Sestrin2 Gene Overexpression Contributes To Regulation Of Mitochondrial Fission In Oxygen-Glucose Deprivation/Reperfusion Model Of SH-SY5Y Cells

ObjectiveIn the present study,oxygen-glucose deprivation and reperfusion?OGD/R?-treated SH-SY5Y cells were used as a cerebral ischemia/reperfusion model in vitro to investigate the role of Sestrin2 in mitochondrial fission and its possible regulatory mechanism.Methods Sestrin2?SESN2?gene overexpression lentiviral vectors were constructed to overexpress SESN2 in SH-SY5Y cell lines.Oxygen-glucose deprivation and reperfusion?OGD/R?-treated SH-SY5Y cells were used as a cerebral ischemia/reperfusion model.The experiment was divided into two parts.Cells of the first part were grouped as follows:normal control group,OGD/R group,Vector group,SESN2 overexpression group.The cells in the later three groups were subjected to oxygen-glucose deprivation?OGD?for 4h followed by restoration of O₂supply for 18h.The cell survival rate was detected by CCK-8 assay.The protein levels of SESN2,Drp1,Fis1,Bcl-2,Bax,Keap1 and Nrf2 were measured by Western blot.The ultrastructure of mitochondria was observed by transmission electron microscopy.The nuclear translocation of Nrf2 was detected by immunofluorescence staining.Cells of the second part were grouped as follows:SESN2 overexpression group,Brusatol+OE-SESN2 group,DMSO+OE-SESN2 group.The cells of these groups were subjected to oxygen-glucose deprivation?OGD?for 4h followed by restoration of O₂ supply for 18h.The cells of Brusatol+OE-SESN2 group were pretreated with normal medium containing Brusatol?final concentration 100nM?for 4h before OGD/R treatment.The cells of DMSO+OE-SESN2group were pretreated with normal medium containing DMSO?final concentration 0.1%?for4 h before OGD/R treatment.The protein levels of SESN2,Drp1,Fis1,Keap1 and Nrf2 were measured by Western blot.Results1.As compared with OGD/R group,the cell survival rate and the protein level of Bcl-2 of OE-SESN2 group were higher,while the protein level of Bax was lower.All differences were statistically significant?p<0.05?.These results point to an anti-apoptosis effect by SESN2.2.As compared with OGD/R group,the protein level of Drp1 and Fis1 of OE-SESN2 group were lower.The differences were statistically significant?p<0.05?.The mitochondrial ultrastructure of OE-SESN2 group were more intact.These findings suggested that the SESN2 overexpression may have a cytoprotective effect by suppressing mitochondrial fission.3.Immunofluorescence revealed that the nuclear translocation of Nrf2 was activated when SESN2 was overexpressed.Compared to OGD/R group,the cytosolic protein level of Keap1was lower in OE-SESN2 group,while the nuclear protein level of Nrf2 was higher.The differences were statistically significant?p<0.05?.These findings suggested that SESN2overexpression promoted the activation of Keap1/Nrf2 pathway through the downregulation of Keap1.4.Compared to OE-SESN2 group,the nuclear protein level of Nrf2 of OE-SESN2+Brusatol group was lower,while the protein level of Drp1 and Fis1 were higher.The differences were statistically significant?p<0.05?.These findings suggested that the effect of suppressing mitochondrial fission by SESN2 overexpression was reversed by Brusatol,which indicated that Keap1/Nrf2 pathway was involved in the regulation of SESN2 on mitochondrial fission.Conclusion Overexpression of SESN2 could attenuate oxygen-glucose deprivation/reperfusion?OGD/R?-induced apoptosis of SH-SY5Y cells.The mechanism of its protective effects may be suppressing the mitochondrial fission.What's more,Keap1/Nrf2 pathway was involved in the regulation of SESN2 on mitochondrial fission.