Effects of 3-phenyl-propenal on the expression of toll-like receptors and downstream signaling components on raw264.7 murine macrophages.

3-phenyl-propenal is one of the principle compounds isolated from Guizhi (Ramulus Cinnamomi), the principal drug in Guizhi-Tang (GZT), a famous traditional Chinese medical formula. The aim of the present study was to investigate the effects of 3-phenyl-propenal on the expression of toll-like receptor 3 (TLR3), TLR4 and the downstream signaling components on Raw264.7 murine microphages. Raw264.7 cells were cultured in RPMI-1640 medium containing LPS (lipopolysaccharide) or poly (I:C) in the presence or absence of 3-phenyl-propenal. After 24-hour incubation, the medium was collected and the amount of TNF-alpha and IFN-beta was measured by ELISA. mRNA expression of TLR3, TLR4, myeloid differentiation factor (MyD88), TRAF-6 (tumor necrosis factor receptor-associated), TRAM (toll-like receptor-associated molecule) and TRIF (TIR domain-containing adaptor inducing IFN-beta) were analyzed by real-time PCR with SYBR green dye. Protein expression of TLR3 and TLR4 was analyzed by Western blotting and that of MyD88 and TRAF-6 was analyzed by immunofluorescence assay. The results indicate that LPS increased the expression of TLR4, MyD88, TRAF-6, TRAM and TRIF, but had no influence on TLR3, while poly (I:C) up-regulated the expression of TLR3, MyD88, TRAM and TRIF. 3-phenyl-propenal significantly decreased the expression of LPS-induced TLR4, MyD88, TRAF-6, while possessing no effect on LPS-induced TRAM and TRIF expression in Raw264.7 cells. When cells were stimulated by poly (I:C), 3-phenyl-propenal significantly decreased TLR3 and MyD88 expression. In conclusion, 3-phenyl-propenal blocked the over-expression of TLR3, TLR4, their downstream signaling components MyD88 and TRAF-6, which indicate that it had an antagonistic effect on TLR3 and TLR4.