| Objective To explore the effects and mechanisms of mammalian target of rapamycin-cell division cycle 42(m TOR-Cdc42)signaling pathway on fine particulate matter with a mean aerodynamic diameter less than 2.5μm(PM2.5)deterioration of impaired phgocytosis of alveolar macrophage(AM) in chronic obstructive pulmonary disease(COPD) mice.Methods Fifty SPF 8-week-old BALB/c mice were divided randomly into normal control(A)group, COPD(B) group, PM2.5 COPD(C) group, EGCG+COPD(D) group, and EGCG+PM2.5COPD(E) group with ten in each group. Using continuous expose to cigarette smoke 90 days established mouse model of COPD. Acquisition Lanzhou City atmospheric PM2.5, group C and E were exposure to PM2.5 at nominal 720?g/m3 sustained atomizing inhaled 90 days, group A, B and D were given equal volume of saline inhalation. Using discontinuous density gradient centrifugation to separate mice AM, adjust the cell number 2×106cells/ml, group D and E were given a final concentration of 40μmol/L EGCG, incubate for 24 h. Using mice non-invasive body plethysmography to test the mouse lung function in each group, taken the left lower lobe,pathological were observed. Using flow cytometry to detect AM phagocytosis of fluorescein isothiocyanate-labeled Escherichia coli(FITC-E.coli) ability. Using real-time polymerase chain reaction(RT-PCR) and Western blot(WB) to detect m RNA and protein expression. G-LISA kit was used to test the activity of Cdc42 of AM. Using laser scanning confocal microscope to observe cytoskeletone.Result(1)The results of lung function and histopathology in COPD model:After exposure to cigarette smoke, the pulmonary function of peak inspiratory flow(PIF), peak exspiratory flow(PEF) and 50% tidal volume expiratory flow(EF50)in group B(7.5±1.0, 4.67±0.6, 3.29±0.43)were significantly lower than those in group A(11.61±1.12, 7.63±0.44, 5.05±0.37)(all P<0.01).Those from C group(4.96±0.91, 3.34±0.48, 2.14±0.29)were lower than those in group B(all P<0.01).Lung tissue can be seen bronchitis and emphysema clearly in group C, more serious than in groupB, while no such changes in group A.(2)The phagocytosis of AM:The mean fluorescence intensity( MFI) and AM phagocyttosing flurescein isothiocyanate-labeled Escherichia coli(FITC-E.coli)(AM%) in group B were(4060±590, 28.65±1.26),significantly lower than those in group A(9190±988, 67.50±4.56)(all P<0.01).Those in group C(2932±700, 18.41±1.48) significantly decreased than those in group B(all P<0.01). Those in group D(5046±732, 39.07±1.33)significantly increased than those in group B(all P<0.01). Those from group E(4018±578, 28.14±1.35)significantly increased than in from group C(all P<0.01).(3)AM m TOR m RNA, protein and activity(p-m TOR): The m TOR m RNA, protein and activity in group B were(2.62±0.46)(1.30±0.52)(1.46±0.43), significantly higher than those in group A(1.00±0.00)(0.48±0.27)(0.58±0.26)(all P<0.01).Those in group C(3.61±0.52)(2.50±0.47)(2.66±0.52)significantly increased than those in group B(all P<0.01). Those in group D(1.83±0.43)(0.91±0.31)(0.97±0.31)significantly decreased than those in group B(P<0.01, P<0.05). Those in group E(2.84±0.49)(1.84±0.43)(1.90±0.44)significantly decreased than those in group C(all P<0.01).(4) AM Cdc42 m RNA,protein and activity: The Cdc42 m RNA, protein and activity in group B were(2.56±0.50)(1.61±0.37)(0.46±0.09),significantly higher than those in group A(1.00±0.00)(0.67±0.22)(0.30±0.07)(all P<0.01).Those in group C(3.56±0.54)(2.70±0.48)( 0.62±0.10) significantly increased than those from group B(all P<0.01). Those in group D(1.77±0.44)(1.00±0.30)(0.37±0.08)significantly decreased than those in group B(P<0.01,P<0.05). Those in group E(2.67±0.48)(2.00±0.57)(0.52±0.06) significantly decreased than those in group C(all P<0.01).(5) The cytoskeleton of AM:There can be seen obvious filopodia and lamellipodia around AM in Group B. In group C cells showed irregular in shape, filopodia extending messy of varying lengths and increased lamellipodia area around AM. In group D showed a small amount of filopodia and lamellipodia around AM. In group E cells looks round with a small amount of filopodia but arranged relatively neatly, part of cell contact appears membrane ruffing. No such changes in group A.(6)Correlation analysis: Negative correlations were existed between m TOR,Cdc42 m RNA, protein, activity and MFI(p<0.01, 0.05),while positive correlations were existed between m TOR m RNA, protein, activity and Cdc42 m RNA, protein, activity in each group(p<0.01, 0.05).Conclusion 1. Continuous expose to cigarette smoke can be successfully established the model of COPD mice; 2. Phagocytosis of AM from COPD mice was damaged, PM2.5 deteriorate the phagocytosis impaired; 3. m TOR-Cdc42 signal pathway is activated of AM in COPD mice,PM2.5 facilitate this signaling pathway; 4. m TOR-Cdc42 signal pathway induced cytoskeletal inappropriately rearrangement is related to impaired phagocytosis of AM; 5. EGCG can inhibit COPD m TOR-Cdc42 signaling pathway and enhanced phagocytosis of AM in COPD mice. |
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