Effect Of Chronic Exposure To PM_2.5 On Mucociliary Clearance In Rats And The Intervention Of N-acetylcysteine

Objective:We investigated the effect of chronic PM_2.5 exposure on mucociliary clearance,and analyzed the effect of N-acetylcysteine on it.Methods:1 Experimental grouping:48 male SD rats were randomly divided into three groups:air-purification control group（FA group）;exposure to PM_2.5group（PM group）;exposure to PM_2.5 group plus N-acetylcysteine group（PM+NAC group）.According to the different times of three months and six months,each group was divided into two subgroups（FA3 group and FA6group,PM3 group and PM6 group,PM3+NAC group and PM6+NAC group）.2 The number of total cells and classification in the bronchoalveolar lavage fluid.3 Lung tissue and tracheal tissue were stained with HE.4 The changes of cilia ultrastructure were observed by scanning electron microscope.5 Mucous cells metaplasla were visualized by Alcian blue-periodic acid chiff staining（AB-PAS）.6 The gene expression and protein expression of MUC5AC and AQP5 in lung,which were respectively measured by revers transcriptase polymerase chain reaction（RT-PCR）,immunohistochemistry and western blot.Results:1.The PM_2.5 concentration:The statistical results showed that the PM_2.5concentration in FA group was significantly lower than that of PM group and outdoor PM_2.5 group.The PM_2.5 concentration between PM group and outdoor PM_2.5 group had no significant difference.2.BALF cytology count:WBC and neutrophils count and percentage of neutrophils in PM groups were higher than PM plus NAC groups and FA group,PM plus NAC groups were higher than FA group.3.The histological manifestation of tracheal tissue and lung tissue in different groups（HE）:The results of lung histopathologic injury score showed that the histopathological grades of PM group was higher than PM plus NAC group and FA group,FA3 group compared with PM3 NAC group,the difference was not significant,PM6 plus NAC group was higher than FA6group,FA6 group was higher than FA3 group,PM6 group was higher than PM3 group group.4.Scanning electron micrograph obervating rat trachea cilia ultrastructure changes:Scanning electron micrograph of rat trachea cilia ultrastructure in FA groups were intact.The damage of cilia ultrastructure in the PM group and the PM plus NAC group was more severe than that in the FA group,and the PM plus NAC group had less damage than the PM group.5.Mucous cells metaplasla were visualized Alcian blue-periodic acid chiff staining（AB-PAS）:The results of AB-PAS showed that the positive staining area of PM group was higher than PM plus NAC group and FA group,the positive staining area of PM6 group was higher than PM3 group,PM6 plus NAC group was higher than FA6 group.6.The expression of MUC5AC protein and AQP5 protein were detected by immunohistochemistry:MUC5AC protein expression in FA group and PM plus NAC group was decreased compared with PM group,AQP5 protein positive staining area increased.MUC5AC protein expression was not significantly different between FA3 group and PM3 plus NAC group,AQP5protein positive staining area increased.Compared with the PM6 plus NAC group,the expression of MUC5AC protein in FA6 group decreased significantly,increased the expression of AQP5 protein.Compared to the PM6group,PM3 group had significantly higher protein expressional level of AQP5in lung,but lower protein expressional level of AQP5.7.The expression of MUC5AC protein and AQP5 protein in lung tissue of rats were detected by Western-blot:MUC5AC protein expression in FA group and PM plus NAC group was significantly decreased compared with PM group,AQP5 protein expression increased significantly.Compared with the PM3 plus NAC group,the expression of MUC5AC protein in FA3 group decreased significantly,increased the expression of AQP5 protein.Compared to the PM6 plus NAC group,FA6 group had significantly lower protein expressional level of MUC5AC in lung,but higher protein expressional level of AQP5.Compared to the PM6 group,the expression of MUC5AC protein in PM3 group decreased significantly,increased the expression of AQP5 protein（P<0.05）.8.The expression of MUC5ACmRNA and AQP5mRNA expression in the lung tissues:compared with PM group,the expression of MUC5ACmRNA in FA group and PM plus NAC group was significantly decreased,but it increased the expression of AQP5 protein.The expression of MUC5ACmRNA in FA3 group decreased compared with PM3+NAC group,AQP5 protein expression increased significantly.The mRNA level of MCU5AC gene in the lung tissues of PM6 plus NAC groups were significantly decreased than FA6group,but it increased the expression of AQP5 mRNA.Compared to the PM6group,the expression of MUC5AC mRNA in PM3 group decreased significantly,increased the expression of AQP5 mRNA.9.Correlation analysis between MUC5AC and AQP5:Compared with FA group,The expressions of MUC5AC mRNA in PM3 group and PM6 group were significantly increased,but it decreased the expression of AQP5mRNA,which were negative correlated.Compared with PM plus NAC group,the expression of AQP5 mRNA was significantly decreased and the expression of MUC5AC mRNA was significantly increased in PM group,which were negative correlated.Conclusion:1.Chronic PM_2.5 exposure can cause damage to MCC（mucociliary clearance）,reducing the total number of ciliated cells,lodging,elongation,shortening,disorder,apoptosis,excessive mucus secretion.2.NAC can reduce the damage of MCC system induced by PM_2.5exposure in rats.The mechanism may be that NAC can up-regulates the expression of AQP5 and down-regulates the expression of MUC5AC.