Fig. 7

S1P/S1PR3/p38 pathway contributes to Sphk1-related p65 activation. For in vivo experiments, rats were sacrificed and specimens were taken at 3 days after SCI. n = 3 per group. a Representative Western blots and quantitative data for S1PR3, p-p38, p38, and GAPDH expression in each group of rats. S1PR3 expression and p38 phosphorylation were enhanced in the SCI group and were inhibited by PF543 treatment. b Double-staining for S1PR3 (green)/Iba-1 (red) in injured spinal cord tissue from each group of rats (scale bar: 100 μm). For in vitro studies, HAPI cells were pretreated with PF543 for 12 h and CAY10444 for 2 h or SB203580 for 2 h, followed by washing three times and treatment with LPS alone or LPS plus S1P for 24 h, n = 3 independent experiments. c Representative Western blots and quantitative data for p-p65, p65, p-p38, p38, IκB, and GAPDH expression in each group of microglia. Both Sphk1 and S1PR3 activity influenced the activation of p65 and p38. d Immunofluorescence staining for p-p38 in each of group microglia (scale bar: 100 μm), PF543 pretreatment decreased p38 phosphorylation, and inhibition of S1PR3 strengthened the inhibitory effect. e Western blot analysis of p-p65, p65, p-p38, p38, IκB, and GAPDH expression in each group of microglia. S1P enhanced the activation of p65 and p38 induced by LPS stimulation, the effect was reversed by SB203580. *P < 0.05 and **P < 0.01