Protective effect of transcription factor POU2F2 on ferroptosis in post-ischemic stroke epilepsy by activating Sestrin2 and the underlying mechanism
Objective To investigate the effect of knockdown of POU transcription factor family level 2 homology frame 2(POU2F2)on post-ischemic stroke epilepsy(PISE)and the underlying mechanism.Methods Binding sites in the promoter regions of POU2F2 and Sestrin2(Sesn2)were predicted using bioinformatics methods.Male Wistar rats were randomly divided into Sham,PISE,PISE+LV-shNC and PISE+LV-shPOU2F2 groups,and a four-vessel occlusion(4-VO)method was used to establish the PISE model.Primary neurons were isolated from the cerebral cortex of healthy rats.The in vitro PISE model was constructed by magnesium-free treatment,and cells were treated with blank control,PISE,PISE+shNC transfection,PISE+shPOU2F2 transfection and PISE+shPOU2F2 transfection+Sesn2 groups.Quantitative real-time polymerase chain reaction(qRT-PCR)was performed to detect mRNA levels of POU2F2 and Sesn2.Western blot was used to detect protein levels of POU2F2,Sesn2 and glutathione peroxidase 4(GPX4).Reactive oxygen species(ROS),lipid ROS,superoxide dismutase(SOD),malondialdehyde(MDA),glutathione(GSH)and lactate dehydrogenase(LDH)levels were measured using commercial kits.The effect of POU2F2 on the promoter transcriptional activity of Sesn2 was examined by dual-luciferase reporter assay and chromatin immunoprecipitation(ChIP).Results Using the JASPAR database,there were binding sites in the Sesn2 promoter region for the transcription factor POU2F2.The results of qRT-PCR and Western blot showed mRNA and protein expressions of POU2F2 and Sesn2 were significantly higher in rats and primary cortical neurons of PISE group than those of control group(P＜0.05).The expression of POU2F2 and Sesn2 mRNA and protein was decreased in the PISE+shPOU2F2 group compared with the control group(P＜0.05).Compared with the control group,brain tissue ROS production,primary cortical neuron lipid ROS accumulation,LDH activity and MDA content were significantly increased,and GSH content and GPX4 protein expression were significantly decreased in the PISE group.Compared with the PISE+shNC group,silencing of POU2F2 further increased brain tissue ROS production and primary cortical neuron lipid ROS accumulation,LDH activity and MDA content,and decreased GSH content and GPX4 protein expression(P＜0.05).Dual luciferase reporter gene technology and ChIP assay confirmed that POU2F2 regulates the activity of Sesn2 promoter.After overexpression of Sesn2,neuronal lipid ROS accumulation,LDH activity and MDA content were decreased and GSH activity was increased in the PISE+shPOU2F2+Sesn2 group compared with the PISE+shPOU2F2 group(P＜0.05).Conclusion PISE induces compensatory increases in POU2F2 and Sesn2 levels,and knockdown of POU2F2 exacerbates PISE by downregulating Sesn2 to promote ferroptosis,which provides a new perspective on the therapeutic mechanism of PISE.
POU transcription factor family level 2 homology frame 2(POU2F2)post-ischemic stroke epilepsySestrin2(Sesn2)ferroptosis
万方数据
维普
