Fig. 6

Sphk1-regulated neuroinflammation is related to S1P-induced NF-κB activation. HAPI cells were pretreated with PF543 for 12 h or BAY11-7085 for 2 h, followed by washing three times and treatment with LPS alone or LPS plus S1P for 24 h. a Representative Western blots and quantitative data for iNOS, COX-2, TNF-α, IL-6, and GAPDH expression. PF543 pretreatment attenuated LPS-induced M1 microglia polarization and proinflammatory cytokine production, effects that were reversed by addition of exogenous S1P. b Immunofluorescence staining for p65 was observed under confocal microscope (scale bar: 10 μm), and nuclear-to-cytoplasmic ratio of p65 was quantified. LPS-induced p65 nuclear translocation was suppressed by PF543 pretreatment and restored by addition of exogenous S1P. c Western blot analysis of p-p65, p65, IκB, and GAPDH expression in HAPI cells. Adding exogenous S1P amplified the NF-κB activation and inflammatory response, and BAY11-7085 significantly eliminated the amplification. d Immunofluorescence staining for TNF-α in each group of microglia (scale bar: 100 μm). e The secretion of TNF-α was also measured by ELISA assay. S1P promoted proinflammatory mediator release, and the effect was eliminated by BAY11-7085. n = 3 independent experiments. *P < 0.05 and **P < 0.01