| Parkinson’s disease(PD)is a sort of the central nervous system degeneration characteristic by the significant reduction of dopamine content in striatal and the loss of dopaminergic neurons in the nigrostriatal area.Some studies have shown that Parkinson’s symptoms of the levels of striatal dopamine decreased by about 80% and dopamine neurons lost by about 50% in the substantia nigra.The main clinical features of PD are rest tremor,bradykinesia,muscle stiffness and postural reflex damage,gravely influencing the patient’s work and livelihood.In China,the prevalence of PD is1.7% in people over the age of 65,and the prevalence is similar to which of the developed countries in the world.At present,the mechanism of PD is not clear,studies have shown that PD is related to genetic,environmental factors,infection,aging,oxidative stress,free radicals,nerve growth factor deficiency and mitochondrial dysfunction and so on.PD is the consequence of diverse mechanisms synergies.In recent years,Ferroptosis is of great concern.Studies suggest that Ferroptosis may be associated with neurodegenerative diseases(Parkinson’s disease,Alzheimer’s disease,etc).In order to explore the role of Ferroptosis in PD,this study uses vitro Parkinson’s disease model builded by 1-methyl-4-pheny1-pyridinium(MPP~+)to explore whether Ferroptosis to occur and the molecular mechanisms of Ferroptosis,and in the process,the effect of Ferroptosis on the expression of DJ-1 provides some theoretical basis for the molecular mechanism of Parkinson’s disease.Objective: To investigate the occurance and molecular mechanism of MPP~+-induced Ferroptosis in PC12 cells.Methods: MTT assay to detect cell viability rate after treatment with MPP~+ and Ferrostatin-1 pretreatment with MPP~+ for different times;Leakage rate of LDH was measured using Lactate dehydrogenase(LDH)kit;The effects of pretreatment with different inhibitors on the model group,the expression of caspase-3 and the contents of glutathione(GSH)in the cells were measured by microplate reader;The autophagy rate was measured by flow cytometry after using the Mondansylcadverin(MDC)staining;H2DCF-DA staining was used to detect the production of reactive oxygen species(ROS)by flow cytometry;C11-BODIPY fluorescent probe was used to test the production of lipid ROS by flow cytometry;The activities of SOD was measured by SOD kit by microplate reader and The activities of MDA was measured by MDA kit by microplate reader;The change of the morphology of mitochondria were tested by transmission electron microscopy;Western blotting was used to detect the expression of Ferroportin1(FP1),Prostaglandin-endoperoxide synthase 2(Ptgs2),Glutathione Peroxidase 4(GPX4)and DJ-1 protein after treatment of Ferrostatin-1 in PC12 cells.Reverse transcription PCR could detect the expression of Ptgs2 and DJ-1.Results: 1.Ferrostatin-1 protected against MPP~+-induced injury.(1)MTT assay showed that MPP~+ markedly inhibited proliferation of PC12 cells(P<0.01)in a certain period of time and concentration.The cell viability was markedly increased and cell death was prevented by Ferrostatin-1(P<0.01),after using Ferrostatin-1 pretreatment.(2)The leakage of LDH distinctly increased after treatment of MPP~+ compared with that of control group(P<0.01)in PC12 cells.Ferrostatin-1 strikingly prevented MPP~+-induced increase of the level of LDH leakage(P<0.01).2.The presence of Ferroptosis in PC12 cells induced by MPP +(1)In the five inhibitors,Z-VAD-FMK,3-Methyladenine(3-MA),Ferrostatin-1 and Defetoxamine(DFO)were able to increase cell viability to a certain extent(P<0.05),whereas Necrostatin-1 did not(P>0.05).(2)MDC staining was used to detect autophagy.The results showed that MPP~+-induced autophagy in PC12 cells,and the autophagy rate was not reduced after pretreatment with Ferrostatin-1(P>0.05).(3)The expression of caspase-3 was detected by microplate reader.The results showed that the expression of caspase-3 was significantly increased after MPP~+ injured PC12cells(P<0.01);The expression of caspase-3 was not significantly decreased after pretreatment with Ferrostatin-1(P>0.05).(4)The results showed that Ferrostatin-1 could inhibit the production of cytoplasm and lipid ROS by H2DCF-DA fluorescence staining and C11-BODIPY fluorescence probe(P<0.05).When pretreatment with Ferrostatin-1 in PC12 cells,intracellular the level of MDA was markedly decreased(P<0.01),SOD activity was strikingly increased when compared with the MPP~+ group(P<0.01)and GSH level was increased(P<0.05).These results suggested that Ferrostatin-1 inhibited MPP~+-induced oxidative stress.(5)Western blot analysis turned out that the expression of FP1 was decreased in MPP~+-induced PC12 cells,while DFO increased the expression of FP1 in a dose-dependent manner(P<0.01).(6)By means of transmission electron microscope observed MPP~+-induced PC12 cell injury.It turned out that mitochondria morphology became smaller and the density of the mitochondrial membrane was increased.After the pre-protection of Ferrostatin-1,mitochondrial morphology closed to normal,the density of the mitochondrial membrane was decreased.3.Molecular mechanism of MPP~+-induced Ferroptosis in PC12 cells(1)Western blot results indicated that the expression of Ptgs2 protein was significantly increased after MPP~+-induced PC12 cells(P<0.01),and the expression of Ptgs2 protein was significantly decreased after the pre-protection of Ferrostatin-1(P<0.01).(2)Western blot results manifested that the expression of GPX4 protein was significantly decreased after MPP~+-induced PC12 cells(P<0.01),and the expression of GPX4 protein was significantly increased after the pre-protection of Ferrostatin-1(P<0.05).(3)Western blot results declared that the expression of DJ-1 protein was significantly decreased after treatment of MPP~+ in PC12 cells(P<0.01),and the expression of DJ-1protein was significantly increased after the pre-protection of Ferrostatin-1(P<0.05).(4)RT-PCR results demonstrated that the expression of Ptgs2 gene was significantly increased in PC12 cells after treatment of MPP~+(P<0.01),and the expression of Ptgs2 gene was significantly decreased after the pre-protection of Ferrostatin-1(P<0.01).(5)RT-PCR results stated that the expression of DJ-1 gene was significantly reduced after PC12 cells were dealed with MPP~+(P<0.01),and the expression of DJ-1 gene was significantly increased after the pre-protection of Ferrostatin-1(P<0.01).Conclusion: The results suggest that MPP~+ cause Ferroptosis by intracellular oxidative stress and increase iron content,and Ferrostatin-1 inhibits the occurrence of Ferroptosis.The molecular mechanism of Ferroptosis may be related to the up-regulation of Ptgs2 expression and the down-regulation of GPX4 and FP1 expression.The occurrence of Ferroptosis is closely related to the expression of DJ-1. |
