Actin-related Protein 2/3 Complex On Phagocytosis Defect Of Alveolar Macrophages In A Mouse Model Of Chronic Obstructive Pulmonary Disease

Background and objective Decreased phagocytosis of alveolar macrophages(AM)in mice with chronic obstructive pulmonary disease(COPD),and this decrease in phagocytosis is associated with abnormal rearrangement of AM cytoskeleton;actin-associated protein(Arp)2/3 complex can positively regulate F-actin and affect cytoskeleton arrangement.However,there are few studies on AM phagocytosis of COPD and the effect of Arp2/3 complex on F-actin on cytoskeleton arrangement.Therefore,this study used the AM of the chronic obstructive lung mice as a research model to investigate the role of the Arp2/3 complex in AM phagocytosis in mice with chronic obstructive lung disease.Methods A model of COPD was established by simple cigarette smoke exposure method.Twenty 8-week-old SPF BALB/c male mice were established and 20 mice of the same condition were used as healthy controls.EMMS were used for lung function test after modeling.The mice were sacrificed by cervical dislocation and the lung tissue was taken and HE staining.AM was separated and divided into healthy control group,chronic obstructive pulmonary disease group,healthy Arp2/3 complex inhibitor(CK666)group and chronic obstructive pulmonary CK666 group.The optimal concentration and time of CK666 were determined by thiazole blue colorimetric method(MTT).The final concentration of CK666 and slow-resistance lung CK666 was added to the final concentration of 10?mol/L CK666 for 24 h.Flow cytometry was used to measure AM phagocytosis.The ability of fluorescein-labeled E.coli(FITC-E.coli)was expressed as mean fluorescence intensity(MFI)and percentage of phagocytic FITC-E.coli positive cells(% phagocytosis);Western Blot assayed AM The expression of Arp2 and F-actin protein was detected by laser confocal microscopy.The average optical density of AM Arp2,F-actin,phagocytic FITCE.coli and the colocalization of Arp2 and F-actin were observed.Observation of the morphology of AM phagocytosis by scanning electron microscopy.Results(1)Pulmonary function and pathological changes in mice: PIF,PEF and EF50(6.56±0.25;4.26±0.23;3.20±0.15)in mice with chronic obstructive pulmonary disease were lower than those in healthy control group(10.97± 0.49;7.19 ± 0.24;5.74 ± 0.25)(both P < 0.01).Pathological changes: The emphysema of the mice with chronic obstructive pulmonary disease changed significantly,but the lungs of healthy control mice showed no abnormalities.(2)Detection of AM phagocytosis: AM MFI and phagocytosis in the chronic obstructive pulmonary group [(4702±243),(32.21±1.66)%] were lower than the healthy control group [(8684±234),(65.88±1.77)%,P<0.01];healthy CK666 group [(7446±236),(50.09±1.64)%] were lower than healthy control group(both P<0.01);chronic obstructive pulmonary CK666 group [(3597±307),(22.09±1.89)%] were lower than the chronic obstructive pulmonary disease group(both P<0.01).(3)AM Arp2 and F-actin expression: The slow-resistance lung group(0.51±0.03,0.46±0.03)was lower than the healthy control group(0.81±0.04,0.72±0.04,both P<0.01);healthy CK666 group(0.26)±0.02)F-actin was lower than healthy control group(P<0.01);slow-resistance lung CK666 group(0.63±0.03)F-actin was lower than chronic obstructive pulmonary disease group(P<0.01).(4)Determination of average optical density values of AM Arp2,F-actin and FITC-E.coli: The slow-resistance lung group(34±0.56,62±0.70,41±0.33)was lower than the healthy control group(143±1.90,146±3.05,189±2.60,all P<0.01);healthy CK666 group(137±1.35,157±1.00)F-actin,FITC-E.coli were lower than healthy control group(both P<0.01);slow resistance F-actin and FITC-E.coli in the lung CK666 group(38±1.04,41±0.33)were lower than those in the chronic obstructive pulmonary disease group(all P<0.01).Determination of Arp2 and F-actin MOC: Montessori colocalization coefficient(MOC)(0.395±0.014)in the chronic obstructive pulmonary group was lower than that in the healthy control group(0.880±0.002,P<0.01);healthy CK666 group(0.737±0.031)All were lower than the healthy control group(P<0.01);the chronic obstructive pulmonary CK666 group(0.297±0.006)was lower than the chronic obstructive pulmonary disease group(P<0.01).(5)AM morphology: healthy control group AM extended dense and long filiform pseudopods,cell surface protrusions and folds;healthy CK666 group AM filopodia protrusion decreased and shorter,surface protrusion folds decreased;slow In the obstructive pulmonary group and the slow-resistance CK666 group,the AM filopodia protruded short or missing,and the surface protrusion wrinkles were significantly reduced.(6)Correlation analysis: After basal state and CK666 intervention,AM Arp2 expression was positively correlated with F-actin protein expression,and AM Arp2 and F-actin protein expression and MOC were positively correlated with MFI.Conclusion Cigarette smoke exposure can successfully establish a mouse model of chronic obstructive pulmonary disease;AM phagocytic function is low in COPD mice;Arp2/3 complex positively regulates F-actin and interacts with cytoskeleton rearrangement to affect AM phagocytosis;the decrease of Arp2/3 complex activity leads to the decrease of its interaction with F-actin,abnormal cytoskeleton rearrangement,significant decrease of AM phagocytosis in COPD mice,and AM in COPD mice is more sensitive to the decrease of Arp2/3 complex activity.