The Role And Molecular Mechanism Of Ferroptosis In TOCP-induced Toxicity Of Human Neuroblastoma SK-N-SH Cells

Objective: Combining with cell in vitro experiments to explore the role and mechanism of ferroptosis in TOCP-induced neuronal toxicity.Methods: 1.Using human neuroblastoma SK-N-SH cells as the research model.The cells were treated with gradient concentration TOCP alone for 48 h or pretreatment with ferroptosis inhibitors DFO(100 μM),Fer-1(10 μM)for 6 h before TOCP(0.75 m M)treated for 48 h.Then cell viability of SK-N-SH was detected by using the CCK-8 method;2.Flow cytometry combined with an inverted fluorescence microscope was used to detect the lipid peroxidation status and mitochondrial membrane potential in SK-N-SH cells after TOCP exposure;3.Using a fluorescence spectrophotometer to detect the content of MDA in SK-N-SH cells after TOCP treatment,and determine the content of SOD and GSH-GSSG by microplate reader method;4.Western Blot combined with immunofluorescence to detect the protein expression of SLC7A11,GPX4,ACSL4,FTH1,NCOA4,LC3II/I,P62,etc.5.Giemsa staining was used to observe the cell morphology after TOCP treatment,and the ultrastructure of cells and mitochondria was observed by transmission electron microscope(TEM).Results: 1.Compared with the control group,ferroptosis inhibitors DFO(100 μM)and Fer-1(10 μM)can prevent the TOCP-induced decrease in SK-N-SH cell viability.2.TOCP exposure can change the morphology of SK-N-SH cells,shrink the cytoplasm,reduce the nucleus,damage the morphology and function of mitochondria,cause the mitochondria to shrink and round,the cristae disappear or decrease,the formation of dense electronic clumps,and the membrane Rupture and decrease of mitochondrial membrane potential.3.TOCP can induce lipid peroxidation in SK-N-SH cells,reduce the ratio of SOD,GSH/GSSG,and increase the content of MDA.The pretreatment of ferroptosis inhibitors DFO(100 μM)and Fer-1(10 μM)can reduce Its degree of lipid peroxidation.4.TOCP exposure can generate a large amount of ROS and attack PUFAs on the membrane,causing lipid peroxidation of PUFAs.At the same time,TOCP can activate ACSL4,synthesize more PUFAs,and promote the formation of lipid peroxides in SK-N-SH cells.Pretreatment with ferroptosis inhibitors DFO(100 μM)and Fer-1(10 μM)can reduce the expression of ACSL4.5.TOCP can activate NCOA4 and P62,promote the binding of FTH1 and LC3 II and then transfer the complex to autophagosomes and lysosomes,where autophagy degradation of ferritin occurs,and the stored ferrous ions are released to disrupt the iron homeostasis balance of SK-N-SH cells.This process is also known as ferritinophagy,which can intensify intracellular oxidative stress and lipid peroxidation.However,pretreatment with ferroptosis inhibitors DFO(100 μM)and Fer-1(10 μM)can alleviate the ferritinophagy process.6.TOCP inhibited the transport of cystine by x CT by inhibiting the expression of SLC7A11,resulting in insufficient raw materials for the synthesis of GSH and GPX4,resulting in a decrease in the ability of GPX4 to remove lipid peroxides,leading to ferroptosis in SK-N-SH cells,and ferroptosis inhibitors DFO(100 μM)and Fer-1(10 μM)pretreatment can suppress this trend.Conclusion.TOCP can induce SK-N-SH cell ferroptosis by promoting the ferritinophagy process.