| Background and Objective The phagocytosis of alveolar macrophages(AM)in chronic obstructive pulmonary disease(COPD)is declining and is associated with abnormal rearrangement of the cytoskeleton.Toll-like receptor(TLR)4 may be involved in AM cytoskeleton rearrangement and phagocytosis by modulating phosphatidylinositol 3-kinase(PI3K)-Ras-associated C3 botulinum substrate(Rac)1 signaling pathway.This kind of research was rarely reported.AM is difficult to obtain,it is difficult to subculture,and is not easy to use RNA interference(RNAi)and other technologies to intervene.The mouse macrophage cell line RAW264.7 retains macrophage phagocytosis and adhesion functions,can be stably passaged and can undergo technical interventions such as RNAi.Therefore,this study used RAW264.7 cells as a research object to investigate the effects of TLR4-PI3K-Rac1 signaling pathway on macrophage cytoskeletal rearrangement and phagocytosis.Methods RAW264.7 was divided into blank group,negative control group and TLR4-RNAi group.Three plasmids carrying the TLR4 short hairpin RNA(sh RNA)and a negative control plasmid carrying non-significant sh RNA sequences were constructed and transfected into RAW264.7 cells by lipofection.A TLR4-sh RNA with the highest transfection efficiency was screened,the TLR4-sh RNA and sh RNA of non-significant control sequences were carried by lentivirus for subsequent transfection.The lentivirus carrying TLR4-sh RNA and nonsense control sequence were transfected into TLR4-RNAi group and negative control group,respectively.The blank group was not transfected.The efficiency of silencing TLR4 gene was detected by Western blot.Real-time fluorescence quantitative PCR was used to detect the expression of PI3 K and Rac1 m RNA in each group.The expressions of PI3 K,p-Rac1 and Rac1 protein were detected by Western blot.Cytoskeleton was observed by laser scanning confocal microscopy.Mean fluorescence intensity(MFI)and the positive percent of RAW264.7 cells engulfing flurescein isothiocyanate-labeled Eseherichina coli(FITC-E.coli)(RAW264.7 cells%)were detected by flow cytometry.Results(1)Transfection Efficiency and Silence Efficiency Detection : TLR4-sh RNA lentivirus can successfully transfected RAW264.7 cells and the transfection efficiency is more than 80%.The silencing efficiency of TLR4 gene was(63 ± 4)%.(2)TLR4 m RNA and protein expression in each group : TLR4 m RNA and protein expression in TLR4-RNAi group(0.20 ± 0.03,0.37 ± 0.04)were lower than those in the blank group and the negative control group [(1.00 ± 0.00,1.00 ± 0.05)and(0.98 ± 0.09,0.97 ± 0.07)](all P<0.01),There was no significant difference between the negative control group and the blank group.(3)PI3K m RNA and protein expression in each group: The expression of PI3 K m RNA and protein in TLR4-RNAi group(0.64 ± 0.06,0.75 ± 0.06)were lower than those in the blank group and the negative control group [(1.00 ± 0.00,1.00 ± 0.08)and(0.98 ± 0.05,1.00 ± 0.09)](all P<0.01),There was no significant difference between the negative control group and the blank group.(4)Rac1 m RNA and protein expression in each group: The expression of Rac1 m RNA,protein and p-Rac1 protein in TLR4-RNAi group(0.75 ? 0.04,0.76 ? 0.01,0.74 ? 0.05)were lower than those in the blank group and the negative control group[(1.00 ? 0.00,1.02 ? 0.05,1.00 ? 0.06)and(0.96 ? 0.10,1.07 ? 0.09,1.00 ? 0.06)](all P<0.01),There was no significant difference between the negative control group and the blank group.(5)Cytoskeleton of RAW264.7s: In the TLR4-RNAi group,the pseudopods of the TLR4-RNAi group were short and stiff,with the impaired capacity of engulfing FITC-E.coli.Blank group and negative control group RAW264.7 cells extend pseudopodia well,with strong capacity to engulfing FITC-E.coli.(6)The ability of each group of cells to engulf FITC-E.coli: The MFI and RAW264.7 cells% of TLR4-RNAi group[(7453 ? 564),(70.20?2.27)%] were lower than those in the blank group and the negative control group[(11733 ? 935),(79.97 ? 1.07)% and(10983 ? 954),(79.60 ? 1.17)%](all P<0.01),MFI and phagocytosis% in the negative control group were not statistically different from the blank group.(7)Correlation analysis: In the basic state and after intervention with RNAi,TLR4 mRNA and protein were positively correlated with PI3 K,Rac1 m RNA and protein,and p-Rac1 protein(all P<0.05),PI3 K m RNA and protein expression was positively correlated with Rac1 m RNA,protein and p-Rac1 protein(all P<0.05),TLR4,PI3 K,Rac1 m RAN and protein and p-Rac1 protein were positively correlated with MFI and RAW264.7%(all P<0.05).Conclusion TLR4-sh RNA plasmid and lentivirus can successfully transfect RAW264.7 cells and silence the TLR4 gene.The TLR4-PI3K-Rac1 signaling pathway regulates macrophage phagocytosis.Silencing TLR4 gene can inhibit the PI3K-Rac1 signaling pathway,resulting in disordered arrangement of macrophages and impaired phagocytosis. |
PDF Full Text Request