Damaging Effect Of Iron Overload On MO3.13 Oligodendrocytes And Its Possible Mechanism

Objective:Parkinson’s disease(PD)is a common neurodegenerative disease,and its main pathological feature is the selective loss of dopaminergic neurons in the substantia nigra(SN).The etiology and mechanism of PD are not yet clear,but iron deposition is a key factor in the pathogenesis of PD.Iron plays important roles in the central nervous system,including myelin production,mitochondrial respiration,neurotransmission and metabolism,DNA synthesis,and more.When iron is present in excess in cells and tissues,it disrupts redox homeostasis and catalyzes excessive production of reactive oxygen species,leading to oxidative stress.In the central nervous system,oligodendrocytes require sufficient iron to form myelin sheaths and are the most iron-rich cells in the brain.Studies have shown that oligodendrocytes re very sensitive to oxidative stress,and iron load is more likely to cause damage to oligodendrocytes,and PD patients have demyelinating changes in the brain.However,the damaging effect of iron loading on oligodendrocytes and its possible mechanism remain unclear.The purpose of this study was to investigate the damaging effect and mechanism of ferric ammonium citrate(FAC)on MO3.13 oligodendrocytes.Methods:The viability of MO3.13 oligodendrocytes was detected by Cell Counting Kit-8(CCK8)kit,the expression changes of ferroptosis-related proteins and apoptosis-related proteins in cells were detected by western blot,and the levels of reactive oxygen species in cells were detected by flow cytometry.Iron assay kits were used to detect changes in intracellular iron content,and transmission electron microscopy was used to observe changes in organelle morphology.Results:1.Damaging effect of iron overload on undifferentiated MO3.13 oligodendrocytes(1)The effects of different concentrations of FAC on the viability of undifferentiated MO3.13 oligodendrocytes were detected by CCK8.Undifferentiated MO3.13 oligodendrocytes were treated with 20 mg/L,50 mg/L,100 mg/L,200 mg/L,500 mg/L,1000 mg/L,2000 mg/L FAC for 24 hours,and the cell viability did not change significantly at 20 mg/L,50 mg/L,100 mg/L concentrations,but the cell viability decreased significantly when treated with FAC of 200 mg/L,500 mg/L,1000 mg/L and 2000 mg/L(P < 0.001).In this experiment,FAC concentrations of 100 mg/L,200 mg/L and 500 mg/L were selected for subsequent experiments.(2)Undifferentiated MO3.13 oligodendrocytes were treated with 100 mg/L,200 mg/L and 500 mg/L FAC for 24 hours.The total iron concentration increased in 100 mg/L,200mg/L,and 500 mg/L FAC treatment groups compared with the control group(P < 0.001),total iron concentration increased in 500 mg/L FAC treatment group compared with the 100mg/L or 200 mg/L FAC treatment group(P < 0.001).The ferrous iron ion concentration increased in 100 mg/L,200 mg/L,and 500 mg/L FAC treatment groups compared with the control group(P < 0.001),ferrous iron ion concentration increased in 500 mg/L FAC treatment group compared with the 100 mg/L or 200 mg/L FAC treatment group(P < 0.001,P < 0.01).The ferric iron ion concentration increased in 100 mg/L,200 mg/L,and 500mg/L FAC treatment groups compared with the control group(P < 0.05,P < 0.001,P <0.001),ferric iron ion concentration increased in 500 mg/L FAC treatment group compared with the 100 mg/L or 200 mg/L FAC treatment group(P < 0.001).(3)Undifferentiated MO3.13 oligodendrocytes were treated with 100 mg/L,200 mg/L and 500 mg/L FAC and 20 μmol/L erastin for 24 hours,and the ferroptosis inducer erastin was used as positive control.The expression of Glutathione peroxidase 4(GPX4)in undifferentiated MO3.13 cells decreased significantly when treated with 100 mg/L,200mg/L,500 mg/L FAC and erastin compared with the control group(P < 0.05,P < 0.01,P<0.05,P < 0.001).(4)Undifferentiated MO3.13 oligodendrocytes were treated with 100 mg/L,200 mg/L,500 mg/L FAC and 20 μmol/L erastin for 24 hours,the intracellular reactive oxygen species increased significantly compared with the control group(P < 0.05,P < 0.05,P < 0.001,P< 0.01).