The Relationship Between PI3K-mTOR-RhoA Pathway Regulating Cytoskeleton Rearrangement And Phagocytic Capacity Of Macrophages

Background and objective: Chronic obstructive pulmonary disease(COPD)has a high incidence and mortality,and the burden of disease is high,but the specific pathogenesis of COPD is unclear.Some studies have found that decreased phagocytic function of alveolar macrophages(AM)is one of the causes of acute exacerbation of COPD,and phagocytosis is mainly related to cytoskeleton rearrangement,another study also found that phosphatidylinositol 3-kinase(PI3K)-mammalian target of rapamycin(mTOR)-Ras homologue family member A(RhoA)signaling pathway is involved in the regulation of cytoskeleton rearrangements,but the relationship between PI3K-mTOR-RhoA signaling pathway regulateing cytoskeleton rearrangement and macrophage phagocytosis is reported rarely,Our study used RNA interference technology(RNAi)to make the PI3 K and mTOR gene silenced which belong to the PI3K-mTOR-RhoA signaling pathway and to investigate the relationship between PI3K-mTOR-RhoA signaling pathway regulating cytoskeleton rearrangement and macrophage phagocytosis and the specific mechanisms.Methods: The mouse macrophage cell line RAW264.7 cells were cultured and divided into four groups: blank group(cells were not transfected),negative control group(cells were transfected with random sequence small interference RNA,shRNA),PI3 K geneing silence group(PI3K-RNAi group),mTOR geneing silence group(mTOR-RNAi group).The gene silencing efficiency were detected by Western blot,The mRNA of PI3 K,mTOR and RhoA were measured by real-time polymerase chain reaction,and the protein expression of PI3 K and mTOR and RhoA were detected by Western blot.Mean fluorescence intensity(MFI)and percentage of RAW264.7 cells engulfing fluoresceine isothiocyanate-labled escheruchia coli(FITC-E.coli)were detected by flow cytometry.The changes of cytoskeleton were observed by laser scaning confocal microscopy.Results:1.The gene silencing efficiency of PI3 K and mTOR is(62±7)% and(61±8)%.2.The mRNA and protein expression of PI3 K and mTOR and RhoA: The mRNA,protein expression of PI3 K,mTOR,RhoA and p-RhoA of PI3K-RNAi group [(0.29±0.07,0.38±0.02),(0.54±0.13,0.72±0.02),(0.59±0.06,0.74±0.03),0.68±0.02]were significantly lower than those in blank group[(1.00±0.00,1.00±0.04),(1.00±0.00,1.00±0.04),(1.00±0.00,1.00±0.04),1.00±0.06]and negative control group[(0.98±0.05?1.00±0.04),(1.01±0.11?1.01±0.08),(0.99±1.0?1.00±0.04),1.03±0.07)](all ?<0.01),The mRNA,protein of mTOR in mTOR-RNAi group(0.30±0.08,0.39±0.02)were lower than those in blank group and negative control group(all ?<0.01),while The mRNA,protein of RhoA and p-RhoA(1.40±0.21,1.54±0.04,1.33±0.01)were higher than those in blank group and negative control group(all ?<0.01).3.Morphological changes of cytoskeleton: The cell morphology of PI3K-RNAi group was stiff,the filopodia were short and messy,the number of stress fibers was decreased compared with the blank group;the cell deformation of mTOR-RNAi group was observed more obviously,more slender and more densely,the number of stress fibers were increased significantly compared with the blank group;The blank group and the negative control group cells deformed obviously,extended longer pseudopodia and more stress fibers.4.The cell phagocytic ability of FITC-E.coli: the MFI and RAW264.7 cell s% of the PI3K-RNAi group[7435±705,(70.73±2.66)]% were lower than those in blank group[11733±935,(79.97±1.07)%]and negative control group[10983±954?(79.60±1.17)%](all ?<0.01),while those parameters in mTOR-RNAi group[18583±1090,(87.72±1.58)%] were higher than the blank group and negative control group(all ?<0.01).5.Correlation analysis: Before or after transfection,positive correlations were existed between mRNA and protein of PI3 K and mTOR(all P<0.05),positive correlations were also existed between mRNA and protein of PI3 K and RhoA and p-RhoA(all P<0.05),while negative correlations were existed between mRNA and protein of mTOR and RhoA and p-RhoA(all ?<0.05).positive correlations were existed between PI3 K mRNA,protein and MFI and phagocytic%(all ?<0.05),positive correlations were also existed between RhoA mRNA,protein,p-RhoA protein and MFI and phagocytic%(all ?<0.05).while negative correlations were existed between mTOR mRNA,protein and MFI and phagocytic%(all ?<0.05).Conclusion:1.The method of lentivirus transfection can successfully make gene of PI3 K and mTOR been silenced;2.The cytoskeleton rearrangements was associated with the phagocytic ability of macrophages;3.PI3K-mTOR-RhoA signaling pathway regulating the cytoskeleton rearrangements.4.PI3 K positively regulates the mTOR-RhoA pathway,promotes cytoskeleton rearrangement correctly and enhances macrophage ability of phagocytosis.While mTOR negatively regulates the pathway,impedes cytoskeleton rearrangement and attenuates macrophage ability of phagocytosis.