Hydrogen Sulfide Inhibits Formaldehyde-induced Ferritinophagy-ferroptosis In HT22 Cell

[Background and Objective]Formaldehyde（FA）has neurotoxicity.Our previous studies have shown that hydrogen sulfide（H₂S）antagonizes FA-induced neurotoxicity,but the mechanisms underlying the protection of H₂S against FA-induced neurotoxicity are poorly understand.Resently,our laboratory found that FA-induced neurotoxicity is closely related to ferroptosis.Ferritin is an important iron storage protein in organism,which can release iron through ferritinophagy.However,excessive ferritinophagy induces ferroptosis.Therefore,we will explore the protective mechanism of H₂S against FA-induced neurotoxicity from the ferritinophagy-ferroptosis pathway.[Methods]Analysis of co-localization of NCOA4 with LC3B or FTH1 used fluorescence double labeling;Fluorescence microscope observed GFP-LC3 spot in HT22 cells transfected with mRFP-GFP-LC3;The cell viability in HT22 cells was detected by trypan blue staining;The cell death was detected by FITC/PI or 7AAD/PE stained flow cytometry;The contents of MDA,GSH,and 4-HNE in HT22 cells were detected by Enzyme-linked immunosorbent assay（Elisa）;The contents of Lipid oxygen were detected by BOOIPYTM C11;The content of Fe²⁺in HT22cells was evaluated by Ca-AM and DFO;The expressions of ferritinophagy associated proteins（LC3II/I,p62/SQSTMI,FTH1,and NCOA4）were detected by Western Blot.[Results]1.FA induced ferroptosis in HT22 cellsTreatment of HT22 cells with FA（50,100,and 200μM,24 h）significantly increased the death rate,decreased the intracellular GSH content,increased the intracellular contents of lipid ROS,MDA,4-HNE,and free Fe²⁺,suggesting that FA causes ferroptosis in HT22 cells.2.DFO prevented FA-induced ferroptosis in HT22 cellsThe pre-treatment with DFO（10μM,30 min）eliminated FA（100μM,24 h）-induced the increase in the death rate,decrease in the intracellular GSH content,and increases in the contents of lipid ROS,MDA,4-HNE,and free Fe²⁺in the HT22 cells,which indicated that the excessive release of iron mediates FA-induce ferroptosis in HT22 cells.3.FA induced ferritinophagy in HT22 cells.Treatment of HT22 cells with FA（50,100,and 200μM,24 h）notably promoted the co-localization of NCOA4 with LC3B or FTH1,increased the fluorescence spot of mRFP-GFP-LC3,upregulated the expressions of autophagy-associated protein LC3II/I and p62,decreased the expression of FTH1 and upregulated the expression of NCOA4,which indicated that FA induces ferritinophagy in HT22 cells.4.NCOA4 shRNA reverses FA-induced ferroptosis in HT22 cells.The pre-treatment with NCOA4 shRNA eliminated FA（100μM,24h）-induced increase in the death rate,decrease in the intracellular GSH content,and increases in the contents of lipid ROS,MDA,4-HNE,and free Fe²⁺in HT22 cells,which indicated that FA-induced ferroptosis is mediated by ferritinophagy.5.H₂S prevents FA-induced ferritinophagy in HT22 cells.Pretreatment H₂S（100,200,and 400μM,for 30 min）cancelled FA（100μM,24 h）-induced co-localization of NCOA4 with LC3B or FTH1,increase in the fluorescence spot of mRFP-GFP-LC3,upregulation of LC3II/I and p62 protein,decrease in the expression of FTH1,and upregulation of NCOA4 expression in HT22 cells,which indicated that H₂S antagonizes FA-induced ferritinophagy in HT22 cells.6.H₂S alleviates FA-induced ferroptosis in HT22 cellsPretreatment H₂S（100,200,and 400μM,for 30 min）inhibited FA（100μM,24 h）-induced increase in the death rate,decrease in the intracellular GSH content,and increases in the contents of lipid ROS,MDA,4-HNE,and free Fe²⁺in HT22 cells,which indicated that H₂S antagonizes FA-induced ferroptosis in HT22 cells.[Conclusion]1.FA induces ferroptosis in HT22 cells via promoting ferritinophagy.2.H₂S inhibits FA-induced ferritinophagy-ferroptosis in HT22 cells.