• S T Hu, Q Xia, X L Zeng, H R Bao, and X J Liu.
• Department of Gerontal Respiratory Medicine, the Frist Hospital of Lanzhou University, Lanzhou 730000, China.
• Zhonghua Jie He He Hu Xi Za Zhi. 2017 Jul 12; 40 (7): 520-526.

AbstractObjective: To investigate effects of Phosphoinositide3-Kinases (PI3Kδ)-Ras homolog gene family member A(RhoA) pathway on phagocytosis deficiency of alveolar macrophages (AMs) in a mouse model of chronic obstructive pulmonary disease (COPD). Methods: Twenty mice were exposed to cigarette smoking to establish the COPD model, with 20 mice as the control group. AMs were isolated from lung tissue by discontinuous density gradient centrifugation and then divided into a healthy control group, a COPD group, a healthy IC87114 group and a COPD IC87114 group. The culture of IC87114 group was mixed with a final concentration of 1 nmol/L IC87114 for 24 hours. Mean fluorescence intensity (MFI) and the positive percent of AMs engulfing flurescein isothiocyanate-labeled Escherichina coli (FITC-E.coli) (AM%) were detected by flow cytometry. Real-Time PCR(RT-PCR)and Western blot were applied to detect mRNA and protein. G-LISA RhoA Kit was used to detect the activity of RhoA, and laser scanning confocal microscopy was used to observe the cytoskeleton structure of AMs. Results: Phagocytosis of AM: MFI and AM %in the COPD group [(4 512±517), (32.2±4.6)%] were decreased than those in the healthy control group [(9 857±1 042), (68.0±4.0)%, all P<0.01]. Compared with the COPD group, MFI and AM% in the COPD IC87114 group [(6 894±472), (50.6±2.1)%] were increased (all P<0.01). The expressions of mRNA and protein of PI3Kδ in the COPD group (3.14±0.54, 0.84±0.08) were increased than those in the healthy control group (1.00±0.00, 0.57±0.07) (all P<0.01). Compared with the COPD group, the expressions of mRNA and protein of PI3Kδ in the COPD IC87114 group (1.52±0.28, 0.66±0.13) were decreased (all P<0.01). The RhoA mRNA, protein and activity in the COPD group (0.70±0.07, 0.41±0.10, 0.70±0.06) were decreased compared to those in the healthy control group (1.00±0.00, 0.56±0.09, 1.19±0.09) (all P<0.01). Compared with the COPD group, the expression of mRNA, protein and activity of RhoA in the COPD IC87114 group(0.91±0.08, 0.48±0.06, 0.86±0.06) were increased (P<0.01, P<0.05). Cytoskeleton of AM: The pseudopods of the healthy control group and the healthy IC87114 group extended well, and the ability of phagocytosing FITC-E.coli was intact, but there were some defects in the COPD group. Compared with the COPD group, the COPD IC87114 group was better, both in phagocytosing and extending of pseudopods. Negative correlations existed between the mRNA, protein of PI3Kδ with mRNA, protein and activity of RhoA. Negative correlations also existed between the mRNA, protein of PI3Kδ with MFI, but positive correlations between RhoA and MFI were observed in all groups. Conclusion: The phagocytosis of AMs in COPD mice was defective, with abnormal rearrangement of the cytoskeleton. PI3Kδ negatively regulated RhoA, while PI3Kδ over activation resulted in decreasing activity of RhoA and then induced abnormal cytoskeleton rearrangement in AMs, which led to phagocytosis deficiency.IC87114 inhibited PI3Kδ activation, improved the activity of RhoA and partly recovered phagocytosis of AMs.

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