Effect Of MTOR-Cdc42 Signaling Pathway On Phagocytosis Deficiency Of Alveolar Macrophages In Chronic Obstructive Pulmonary Disease Mice

Objective: To investigate the effect and mechanism in phagocytosis deficiency of alveolar macrophages by m TOR-Cdc42 signaling pathway in chronic obstructive pulmonary disease.Methods: 40 SPF level 8-week-old male BALB/c mice were divided into control group, healthy rapamycin group, COPD group and COPD rapamycin group.Mouse were exposed to cigarette smoke for 90 days and than observed the lung function and pathological changes of mice in each group. After the AMs were isolated after the end of cigarette smoke exposure and intervened with 10nmol/L rapamycin in 24 h. Flow cytometry method was applied to detect the ability of alveolar macrophages engulfed FITC-E.coli; Real-time quantitative polymerase chain reaction(RT-PCR) was applied to detect m TOR, p-m TOR and Cdc42 m RNA expression of AM; Western blot was applied to detect m TOR, p-m TOR, Cdc42 protein expression in the AM; Using the G-LISA assay detect the activity of Cdc42;Laser scanning confocal microscopy was used to observe the cytoskeleton structure of AM.Results :(1) The results of pulmonary function tests and morphology of brochus and lung tissue in COPD model : COPD group peak inspiratory flow and peak expiratory flow and 50%tidal volume expiratory flow(7.5±1.0;4.67±0.6;3.29±0.43) was significantly lower than the healthy control group(11.61±1.12;7.63±0.44;5.05±0.37)(P<0.01); Significant small airway inflammation and emphysema were seen in the lung tissue of COPD group comparing with the healthy control group.(2)The phagocytosis of alveolar macrophage: Mean fluorescence intensity and percent positive swallowed: COPD group( 4060±590; 28.65% ± 1.26%) was significantly lower than the healthy control group(9190±988;67.50%±4.56%)(P<0.01); COPD rapamycin group(4856±762; 38.31%±1.71%) was significantly higher than COPD group(P<0.05 or P<0.01); Healthy rapamycin group(9207±967;67.91%±3.64%) compared with healthy control group was not statistically significant.(3)The m RNA expression of m TOR and Cdc42 in AM: COPD group(2.62±0.46; 2.56±0.50) compared with the healthy control group(1.00±0.00; 1.00±0.00) were significantly increased(P <0.01); COPD rapamycin group(1.40±0.36; 1.38±0.34) compared with COPD group were decreased(P<0.05); healthy rapamycin group(0.94±0.37; 0.90±0.34) compared with healthy control group was not statistically significant.(4) The protein expression of m TOR, p-m TOR and Cdc42 in AM: COPD group(1.3047 ± 0.5188; 1.4645±0.4330; 1.6121±0.3749) compared with the healthy control group( 0.4829 ±0.2674;0.5798±0.2552; 0.6671±0.2168) were significantly increased(P<0.01); COPD rapamycin group(0.8996 ± 0.6634; 0.9208 ± 0.2771; 0.9143 ± 0.4764) compared with COPD group were decreased(P<0.05 or P<0.01); Healthy rapamycin group(0.4725 ±0.2845; 0.5592 ±0.2176; 0.6368 ± 0.22) compared with healthy control group was not statistically significant.(5)The activity expression of Cdc42 in AM: COPD group(0.4591±0.0853) compared with the healthy control group(0.2995±0.0674) was significantly increased(all P<0.01); COPD rapamycin group(0.3616±0.06) compared with COPD group was decreased(P<0.05); Healthy rapamycin group(0.2668±0.0572) compared with healthy control group was not statistically significant.(6)The changes in Cytoskeleton of AM: There can be seen obvious filopodia and lamellipodia around AM in COPD group. In COPD rapamycin group cells looks round with a long filopodia but arranged relatively neatly, No such changes in healthy control group and healthy rapamycin group.(7)Correlation analysis:The m RNA, protein of m TOR and Cdc42 and protein of p-m TOR, activity of Cdc42 in all group were negatively correlated with the mean fluorescence intensity(P<0.05 or P<0.01); The m RNA, protein, activity of m TOR in all group were positively correlated with the m RNA, protein, activity of Cdc42(P<0.05 or P<0.01).Conclusion: Continuous expose to cigarette smoke can be successfully established the model of COPD mice; Phagocytosis of AM from COPD mice was impaired; m TOR-Cdc42 signaling pathway was activated in alveolar macrophage of COPD; The activation of m TOR-Cdc42 signaling pathway can induced cytoskeleton anomaly rearrangemen result in phagocytosis deficiency of alveolar macrophage in COPD; Rapamycin can inhibition m TOR-Cdc42 signaling pathway partly recover phagocytosis of alveolar macrophage in COPD.