Inhibition Of RAW264.6 Macrophage Inflammatory Response By Small Haparin RNAi Targeting TLR4

Background Recently, a family of receptor proteins, the Toll-like receptors (TLRs), have been identified in mammals. TLRs mediate cellular responses to a large array of microbial ligands. At present, 13 different TLR proteins have been cloned. TLR2 is the receptor for a variety of microbial ligands, including gram-positive bacteria, peptidoglycan, yeast zymosan, and mycobacterial ara-lipoarabinomannan (araLAM). TLR4 is a receptor for gram-negative bacteria, LPS, and some viruses. TLR4 and TLR2, like other TLR family members, have a conserved intracellular signaling motif. This signaling motif, which is also found in the intracellular domain of the IL-1 receptor (IL-1R), is responsible for nuclear factor-kappa B (NF-κB) activation/translocation after TLR or IL-1R receptor engagement and is an essential signaling pathway for IL-6 and tumor necrosis factor-alpha (TNF-α) secretion.Exaggerated production of cytokines leads to coagulation disorder, tissue injury and finally multiple organ failure, the clinical hallmark of sepsis. TLR4 is a gate of the downstream of inflammatory cascade for LPS signaling, and TLR2 may be involved with this process. Therefore the research inbiting the effect of TLRs can help us look for a triggering point that controls the response from the origin of inflammation, thus attain to adjust to control the excesssive inflammtory disease of the body effectively.Objectives To construct a expression vector carrying small hairpin RNA for Toll-like receptor 4 mRNA and a reporter gene of enhanced green fluorescence protein (EGFP) and study the inhibition of cytokine release by RAW264.7 cell induced by lipopolysaccharide (LPS) stimulation through transient transfection and expression of shRNA targeting TLR4 gene via the RNAi mechanism; and observe the expression of Toll-like receptor 2 following small haparin RNAi Targeting TLR4 through stable transfection under the condition of LPS stimulation in macrophage cell line RAW 264.7.Methods The reporter gene plasmid pEGFP-H1/siRNA, which contains Bbs site and reporter EGFP gene. Then an oligo nuclear haparin sequence targeting TLR4 gene was designed by internet tool and inserted into the plasmid pEGFP-H1/siRNA forming plasmid pEGFP-H1/TLR4. Then the plasmid of pEGFP-H1/TLR4 were transfected into RAW264.7 cells. The efficiency of transfection were detected by observe the expression of EGFP (Enhanced Green Fluorescent Protein, EGFP).After transient transfection of pEGFP-H1/TLR4 into RAW264.7 cells, Cells after 24 hour of transfection were given the pressure of selective medium (G418) to get stable cell lines. Cells were divided into 3 groups: Blank group, LPS group, TLR4-siRNA group. The expression of TLR2mRNA was determined by reverse transcription polymerase chain reaction (RT-PCR). The variation of NF-κB expression in three groups was analyzed by western blot. TNF-αproduction of the supernatants was measured with the ELISA(Enzyme-Linked Immunosorbent Assay) following the protocol presented by manufacturer.Results The constructed pEGFP-H1/TLR4 carrying hairpin RNA for TLR4 gene and reporter EGFP gene were proven to be right by restriction endonuclease analysis. The expression of EGFP gene were (45.25±9.23)% and after transfection of the plasmid pEGFP-H1/ TLR4-siRNA, the expression of TLR2 mRNA and NF-κB in TLR4-siRNA group were decreased compared to that in LPS group, as well as the level of TNF-αin the supernatants.Conclusions Short hairpin RNA (shRNA) targeting TLR4 gene could inhibited the TNF-αrelease by RAW264.7 cells evoked by LPS, and also significantly inhibited the expression of TLR2. It suggested that TLR4 might modulate expression of TLR2.