The Role Of Sphingomyelin Synthase1in Oxidative Stress Induced N2a Cell Apoptosis And The Underlying Mechanism

To investigate whether SMS1was involved in the process of H2O2induce N2a cell apoptosis and to explore the underlying mechanism.Methods:(1) The effect of H2O2on the morphological change of N2a cell was observed. N2a cell was treated with different concentrations of H2O2for24h, and the cellular proliferation capacity was detected by MTT assay.(2) The cell was treated with different concentration of H2O2(50μM,100μM,150μM), and the expressions of SMS1were determined by Western-Blot and Real-Time PCR.(3) To study the role of SMS1in H2O2induces N2a cell apoptosis. Cultured N2a cell were randomly divided into four groups:①control group:cells were incubated in high glucose DMEM containing1%fetal bovine serum for24hours;②H2O2treatment group:cells were incubated in high glucose DMEM containing150μM H2O2for24hours;③H2O2+SMS1inhibitor (D609) group:cells were pretreated with50μM D609for3hours prior to H2O2exposure;④D609group:cells were pretreated with50μM D609for3hours before incubating in high glucose DMEM containing1%fetal bovine serum for24hours. The expression of SMS1was assessed by western blot and real time-PCR; The expression of p-ERK were determined by western blot. The rate of cell apoptosis was measured by Hoechst staining and flow cytometry. The concentration of ceramide in each group was detected by LS-MS analysis.Results:(1) The morphology of N2a cell was changed in H2O2treated group. The MTT assay result shows that the cell activity was decreased as the concentration of H2O2was increased.(2) The expression of SMS1was unregulated by H2O2in a dose-depended manner, both in mRNA and protein level. The protein expression of SMS1was highest when treated with H2O2(150uM).(3) Compared with the control group, H2O2(150μM) significantly increased the expression of SMS1, the content of ceramide, the expression of p-ERK and the cell apoptosis. Inhibiting the SMS1by D609, the content of ceramide and the cell apoptosis percentage were increased, whereas expression of p-ERK was decreased.Conclusion:Thus the present study has demonstrated that SMS1may be exert protective benefit in the process of the H2O2-induced N2a cell apoptosis. The results indicate that SMS1inhibit H2O2-induced N2a cell apoptosis by decrease the accumulation of ceramide. Its effects on ceramide and ultimately on p-ERK expression define the protective role of SMS1against H2O2-induce neurocyte apoptosis.