Study On The Possible Mechanism Of Inhaled Fine Particles Affecting Th1/Th2 Immune Imbalance In Asthma Mice Through Notch Signaling Pathway

Background:The occurrence and development of bronchial asthma（asthma for short）is closely related to Th1/Th2 immune imbalance.Fine particulate matter（PM2.5）can cause Th1/Th2 immune imbalance and is an important cause of acute asthma attacks.Notch signaling pathway is closely related to the pathophysiological processes such as immune imbalance and airway remodeling in chronic airway inflammatory diseases.The relationship between asthma Th1/Th2 immune imbalance and Notch signaling pathway aggravated by PM2.5 is not yet clear.Objective:In this study,asthmatic mouse model was given aerosol inhalation of PM2.5（510g/m³）to isolate spleen T lymphocytes,and the expression of Notch1receptor and its downstream Hes1 in spleen T lymphocytes Notch signaling pathway was detected.Th1 and Th2 subsets of spleen T lymphocytes;Serum and bronchoalveolar lavage fluid（BALF）interferon（IFN）-γand interleukin（IL）-4concentrations,to explore the regulatory mechanism of Notch signaling pathway in PM2.5 mediated Th1/Th2 immune imbalance in asthmatic mice,so as to clarify the effect of PM2.5 on acute asthma attack.Methods:Forty SPF 8-week-old female BALB/c mice were divided into healthy control group,healthy PM2.5 group,asthma group and asthma PM2.5 group according to random number table method,with 10 mice in each group.PM2.5 near the main traffic line of Tianshui South Road in Lanzhou was collected.The asthmatic mouse model was sensitized and stimulated by chicken ovalbumin（OVA）and aerosolized by inhalation of PM2.5.Lung function of mice was measured by pulmonary function apparatus.The mice were sacrificed by cervical dislocation,and the right lung tissues of the mice were fixed with 4%paraformaldehyde for HE,AB-PAS and Masson staining.The infiltration of inflammatory cells,mucus secretion,airway smooth muscle area and collagen area deposition of the mice were observed.T lymphocytes from spleen of mice were isolated and purified.Flow cytometry was used to detect the proportion of Thl and Th2 cells in CD4⁺T lymphocytes in spleen of mice in each group,and the ratio of Th1/Th2 was calculated.Real-time fluorescence quantitative polymerase chain reaction（RT-PCR）was used to detect the expression of Notch1 receptor and its downstream Hes1 m RNA in T lymphocytes.The expression of Notch1 receptor and its downstream Hes1 protein were detected by Western blot.Orbinal venous blood of mice was collected and serum was separated.BALF of mice was recovered and supernatant was collected.The concentrations of IFN-γand IL-4 in serum and BALF were determined by enzyme-linked immunosorbent assay（ELISA）.Results:（1）Model establishment and behavioral changes of asthma:OVA sensitization and stimulation can successfully establish a mouse model of asthma.Mice in asthma group were scratched,fidgety,shortness of breath,abdominal muscle spasm,incontinence and so on after each atomization.However,healthy control mice behaved normally and breathed smoothly after atomization.（2）Lung function and pathological changes:The forced end-expiratory volume at0.1 second(FEV_0.1),the forced end-expiratory volume at 0.2 second(FEV_0.2),the peak expiratory velocity（PEF）and the flow velocity(EF₅₀)at 50%exhaled tidal volume（0.48±0.05,0.84±0.18,8.32±1.04,4.59±0.37）ml/s in asthma group were significantly lower than those in healthy control group（0.83±0.09,1.36±0.22,12.42±1.22,6.46±0.60）ml/s.Penh value（1.70±0.05）was significantly higher than that of healthy control group（0.52±0.15）（both P<0.01）.After PM2.5 intervention,FEV_0.1,FEV_0.2,PEF and EF₅₀（0.60±0.05,1.01±0.12,9.63±0.70,5.11±0.29）ml/s and（0.32±0.09,0.57±0.14,6.36±0.63,3.04±0.56）ml/s in healthy PM2.5 group and asthma group were significantly lower than those in control group,and Penh values（1.04±0.23）and（2.03±0.33）were significantly higher than those in control group（both P<0.01）.In the asthmatic group,the lumen was narrow,the folds of the airway mucosa increased and lengthened,a large number of inflammatory cells gathered around the trachea and pulmonary vessels,the alveolar septum was widened,the structure was damaged,and the smooth muscle was thickened.The number of goblet cells increased significantly,the normal ciliated epithelial cells decreased significantly,and the airway mucus was abundant.There were a lot of blue collagen deposits around tracheal vessels and obvious fibrosis in subepithelium.There were