Promoting proliferation and inhibiting apoptosis effects of sphingosine-1-phospate on human retinal pigment epithelium cells under the hypoxic condition

Background Sphingosine-1-phospate (S1P) is a bioactive lipid and important messenger molecule in cells.It participates in the regulation of many biological processes, such as cell proliferation, migration, survival, differentiation, apoptosis, etc.Hypoxia is a trigger factor of choriod neovascularization (CNV) and pathological basis of many diseases, and retinal pigment epithelial (RPE) cells are involved in formation of CNV.However, the effects of S1P on proliferation and apoptosis of RPE cells are below understood. Objective This study was to investigate the influence of S1P on proliferation and apoptosis of human RPE cells under hypoxic conditions. Methods Human RPE cells line–D407 cells were cultured and passaged and generation 3-5 cells were used and divided into 6 groups.The cells were regularly cultured in the blank control group using DMEM containing 10% fetal bovine serum.CoCl2 (200.00 μmol/L) was added into the colture medium for 2 hours in the hypooxic group.S1P of different concentrations (0.01, 0.10, 1.00, 10.00 μmol/L) were added in culture medium 2 hours after the affection of 200.00 μmol/L CoCl2.The proliferative values of the cells were detected using WST-1 method as the absorbance (A value) and the proliferative rate of different groups were calculated.The apoptosis of the cells was assayed by Hoechst staining.The results were compared among different groups. Results Cultured cells showed the round-like in shape with clear nuclei and pigment.The proliferative values (A value) was 0.91±0.08, 0.37±0.09, 0.46±0.08, 0.52±0.09, 0.61±0.06, 0.70±0.10 in the blank control group, hypoxic group and 0.01, 0.10, 1.00, 10.00 μmol/L S1P groups, respectively, with a significant difference among the groups (F=21.104, P=0.000), and A values in various S1P groups were higher than those in the hypoxiac group (all at P<0.05).The proliferative rate was gradually raised with the increase of dose of S1P.Hoechst staining exhibited a few apoptosis cells in the blank control group, but in the hypoxic group, a lots of apoptosis cells were seen with the light-blue nuclei and condensable chromatin.However, the number of apoptosis cells was significantly decreased in various concentrations of S1P groups.The apoptosis rates were (1.21±0.08)%, (8.99±0.09)%, (6.60±0.08)%, (5.95±0.09)%, (4.81±0.06)% and (3.96±0.10)% in the blank control group, hypoxic group and the 0.01, 0.10, 1.00, 10.00 μmol/L S1P groups, respectively, with a significant difference among the groups (F=25.070, P=0.000).Compared with the hypoxia group, the cellular apoptosis rates of various S1P groups were lower (all at P<0.05). Conclusions Under the hypoxia condition, S1P can promote the proliferation of human RPE cells and inhibit apoptosis. Key words: Hypoxia; Sphingolipids/metabolism; Retinal pigment epithelial cell/human; Proliferation; Apoptosis; Choroid; Neovascularization