Time and dose effects of lipopolysaccharide on release of inflammatory factors in N9 microglia

Objective To explore the time and dose effects of lipopolysaccharide(LPS)on the release of inflammatory factors in N9 microglia.Methods N9 microglia were treated with different doses of LPS.At different time points of LPS treatment,the levels of tumor necrosis factor alpha(TNF-α) and interleukin-1 beta(IL-1β) in the medium were measured by enzyme-linked immunosorbent assay and nitric oxide(NO) level in the medium was determined by nitrate reductase method.The levels of nuclear factor-kappa B(NF-κB) protein in the cytoplasm and nuclei were detected by Western blot.Results After 6 and 12 hours there were no significant differences of the IL-1β and NO levels in the medium between each LPS group and control group(P0.05).After 24 hours the levels of IL-1β in the medium of 1 000 and 10 000 μg·L-1 LPS groups were higher than those in control group(P0.01).After 30 minutes,TNF-α levels in the medium of all LPS groups had no significant difference compared with those of control group(P0.05).After 6 and 24 hours,the TNF-α levels in the medium of all LPS groups were higher than those of control group(P0.01),and the TNF-α level in the medium of 1 000 μg·L-1 LPS group was the highest.Western blot showed that the levels of NF-κB protein in the cytoplasm and nuclei of all LPS groups were higher than those of control group(P0.01),and 1 000 μg·L-1 LPS group had the highest NF-κB protein expression level whether in cytoplasm or in nuclei.Conclusion 1 000 μg·L-1 LPS is the best dose to activate N9 microglia and induce its inflammatory response.Treatment with LPS for 6 hours mainly promotes the release of TNF-α and for 24 hours significantly increases the release of IL-1β and NO.