no such changes in the healthy control group.The number of eosinophils（EOS）and the percentage of goblet cells in epithelial cells,airway inflammatory cell score and mucus secretion score,the total area of airway wall,smooth muscle area and collagen deposition area in asthma group were significantly higher than those in healthy control group.After PM2.5 intervention,inflammatory cell infiltration was observed around trachea and blood vessels in the healthy PM2.5 group compared with the control group.In the asthma PM2.5 group,the airway lumen was narrowed significantly,the alveolar structure was damaged significantly,the blue collagen deposition around the tracheal vessels was further aggravated,the subepithelial fibrosis was further aggravated,and the PM2.5 deposition could be seen in the airway lumen and the alveolar septum.（3）The proportion of Th1 and Th2 subsets and Th1/Th2 ratio:Th1%and Th1/Th2ratio（27.58±2.86）%and（8.38±0.97）in asthma group were significantly lower than those in healthy control group（34.50±2.25）%and（18.10±3.52）（both P<0.01）.The healthy PM2.5 group（29.29±2.01）%,（10.88±0.67）and asthma PM2.5 group（24.47±2.56）%,（6.23±0.73）were lower than the control group（P<0.05）.Th2%（3.30±0.23）%in asthma group was significantly higher than that in healthy control group（1.96±0.33）%（both P<0.01）.Th2%（2.70±0.18）%and（3.94±0.15）%in healthy PM2.5 group and asthma group were significantly higher than those in control group（both P<0.01）.（4）m RNA expression of Notch1 and Hes1:The m RNA levels of Notch1 and Hes1in T lymphocytes of asthma group（1.54±0.27,1.57±0.44）were significantly higher than those of healthy control group（0.88±0.22,0.76±0.16）（both P<0.01）.The healthy PM2.5 and asthma PM2.5 groups（1.22±0.14,1.12±0.23）and（2.09±0.50,1.93±0.40）were higher than those in the control group,respectively（P<0.05 or P<0.01）.（5）Expression of Notch1 and Hes1 protein:The levels of Notch1 and Hes1 protein in T lymphocytes of asthma group（1.05±0.11,0.94±0.08）were significantly higher than those of healthy control group（0.46±0.08,0.66±0.08）（both P<0.01）.The healthy PM2.5 and asthma PM2.5 groups（0.69±0.06,0.79±0.11）and（1.27±0.16,1.32±0.08）were higher than those in the control group,respectively（P<0.05 or P<0.01）.（6）Levels of IFN-γand IL-4 in serum and BALF:The concentrations of IFN-γin serum and BLAF in asthma group[（280.3±58.6,383.7±55.3）pg/ml]were significantly lower than those in healthy control group[（452.3±109.9,512.8±76.7）pg/ml]（both P<0.01）.The healthy PM2.5 group[（352.7±58.1,451.3±61.8）pg/ml]and asthma PM2.5 group[（195.6±30.6,299.2±29.0）pg/ml]were lower than the control group（P<0.05 or P<0.01）,respectively.The level of IL-4 in serum and BALF of asthma group[（399.1±96.9,412.5±53.6）pg/ml]was significantly higher than that of healthy control group[（208.0±68.1,209.5±26.4）pg/ml]（both P<0.01）.The healthy PM2.5 group[（304.2±38.2,320.6±58.7）pg/ml]and asthma PM2.5 group[（493.3±162.5,507.9±138.8）pg/ml]were higher than the control group（P<0.05 or P<0.01）,respectively.（7）Correlation analysis:m RNA and protein expressions of Notch1 and Hes1 were positively correlated with Th2%and IL-4 concentration after the intervention of basal state and PM2.5.It was negatively correlated with Th1%,Th1/Th2 ratio and IFN-γconcentration.Th1%and Th1/Th2 ratio were positively correlated with IFN-γconcentration.There was a positive correlation between Th2%and IL-4 concentration,and a negative correlation between Th1/Th2 ratio and IL-4 concentration（P<0.05 or P<0.01）.Conclusion:（1）OVA sensitization and stimulation can successfully establish a mouse model of asthma;（2）There was Th1/Th2 immune imbalance in asthmatic mice,and the increase of Th2 cells was the main factor.The levels of IFN-γin BALF and serum decreased,and the levels of IL-4 increased.There was an obvious immune imbalance dominated by Th2 cells in asthmatic mice,which could lead to inflammatory response,and PM2.5 could aggravate the immune imbalance and inflammatory response.（3）The m RNA and protein expressions of Notch1 and Hes1 were significantly increased in spleen T lymphocytes of asthmatic mice,indicating abnormal activation of Notch signaling pathway.PM2.5 can further increase the m RNA and protein expression of Notch1 and Hes1 in asthmatic mice.（4）The m RNA and protein of Notch1 and Hes1 were negatively correlated with Th1/Th2,negatively correlated with BALF and serum IFN-γ,and positively correlated with IL-4.PM2.5 may exacerbate Th1/Th2 immune imbalance in asthmatic mice by activating Notch signaling pathway.