Effects Of PI3K?-RhoA Pathway On Phagocytosis Defect Of Alveolar Macrophages In Chronic Obstructive Pulmonary Disease Mice

Background and objective The phagocytosis of alveolar macrophages(AM)in chronic obstructive pulmonary disease(COPD)mice is defectient,and this disorder of phagocytosis may be related to abnormal rearrangement of AM cytoskeleton.Phosphatidylinositol 3-kinase(PI3K?)is a lipid kinase that affects the cytoskeletal arrangement by specific negative regulation of the Ras homologous gene family member A(RhoA).However,the relationship between PI3K?-RhoA mediated cytoskeletal arrangement with the defectient phagocytosis was raely reported.We take the AM of COPD mice as the research object,explore the mechanism of PI3K?-RhoA pathway in phagocytosis defect of alveolar macrophage(AM)in chronic obstructive pulmonary disease(COPD)mice.Methods Forty SPF level 8-week-old male BALB/c mice,Twenty mice were exposured of cigarette smoking for 90 days to establish COPD model,Twenty mice for control group.Using mice non-invasive pulmonary function instrument to test the mouse pulmonary function in each group,then kill the mice by cervical dislocation and take the lung tissue for HE staining,pathological changes were observed under light microscope.AM were isolated from lung tissue by discontinuous density gradient centrifugation and then divided into health control group,COPD group,health IC87114 group and COPD IC87114 group.The culture of IC87114 group were mixed a final concentration of 1nmol/L IC87114 for 24 hours.Mean fluorescence intensity(MFI)and the positive percent of alveolar macrophage engulfing flurescein isothiocyanate-labeled Escherichina coli(FITC-E.coli)AM(%)were detected by flow cytometry.The PI3K??RhoA mRNA expression of AM was measured by RT-PCR.PI3K??RhoA protein expression of AM was measured by Western blot.The activity of RhoA was measured by G-LISA RhoA Kit.The changes of cytoskeleton structure of AM were observed by Laser scanning confocal microscopy.Results(1)Pulmonary function and pathological changes in mice: the peak inspiratory flow(PIF)?peak expiratory flow(PEF)and 50% tidal volume expiratory flow(EF50)in COPD group(7.06±0.52?4.24±0.29?3.27±0.36)were significantly decreased compared with the healthy control group(10.37±0.76?7.13±0.41?5.04±0.47)(P<0.01);In COPD group,there can be seen significant decreased alveolar numbers,alveolar cavity expansion and fusion in the lung tissue,meanwhile with a large number of inflammatory cell infiltratation.While with no these changes in the healthy control group.(2)Detection of phagocytic capacity in AM:The MFI and AM% in COPD group(4 512±517,32.19±4.57)were significantly decreased than those in healthy control group(9 857±1042?68.53±2.88)(P<0.01),Compared with COPD group,MFI and AM% in COPD IC87114 group(6894±472,50.63±2.12)were increased(all P < 0.01).While Healthy IC87114 group(10097±897?70.99±3.86)have no statistical difference with healthy control group.(3)PI3K? mRNA and protein expression in AM : The mRNA and protein of PI3K? in COPD group(3.41±0.54?0.84±0.08)were higher than those in the healthy control group(1.00±0.00;0.57±0.07)(P <0.01);In COPD IC87114 group(1.52±0.28?0.66±0.13),the above parameters were remarkably decreased compared with those in COPD group(P<0.01);While healthy IC87114 group(0.74±0.07?0.50±0.10)have no statistical difference with healthy control group.(4)RhoA mRNA and protein expression in AM : The mRNA,protein of RhoA in COPD group(0.70±0.07?0.41±0.10)were significantly decreased than those in healthy control group(1.00±0.00;0.56±0.09)(P <0.01);COPD IC87114 group(0.91±0.08?0.48±0.06)compared with COPD group were increased(P<0.01 or P<0.05);The mRNA expression of RhoA in healthy IC87114 group(1.07±0.10)was increased compared with healthy control group(P<0.05),but protein expression have no statistical difference.(5)RhoA activity detection in AM: The activity of RhoA in COPD group(0.70±0.06)was lower than that in healthy control group(1.19±0.09)(P <0.01);COPD IC87114 group(0.86±0.06)compared with COPD group were increased(P<0.01);But healthy IC87114 group(1.25±0.05)have no statistical difference with healthy control group.(6)Changes of Cytoskeleton in AM: The pseudopod of healthy control group and healthy IC87114 group extend well,and the ability of phagocytosing FITC-E.coli is well,but there are some deficiency about these in COPD group.Compared with COPD group,the COPD IC87114 group is better,both in phagocytosing and extending of pseudopod.(7)Correlation analysis: The expression of PI3K? mRNA,protein and RhoA mRNA,protein,activity have a negative correlations(P <0.01);Negative correlations were also existed between the mRNA,protein of PI3K? with MFI in all groups(P<0.01 or P<0.05),but positive correlations between that of RhoA with MFI in all groups(P<0.01 or P<0.05)Conclusions Cigarette smoke exposure can established the model of COPD mice successfully;Phagocytosis of AM from COPD mice was impaired,cytoskeleton abnormally rearrangement;PI3K? negatively regulate RhoA;PI3K? over activated result in the activity of RhoA decreasing and then induce abnormally cytoskeleton rearrangement in alveolar macrophage of COPD which result in phagocytosis defect of AM;IC87114 can inhibit PI3K? activated,improve the activity of RhoA and partly recover phagocytosis of AM.