An experimental study on targeted blockage of NF-κB inflammatory signaling pathway with dsODNs in alveolar macrophages (AMs)

doi:10.3969/j.issn.1002-1949.2014.08.015

Abstract：Objective After determined the optimal transfection concentration and time , evaluate the effectiveness of the inhibition of ( NF-κB)/DNA binding activity by lipofectamine 2000 transfected double -stranded oligodeoxynucleotides ( dsODNs ) in order to test the feasibility of dsODNs-decoy strategy targeted on NF -κB pathway in alveolar macrophages ( AMs ) .Methods AMs were separated from bronchoalveolar lavage and cultured for dsODNs transfection by Lipofectamine2000.The effectiveness of dsODNs/Lipofectamine2000 transfection activity, the optimum concentration ratio and transfection rate in AMs were investigated . Transfection toxicity of Lipofectamine2000 was detected using LDH kits .The expression levels of IL -1α, IL-6, TNF -αmRNA were determined by RT -PCR.Nuclear translocation of p65 after dsODNs/Lipofectamine2000 transfected in AMs was detected by Western -blot.Resu lts The transfection rate was best and cytotoxicity was moderate when the concentration ratio of dsODNs /Lipofectamine2000 was 1∶5.The cell transfection efficiency , fluorescence intensity and cells activity were best at the time spot of 6 hours after transfection.IL-1α, IL-6 and TNF-αmRNA expression were all decreased in the treatment group compared with LPS group (P <0.01).NF -κB p65 translocation to the nuclei was significantly blocked.Conclusion Lipofectamine 2000 is effective in the transfection of dsODNs into AMs and dsODNs-decoy can inhibit the gene expression by blocking the NF -κB pathway.It is viable to use dsODNs-decoy strategy to affect inflammatory in AMs .