(5)Undifferentiated MO3.13 oligodendrocytes were treated with 100 mg/L,200 mg/L,500 mg/L FAC and 20 μmol/L erastin for 24 hours,the intracellular mitochondria showed increased membrane density and decreased mitochondrial cristae to varying degrees.The mitochondrial length of the 100 mg/L,200 mg/L,500 mg/L FAC and erastin groups decreased,compared with the control group(P < 0.01,P < 0.001,P < 0.001,P < 0.001).The mitochondrial length of the 200 mg/L,500 mg/L FAC and erastin groups decreased,compared with the 100 mg/L FAC treatment group(P < 0.001,P < 0.001,P < 0.01).(6)Undifferentiated MO3.13 oligodendrocytes were treated with 100 mg/L,200 mg/L,500 mg/L FAC and 100 nmol/L staurosporine for 24 hours,staurosporine,an inducer of apoptosis was used as positive control.The expressions of Bcl-2/Bax and cleaved caspase-3 in undifferentiated MO3.13 oligodendrocytes treated with 100 mg/L,200 mg/L and 500mg/L FAC had no significant changes compared with the control group.The expression of cleaved caspase-3 increased in staurosporine treated cells compared with the control group(P < 0.001).(7)Undifferentiated MO3.13 oligodendrocytes were pretreated with 200 μmol/L Deferoxamine(DFO)for one hour,and then treated with 100 mg/L,200 mg/L and 500mg/L FAC respectively 24 hours,GPX4 expression increased in DFO and FAC treated group compared with sole FAC treatment group(P < 0.05).(8)Undifferentiated MO3.13 oligodendrocytes were pretreated with 10 μmol/L Ferrostatin-1(Fer-1)for one hour,and then treated with 100 mg/L,200 mg/L and 500 mg/L FAC respectively 24 hours.GPX4 expression increased in Fer-1 and FAC treated group compared with sole FAC treatment group(P < 0.05).(9)The undifferentiated MO3.13 oligodendrocytes were treated with 100 mg/L,200mg/L and 500 mg/L FAC for 24 hours.The phosphorylation level of p53 was not changed when cells were treated with 100 mg/L,200 mg/L and 500 mg/L FAC,compared with control group.The phosphorylation level of extracellular signal-regulated kinases 1 and 2(Erk1/2)was markedly increased when cells were treated with 100 mg/L,200 mg/L and500 mg/L FAC,compared with the control group(P < 0.05,P < 0.001,P < 0.001).The ratio of protein expressions of p-Erk1/2/Erk1/2 increased when cells were treated with 200mg/L and 500 mg/L FAC compared with 100 mg/L FAC treatment group(P < 0.05).2.Damaging effect of iron overload on differentiated MO3.13 oligodendrocytes(1)Undifferentiated MO3.13 oligodendrocytes were treated with 4-b-phorbol-12-myristate-13-acetate for 7 days and differentiated into mature oligodendrocytes,100 mg/L,200 mg/L and 500 mg/L FAC were treated in differentiated MO3.13 oligodendrocytes for24 hours.There was no significant change in GPX4 expression when differentiated MO3.13 oligodendrocytes were treated with FAC at concentrations of 100 mg/L and 200 mg/L,compared with the control group.While the expression of GPX4 was significantly decreased when differentiated MO3.13 oligodendrocytes were treated with 500 mg/L FAC compared with control group(P < 0.01).(2)Differentiated MO3.13 oligodendrocytes with 100 mg/L,200 mg/L and 500 mg/L FAC treated for 24 hours.The expressions of Bax/Bcl-2 protein levels did not change in differentiated MO3.13 oligodendrocytes treated with 100 mg/L,200 mg/L and 500 mg/L FAC compared with the control group.The expressions of cleaved caspase-3 did not change in differentiated MO3.13 oligodendrocytes treated with 100 mg/L,200 mg/L FAC compared with the control group.The expression of cleaved caspase-3 decreased when differentiated MO3.13 oligodendrocytes were treated with 500 mg/L FAC(P < 0.05).Conclusion:Iron overload causes ferroptosis but does not induce apoptosis in oligodendrocytes.The iron chelator DFO and the ferroptosis inhibitor Fer-1 could inhibit the ferroptosis of oligodendrocytes induced by ir on overload.The mechanism of ferroptosis in oligodendrocytes induced by iron overload may be related to Mitogen-activated protein kinase pathway activation rather than p53 pathway activation.This study provides a reliable experimental basis for understanding the mechanism of iron load-induced oligodendrocyte injury and provides new ideas for the treatment of PD demyelination.