Fig 2: S1PR1 activation enhanced NSCs proliferation and neuronal differentiation in hippocampal dentate gyrus (DG) after TBI. (a) Coronal diagram of rat hippocampus, the subgranular zone, granular cells layer, and molecular layer of DG were, respectively, marked by SGZ, GCL, and MOL, the blue pane representing one of microscopic visual fields for counting immunolabeled cells of immunofluorescence (IF). (b) NSCs proliferation in SGZ at 7 days after TBI was assessed by BrdU/SOX2 double-labeling IF. Quantitation analysis showed that, relative to sham group (n = 3), brain injury induced more double-positive cells in TBI+Vehicle group (n = 6). Following the treatment of SEW2871, the number of BrdU+/SOX2+ cells further increased in TBI+SEW group (n = 6). Reversely, administration of VPC23019 resulted in a significant reduction of BrdU+/SOX2+ cells in TBI+SEW+VPC group (n = 6). (c) Neuronal differentiation of NSCs in SGZ at 28 days after TBI was assessed by BrdU/NeuN double-labeling IF. Statistical data indicated that BrdU+/NeuN+ cells increased in TBI+Vehicle group (n = 6) compared with sham group (n = 3). And the double-positive cells increased at even higher level after SEW2871 treatment in TBI+SEW group (n = 6). However, VPC23019 administration caused an evident decrease of BrdU+/NeuN+ cells in TBI+SEW+VPC group (n = 6). (d) and (e) Representative IF microphotographs of hippocampus immunolabeled with BrdU/SOX2 and BrdU/NeuN in all the above four groups at 7 days and 28 days after trauma. Scale bar: 50 µm. * P < 0.05 versus sham group; # P < 0.05 versus TBI+Vehicle group or TBI+SEW+VPC group.

Fig 3: Cognitive function of rats in sham, TBI+Vehicle, TBI+SEW, TBI+SEW+U0126, TBI+VPC, and TBI+VPC+ERN group (n = 3 in sham and n = 5 in other groups) were evaluated by Morris water maze (MWM) test. (a) Escape latency in hidden platform trial exhibited a gradual reduction tendency from 24 to 27 days after trauma. Daily escape latency of TBI+Vehicle group was longer than that of sham group. Use of S1PR1 agonist in TBI+SEW group significantly shorten the latency, but the effect was eliminated in TBI+SEW+U0126 group. In addition, the escape latency of TBI+VPC+ERN group decreased compared with TBI+VPC group. (b, c) Platform crossing times and target quadrant duration in probe trial revealed that, relative to sham group, TBI+Vehicle group presented less times and shorter duration at 28 days after TBI. S1PR1 activation significantly increased the two indexes of TBI+SEW group, but the favorable effect was blocked by U0126 treatment in TBI+SEW+U0126 group. Moreover, the times of platform crossing and duration in target quadrant of TBI+VPC group were lower than those of TBI+VPC+ERN group. (d) Rat swimming speed of the six groups did not show any statistical difference. * P < 0.05 versus sham group; # P < 0.05 versus TBI+SEW group, † P < 0.05 versus TBI+VPC group.

Fig 4: Activation of S1PR1 triggered MEK/Erk pathway phosphorylation in hippocampus at 7 days after TBI. (a) WB was performed to determine hippocampal S1PR1, pMEK, tMEK, pErk, and tErk expression in groups of sham, TBI+Vehicle, TBI+SEW, and TBI+SEW+VPC group (n = 3 in sham and n = 6 in other groups). (b, c, and e) The level of S1PR1, pMEK, and pErk significantly increased after trauma. SEW2871 induced further upregulation of S1PR1, pMEK, and pErk. However, the effect was attenuated by administration of VPC23019. (d, f) Neither S1PR1 agonism nor antagonism had effect on tMEK and tErk expression in hippocampus after TBI. * P < 0.05 versus sham group; # P < 0.05 versus TBI+Vehicle group or TBI+SEW+VPC group.

Fig 5: S1PR1-induced NSCs proliferation and neuronal differentiation in TBI rats were affected by MEK/Erk activity. (a, b) Representative IF microphotographs of hippocampus immunolabeled with BrdU/SOX2 and BrdU/NeuN in TBI+SEW, TBI+SEW+U0126, TBI+VPC, and TBI+VPC+ERN groups at 7 days and 28 days after trauma (n = 6 in each group). (c, d) Statistical analysis showed that, compared to TBI+SEW group, administration of U0126 significantly decreased the number of BrdU+/SOX2+ cells and BrdU+/NeuN+ cells in TBI+SEW+U0126 group. Reversely, the double-labeled cells in TBI+VPC+ERN group were significantly increased compared with TBI+VPC group. Scale bar: 50 µm. * P < 0.05 versus TBI+SEW group; # P < 0.05 versus TBI+VPC group.