Effects Of Actin Depolymerization On Tlrs Expression And Apoptosis In Macrophages

Background: Macrophages,the "sentinel" of human's immune system,which can recognize PAMPs(pathogen-associated molecular patterns)through various types of PRRs(pattern recognition receptors)located on the surface of macrophages.TLRs(Toll-like receptors),the key PRRs,can involve in inflammatory responses through NF-?B(nuclear transcription factor)/MAPK(mitogen-activated protein kinase)signaling pathways.In the inflammatory response mediated by pro-inflammatory factors such as lipopolysaccharide(LPS),TLRs/F-actin(fibrin actin)pathway is widely distributed in macrophages and cancer cells such as lung cancer,but the possible correlation between the macrophages,TLRs,F-actin and tumors has not been fully elucidated.About the TLRs/F-actin pathway,the forward mode research has been reported before,However,the studies on regulating the expression of TLRs in macrophages by interfering with F-actin polymerization have not been reported.Purposes: The purpose of this study was to verify the effests of actin depolymerization on the expression of TLRs and apoptosis in macrophages.The expression level of TLRs in mouse macrophages RAW264.7 treated with different concentrations of cytochalasin D(cytD)was analyzed.The pEGFP-N1-Cofilin-1 plasmid(abbreviated as pEGFP-N1-CFL1)was constructed and transfected into macrophages,then the effects of endogenous over-expression of Cofilin-1 in macrophages on cytoskeleton,TLRs expression and apoptosis of RAW264.7 cells were explored.Methods:1.The recombinant plasmid pEGFP-N1-CFL1 has been constructed.The RAW264.7 cells were divided into three groups: the negative control group transfected with pEGFP-N1 plasmid,the recombinant plasmid group transfected with pEGFP-N1-CFL1 plasmid,and the non-treated blank control group of RAW264.7 cells.Fluorescence microscopy was used to detect the expression of fluorescent protein in each group of cells.2.After the RAW264.7 cells transfected with pEGFP-N1-CFL1 plasmid,Real-Time PCR and Western blot were used to detect the expression levels of Cofilin-1 mRNA and Cofilin-1 protein respectively.The cytoskeleton rearrangement was examined by fluorescence microscopy and the cytoskeleton rearrangement rate was calculated.3.RAW264.7 cells were treated with cytD at concentrations of 0 ?M,0.5 ?M,1.0 ?M,1.5 ?M,2.0 ?M,and 2.5 ?M for 48 h,and the growth state of cells were observed by inverted microscope.The expressions level of TLR2,TLR4 and TLR9 in the 6 cytD treated groups,and the groups before and after the RAW264.7 cells transfected with pEGFP-N1-CFL1 plasmid were analyzed by Western blot.The apoptosis rate of cells transfected with pEGFP-N1-CFL1 plasmid was detected by flow cytometry.Results:1.Enzyme digestion and gene sequencing confirmed that the pEGFP-N1-CFL1 plasmid was successfully constructed.Strong fluorescence was observed in RAW264.7 cells after transfected with pEGFP-N1-CFL1 plasmid.2.After the RAW264.7 cells transfected with the pEGFP-N1-CFL1 plasmid,the transcriptional level of Cofilin-1 mRNA and the expression level of Cofilin-1 protein in RAW264.7 cells has been enhanced significantly(P<0.05).The cytoskeleton rearrangement rate in cells transfected with pEGFP-N1-CFL1 plasmid was significantly lower than that of the other two groups(P<0.05).3.The results of Western blot showed that TLR2 decreased significantly when treated with cytD with final concentration ?1.5 ?M(P<0.05);When the final concentration of cytD ?1.0 ?M,the expression level of TLR4 and TLR9 was significantly decreased(P<0.05);The expression levels of TLRs(TLR2,TLR4,TLR9)in macrophages transfected with pEGFP-N1-CFL1 plasmid were significantly lower than those in control group(P<0.05).The results of flow cytometry showed that the apoptotic level of pEGFP-N1-CFL1 plasmid transfected cells was significantly lower than that of the control group(P<0.05).Conclusions:1.Both exogenous depolymerization factor cytD and endogenous overexpression of Cofilin-1 can induce the depolymerization of F-actin in macrophages.2.The cytD at different concentrations can selectively down-regulate the expression of TLR2,TLR4 and TLR9 in macrophages.3.Over-expression of Cofilin-1 can significantly down-regulate the expression of TLRs in macrophages and decrease the apoptotic rate of macrophages.