The Effect Of Ferritin And Apoferritin In The Protection Against MPP~+-Induced Neurotoxicity In MES 23.5 Cells

Parkinson's disease?PD?is a common neurodegenerative disease,which is characterized by the progressive degeneration of dopamine?DA?neurons in the substantia nigra pars compacta?SNpc?and accompanied by the aggregation of?-synuclein and formation of Lewy bodies.The etiology of PD has not yet been fully clarified until now.It has been reported that the iron levels were significantly increased in the substantia nigra?SN?of PD patients and animal models,and iron levels in the SN were associated with the progression of PD.The disorder of iron metabolism and iron-mediated oxidative stress might be an important pathological mechanism in the degeneration of DA neurons.Ferritin is a large hollow,globular protein composed of two subunits?H and L?,which can store up to 4500 iron atoms in its cavity.H-subunit has ferroxidase activity and is responsible for converting soluble ferrous iron to the storable ferric form.L-subunit contains nucleation sites whose main function is to promote iron mineralization and nuclear formation.There are mainly two forms of ferritin:apoferritin and ferritin.Ferritin plays an important role in maintaining iron homeostasis.Studies have confirmed that there was a large amount of iron deposition in the SNpc of PD patients,while the level of ferritin didn't increase.This might contribute to the increased vulnerability of DA neurons to oxidative stress damage in the SN.Studies have shown that astrocytes express high levels of ferritin and could secrete ferritin to the outside of the cell.However,the role of secreted ferritin in DA neurons and its possible mechanisms in PD are unclear.In this study,we established the PD cell model using 1-methyl-4-phenylpyridinium ion?MPP⁺?,investigated the effect of FAC on ferritin release in astrocytes and observed the effect of exogenous ferritin on MPP⁺-induced cell damage in MES23.5 dopaminergic cells using iron assay kit,ELISA kit,MTT,CCK-8,flow cytometry,western blots and other methods.The results were shown as follows:1.The release of ferritin was significantly increased in primary cultured astrocytes with100?mol/L FAC or FAC+MPP⁺treatment for 24 h,compared with the control group?P<0.001?.And the release of ferritin in FAC+MPP⁺group was decreased compared with FAC group?P<0.001?.2.Content of total Fe,Fe²⁺and Fe³⁺in the culture media of astrocytes and astrocytes were all increased significantly with 100?mol/L FAC treatment for 24 h,compared with the control group?P<0.05?.Content of total Fe and Fe³⁺in astrocytes culture media with FAC treatment was significantly decreased compared with the cell-free FAC treatment group?P<0.01?.3.After treatment with 50,100,150,200,300?mol/L MPP⁺for 24 h,the cell viability of MES23.5 cells decreased significantly compared with the control group?P<0.001?.100?mol/L MPP⁺was selected as the optimum modeling concentration for subsequent experiments.4.After treatment with 50?g/ml or 100?g/ml Apoferritin for 24 h,there was no difference in the mitochondrial transmembrane potential???m?between Apoferritin treated group and the control group in MES 23.5 cells.After treatment with different concentrations of Ferritin for 24 h,there was no difference in the??m between 80?g/ml Ferritin group and the control group.The??m in 100?g/ml Ferritin group was decreased compared with the control group?P<0.01?.50?g/ml Apoferritin and 80?g/ml ferritin were selected as the optimum concentration for subsequent experiments.5.The expression of H-Ferritin in MES23.5 cells treated with Apoferritin or Ferritin was increased significantly,compared with the control group?P<0.05?.6.The cell viability in MPP⁺-treated MES23.5 cells decreased significantly compared with the control?P<0.001?.Apoferritin or Ferritin pretreatment could antagonize MPP⁺-induced decrease of cell viability?P<0.001?.7.MPP⁺treatment could decrease??m of MES23.5 cells?P<0.001?,which could be antagonized by Apoferritin or Ferritin?P<0.001?.8.The ROS level was increased in MPP⁺-treated MES23.5 cells,compared with the control?P<0.001?.Apoferritin or Ferritin pretreatment could inhibit MPP⁺-induced increase of ROS level?P<0.05?.9.The fluorescence intensity of Calcein-AM was decreased in MPP⁺-treated MES23.5cells,compared with the control,which meant the higher LIP level in MPP⁺-treated MES23.5 cells than the control?P<0.001?.Apoferritin or Ferritin pretreatment could antagonize MPP⁺-induced increase of LIP level?P<0.05?.10.LIP level was significant increased in MPP⁺+FAC treated MES23.5 cells,compared with the control?P<0.001?.Apoferritin or Ferritin pretreatment could antagonize MPP⁺+FAC-induced increase of LIP level?P<0.01?.In summary,astrocytes might reduce extracellular iron levels by releasing more Ferritin,which could combine extracellular iron under iron overload condition.In addition,after treatment with MPP⁺in MES23.5 dopaminergic cells,cell viability and??m decreased,and ROS levels increased significantly.Apoferritin or Ferritin pretreatment could protect against MPP⁺-induced cell damage by restoring the cell viability,??m,and decreasing ROS level in MES23.5 cells.Furthermore,Apoferritin or Ferritin might also suppress MPP⁺or MPP⁺+FAC-induced increase of the LIP level by binding with intracellular iron,thereby protecting MES23.5 dopaminergic cells.These results suggest that the imbalance of ferritin release might be involved in nigral iron accumulation and iron-mediated damage to DA neurons in PD.Regulating ferritin levels might be a new strategy for the treatment of PD.