Formaldehyde Induces Ferroptosis In HT22 Cells By Upregulation Of Warburg Effect

[Background and Objective]Formaldehyde(FA)induces neurotoxicity,which participates in the occurrence of neurodegenerative diseases.However,its mechanisms were not yet clarified.Ferroptosis,as a new-regulatory cell death,is closely associated with neurodegenerative diseases.Warburg effect,known as aerobic glycolysis,exists in brain tissue and involves in regulating nerve function.In this study,we used mouse hippocampal neurons HT22 cells to investigate whether FA induces neuronal ferroptosis and determine whether Warburg effect mediates FA-induced ferroptosis in HT22 cells.[Methods]1.The cell viability in HT22 cells was detected by trypan blue staining.2.The cell mortality rate in HT22 cells was evaluated by flow cytometry(FCM)after Annexin/PI staining.3.The contents of GSH,MDA,4-HNE in HT22 cells were detected by Enzyme-linked immunosorbent assay(Elisa assay).4.The level of reactive oxygen species(ROS)in HT22 cells was evaluated by FCM after dichlorofluorescin diacetate(DCFDA)staining.5.The content of Fe2+ in HT22 cells was evaluated by FCM after calcein-acetoxymethlester(Ca-AM)staining.6.The content of lactate in HT22 cells was detected using Lactate Assay Kit II.7.Warburg effect-associated proteins in HT22 cells,including Pyruvate kinase M2(PKM2),Pyruvate dehydrogenase kinase 1(PDK-1),Pyruvate dehydrogenase(PDH)and Lactate dehydrogenase(LDHA),were detected by Western Bloting.[Results] 1.Formaldehyde induces ferroptosis in HT22 cellAfter treatment of HT22 cells with FA(0.1,0.5,1.0 m M)for 6 h,the cell viability of HT22 cells was obviously decreased and the cell mortality of HT22 cells was significantly increased.After treatment of HT22 cells with FA(0.1,0.5,1.0 m M)for 6 h,the content of GSH in HT22 cells was decreased as well as the contents of MDA,4-HNE,and ROS in HT22 cells were increased.After treatment of HT22 cells with FA(0.1,0.5,1.0 m M)for 6 h,the content of Fe2+ in HT22 cells was significantly increased.2.Formaldehyde upreglulates warburg effect in HT22 cellsTreatment of HT22 cells with FA(0.1,0.5,1.0 m M,6 h)up-regulated the expressions of PKM2,PDK-1,and LDHA,down-regulated the expression of PDH,and increased the production of Lactate.3.DCA,an inhibitor of warburg effect,suppresses formaldehyde-induced ferroptosis in HT22 cellsPretreatment with DCA(20 m M,12 h)inhibited FA-induced decrease in the cell viability of HT22 cells and increase in the cell mortality of HT22 cells.Pretreatment of HT22 cells with DCA(20 m M,12 h)blocked FA-decreased GSH content as well as FA-increased MDA,4-HNE,and ROS level in HT22 cells.After pretreatment of HT22 cells with DCA(20 m M,12 h),FA-induced increase in Fe2+ content in HT22 cells was inhibited.[Conclusion]Formaldehyde induces ferroptosis in HT22 cells by upreglulation of Warburg effect.