Dihydroartemisinin modulation of toll-like receptors-4 signaling pathway in lipopolysaccharide-stimulated RAW264.7 murine macrophages

Objective: To investigate how dihydroartemisinin (DHA) modulate Toll-like receptors-4 signaling pathway in LPS-stimulated RAW264.7 murine. Methods: IRF3 was detected in RAW264.7 cells treated with lipopolysaccharide (LPS) (1 μg/ml), lipopolysaccharide and dihydroartemisinin (LPS/DHA) (10 μM) and equal volume of culture medium respectively by using immunocytochemistry staining. IFN-α/β mRNA was extracted from Raw264.7 cells in LPS, LPS/DHA and control group, and then was measured by real-time PCR. The concentration of IFN-β in the supernatants was detected by enzyme linked immunosorbent assay. Evaluation on TRAF6 and IRF3 protein expression was carried out by western blot. Results: LPS increased the expression level of IRF3 while DHA significantly inhibited the effect of LPS. Compared with the control group, the IFN-β mRNA level of LPS group dramatically increased. DHA significantly diminished LPS-induced IFN-β gene expression (**P < 0.01 vs. control group; ##P < 0.01 vs. LPS group). However, though LPS slightly upregulated IFN-α expression, no significant difference was observed among the three groups. The protein level of IFN-β in cells supernatants was increased in LPS group, while DHA decreased the release of IFN-β in LPS/DHA group (**P < 0.01 vs. control group; ##P < 0.01 vs. LPS group). LPS enhanced IRF3 expression and DHA inhibited LPS-stimulated effect in this study. Whereas no statistically significant difference of TRAF6 protein level was observed between LPS group and LPS/DHA group. Conclusion: DHA has potent immunosuppressive effect in vitro by inhibiting TRIF-IRF3 signaling pathway of TLR4 and IFN-β production.