Toll-like receptor 4-related regulation of Ornithogalum caudatum extract on inflammatory responses in LPS activated macrophages

OBJECTIVE To investigate toll-like receptor 4(TLR4)-related the regulation of Ornithogalum caudatum extract(OCE) on inflammatory responses in lipopolysaccharide(LPS) activated macro.phages.METHODS Primary peritoneal macrophage,Raw 264.7,and THP-1 were incubated in 96-well plate for 24 h and treated with OCE of the concentration of 0-400 μg/ml for 4 h.The viability of cells was measured by MTT assay.Specific concentrations of OCE were added into the medium of primary peri.toneal macrophage,Raw 264.7,and THP-1,respectively,then following with lipopolysaccharides(LPS).Cells were harvested and the total cellular protein and nuclear protein were extracted,and the protein content was determined using BCA protein assay Kit.The expressions of TLR4,inducible nitric oxide synthase(iNOS),cyclooxygenase 2(COX-2),α-inhibitor of NF-κB(IκB-α) and nuclear factor-κB(NF-κB) were assayed by Western blot.The expressions of interleukin-1α(IL-1α),interleukin-1β(IL-1β),interleukin-18(IL-18),and tumor necrosis factor-α(TNF-α) were measured by RT-PCR.RESULTS The results of MTT showed that OCE has no cytotoxicity in Raw 264.7 cells between 1.56 μg/ml and 400 μg/ml.Compared with normal group,the expressions of TLR4,iNOS,COX-2,NF-κB and IL-1α,IL-1β,IL-18,TNF-α,the level of nitric oxide(NO) were significantly increased by LPS stimulation,while OCE pretreat.ment reduced these increase induced by LPS.However,OCE pretreatment reversed the reduction of IκB-α after LPS stimulation.CONCLUSION OCE might suppress TLR4 expression and block the inflamma.tion process of NF-κB and iNOS,further decrease the expression of COX-2 and inhibit the release of inflammatory factors.