescence technology was used to measure the distribution and expression of Kallikrein in hASM cells;CCK-8 kit was used to measure the effects of different concentrations of PM2.5(0,6.25,12.5,25,50,100 ?g/ml)on hASM cell viability;Cyto Select TM 48-Well Cell Contraction Assay Kit was used to measure the effects of different concentration levels of PM2.5 on contractile function of hASM cells;Western Blotting was used to measure different concentration levels of PM2.5 on the expression of kallikrein and bradykinin receptor B2 R in hASM cells;Enzyme-linked immunosorbent assay was used to measure effects of PM2.5(12.5 ?g/ml)on expression level of bradykinin in supernatant of hASM cells;fluorescent probe Flu-4 staining was used to measure the effect of PM2.5 on the level of total calcium released by the cytoplasmic calcium pool by flow cytometry.2.Real time PCR was used to measure the effect of PM2.5(12.5 ?g/ml)on the expression level of kallikrein family 1-15 in hASM cells.3.Lipo Rimax,three types of si RNA and scramble si RNA were used to transfect hASM cells.After knocking down the KLK14 gene,hASM cells were incubated with PM2.5(12.5 ?g/ml)for 24 hours.Changes in cell contractile function were observed.There were changes in the expression of kallikrein and in level of bradykinin in the cell supernatant.There were changes in the level of total calcium released by the cytoplasmic calcium pool.The method was the same as 1.4.Statistical analysis: One-way analysis of variance was used to evaluate the differences between different groups.P<0.05 indicates that the difference was statistically significant.Results:1.The distribution and expression of kallikrein in cytoplasm of hASM cells were observed through fluorescence microscopy.2.After incubating hASM cells with different concentrations of PM2.5,PM2.5 was measured within a certain concentration range(0-50 ?g/ml)to promote the proliferation of hASM cells.The most obvious proliferation effect on hASM cells presented when it reached around 12.5 ?g/ml.When PM2.5concentration was greater than 50 ?g/ml,it had a certain toxic effect on hASM cells.At the level of 100 ?g/ml,cell viability was around 50% of the control group.3.After incubating hASM cells with different concentrations of PM2.5,PM2.5 in a certain concentration range(0-50 ?g/ml)was able to promote contractile function of hASM cells.The most obvious effect on hASM cell contraction presented when it reached around 12.5 ?g/ml.When PM2.5concentration was greater than 50 ?g/ml,the contractile funcation of hASM cells was significantly reduced.4.After incubating hASM cells with different concentration levels of PM2.5,PM2.5 promoted the expression of kallikrein in hASM cells within a certain concentration range(0-50 ?g/ml).The highest expression level of kallikrein in hASM cells presented when it reached around 12.5 ?g/ml.When concentration was more than 50 ?g/ml,level of kallikrein in hASM cells was decreased.5.After incubating hASM cells with PM2.5(12.5 ?g/ml),it was observed that the level of bradykinin in cell supernatant of PM2.5 group was significantly higher than that of the control group.Meanwhile,after incubating hASM cells with different concentrations level of PM2.5,PM2.5 promoted the expression of bradykinin receptor B2 R in hASM cells within a certain concentration range(0-50 ?g/ml).The highest expression level of bradykinin receptor B2 R in hASM cells presented when it reached around 12.5 ?g/ml.When PM2.5 concentration was greater than 50 ?g/ml,level of bradykinin receptor B2 R in hASM cells was reduced.6.PM2.5(12.5 ?g/ml)was observed to promote kallikrein family 1-15 and the expression of kallikrein 14 gene after incubating hASM cells.7.After knocking down kallikrein 14 gene and then incubating with PM2.5(12.5?g/ml),it was observed that compared with scramble si RNA group,the contraction-promoting effect of PM2.5 on hASM cells was no longer obvious in si RNA-01 group.The releasing effects of calcium from plasma calcium pool of hASM cells was no longer significantly increased.Meanwhile,the level of bradykinin in the supernatant of hASM cells was no longer significantly increased.Summary: In cell experiments,PM2.5 incubation of hASM cells increased cell contractile function,which is relevant to the activation of the kallikrein-bradykinin signaling pathway.Conclusions:1.PM2.5 is one of the important risk factors of acute attack in patients with asthma.Moreover,it is more meaningful to reduce acute attacks in asthma patients by lower PM2.5.2.PM2.5 can induce AHR,and kallikrein-bradykinin system plays an important role in this process.These findings may provide powerful therapeutic targets for the prevention and treatment of AHR and related diseases.