Mechanisms Of Cytoskeleton And PI3Kδ-RhoA In PM2.5 Deteriorating Phagocytosis Defect Of Alveolar Macrophage In Chronic Obstructive Pulmonary Disease Mice

Background and objective PM2.5 could deteriorate phagocytosis defect of alveolar macrophage(AM)in chronic obstructive pulmonary disease(COPD)mice,but the mechanisms of PM2.5 effected phagocytosis of AM in COPD through PI3Kδ-RhoA and cytoskeleton was rarely reported.In this paper,we explore the mechanisms of cytoskeleton and PI3Kδ-RhoA in PM2.5 deteriorating phagocytosis defect of AM in COPD mice.Methods Fourty SPF 8-week-old BALB/c mice were randomly divided into four groups: health control group,COPD group,health PM2.5 group,COPD PM2.5 group with ten in each group.A mouse model of COPD was established by cigarette smoke exposure,and as the same time of modeling,COPD PM2.5 group and health PM2.5 group were given PM2.5(588 μg/m3)aerosol inhalation for 90 days.The health control group and COPD group were given equal volume of saline inhalation.Using mice non-invasive pulmonary function instrument to test the mouse pulmonary function in each group,which be expressed in peak inspiratory flow(PIF),peak exspiratory flow(PEF)and 50% tidal volume expiratory flow(EF50),then kill the mice and take the lung tissue for HE staining,and pathological changes were observed under light microscope.AM were isolated from lung tissue by discontinuous density gradient centrifugation.Mean flurescein intensity(MFI)and the positive percent of AM engulfed flurescein isothiocyanate labeled Escherichia coli(FITC-E.coli)AM(AM%)were detected by flow cytometer.Extracting the RNA and protein of AM and the mRNA and protein expression were measured by RT-PCR and Western blot.The activity of RhoA were measured by G-LISA Kit.The change of cytoskeleton were observed by laser scanning confocal microscopy.Result(1)Pulmonary function and pathological changes in mice: the pulmonary function of PIF,PEF and EF50 in COPD group(7.06±0.52,4.24±0.29,3.27±0.36)and health PM2.5 group(7.74±0.43,5.21±0.31,3.40±0.32)were obviously lower than health control group(10.37±0.76,7.13±0.41,5.04±0.47)(all P<0.01).The above parameters from COPD PM2.5 group(4.73±0.21,3.43±0.17,2.04±0.14)were significantly lower than those in COPD group(all P<0.01).There can be seen alveolar structure disorder,alveolar wall thinning,alveolar cavity expansion,rupture,alveolar septum rupture,inflammatory cell infiltration,bronchial wall stenosis in lung tissue of mice with COPD group and health PM2.5 group,and alveolar destruction in COPD PM2.5 group was significantly heavier than that in COPD group.In healthy control group,the structure of alveolar wall was complete,there was no cell shedding in airway epithelium,no infiltration of inflammatory cells,almost no effusion in alveolar space and alveolar septum was normal.(2)Detection of phagocytic capacity in AM: The MFI and AM% in COPD group [4512±517,(32.19±4.57)%] and health PM2.5 group [7631±585,(50.78±4.58)%] were decreased than health control group [9857±1042,(68.53±2.88)%],while those in COPD PM2.5 group[3121±393,(21.90±2.58)%] were lower than COPD group(all P<0.01).(3)PI3Kδ mRNA and protein expression in AM: The mRNA and protein of PI3Kδ in COPD group(3.41±0.54,0.84±0.08)and health PM2.5 group(1.52±0.35,0.71±0.11)were higher than health control group(1.00±0.00,0.57±0.07)(all P<0.05).And in COPD PM2.5 group(5.53±0.42,1.17±0.25),the above parameters were remarkably increased as compared to COPD group(all P<0.01).(4)RhoA mRNA and protein expression and activity detection in AM: The mRNA,protein and activity of RhoA in COPD group(0.70±0.07,0.41±0.10,0.70±0.06)and health PM2.5 group(0.84±0.06,0.46±0.11,0.87±0.07)were lower than health control group(1.00±0.00,0.56±0.09,1.19±0.09).And above parameters of COPD PM2.5 group(0.42±0.05,0.31±0.06,0.44±0.04)were significantly lower than COPD group(all P <0.05).(5)Changes in the cytoskeleton of AM: Long and dense filopodia and membrane fold could been seen clearly around the AM of health control group,and cell deform obviously;In COPD group and health PM2.5 group,there can been seen short and sparse filopodia and slightly deformed.Filopodia remarkably decreased and cell rigid in COPD PM2.5 group.(6)Correlation analysis: The mRNA and protein of PI3Kδ were significantly negatively correlated with mRNA,protein and activity of RhoA in each group(all P<0.01).There is negative correlations between mRNA and protein of PI3Kδ and MFI in all groups and positive correlations between mRNA,protein and activity of RhoA and MFI in all groups(all P<0.05).Conclusion Cigarette smoke exposure can be used to establish the model of COPD mice;Phagocytosis of AM in COPD mice was decreased;PM2.5 can deteriorate the phagocytosis of AM from COPD mice and reduce phagocytosis of AM from healthy mice;PI3Kδ is activated and RhoA is inhibited in AM of COPD mice,PM2.5 aggravate this change;PI3Kδ-RhoA and cytoskeletal aberrant rearrangements,which is associated with PM2.5 deteriorating the phagocytosis of AM from COPD mice.