关键词: 脂多糖；Toll样受体4；巨噬细胞移动抑制因子；脐静脉内皮细胞

Abstract:

Objective    To study the effect of ISO-1 ［(S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester］, a specific antagonist of MIF (Macrophage migration inhibition factor),  on TLR4 (Toll-like receptor) activities in HUVEC (Human umbilical vein endothelial cells), and to provide an experimental evidence for  the treatment of  TLR4-related diseases in which ISO-1 is targeted at. Methods   HUVECs were cultured and divided into 4 groups: LPS (lipid polysaccharide) group, ISO-1 pretreatment group, LPS+ISO-1 group and Medium control group.  Effects of ISO-1 on TLR4 expression, TNFα (tumor necrosis factor) and NOS (nitric oxide synthase ) secretion were investigated with RT-PCR, immunofluorescense staining, radioimmunoassay and enzyme-chemical reactions. Results    The expression of TLR4 in HUVEC was obviously inhibited by pretreatment with high concentration of ISO-1, especially in 25 and 50μmol/L groups (P＜0.05).  Low expression of TLR4 was also detected at 0.5, 1, 2 and 3h after the pretreatment with 50μmol/L ISO-1, in which the lowest appeared at 1h (P＜0.05) . Immunofluorescense showed low level TLR4 in  normal HUVEC, however, the expression could be enhanced by LPS (10ng/mL). TLR4 expression, TLR4-induced TNFα and NOS secretion were markedly suppressed 1h later after the pretreatment with 50μmol/L ISO-1, and such inhibition was does-dependent (P＜0.05). The effective time of ISO-1 was short and the maximal was at 1h. Conclusion    ISO-1 can exert the inhibitory effects through down-regulating the TLR4 activities of HUVEC, which suggests that ISO-1 may be  a potential target-agent in the treatment of TLR4-related diseases, such as atherosclerosis and inflammation.

Key words:  Lipid polysaccharide; Macrophage migration inhibition factor; Toll-like receptor; Human umbilical vein endothelial cells