| Objective: To investigate the regulatory pathway of lipid peroxidation damage in ferroptosis and the pathogenesis of Parkinson’s disease(PD)and to analyze the protective mechanism of activating the Keap1-Nrf2-ARE pathway against ferroptosis in dopamine(DA)neurons.Methods:(1)Zebrafish model of PD: zebrafish embryos were acquired48 h after fertilization and divided into control(n=100)and two treatment groups,i.e.MPTP group(n=100,treated by 200 u M MPTP)and Nomi group(n=100,treated by200 u M MPTP+1.5 g/m L nomifensine [Nomi]).Locomotor behavioral tests(movement ability,movement velocity,and movement distance)were performed for zebrafish 5-day after fertilization.Immunofluorescence assay was used to evaluate the alpha-Synuclein(α-Syn)level and tyrosine hydroxylase(TH)expression in the brain and to assess the viability of DA neurons;iron,glutathione(GSH),and oxidation index detection kits were used to measure the levels of iron,GSH,and lipid peroxidation(lipid peroxides [LPO],catalase [CAT],MDA,reactive oxygen species [ROS],and glutathione peroxidase 4[GPX4]).Therefore,the iron deposition could induce damage to DA neurons through the metabolic regulatory mechanisms of lipid peroxidation in ferroptosis at the animal level.(2)MN9D cell model: The cells were divided into control,MPP+,and MPP+Fer groups.CCK-8 assay was used to assess the cell viability and determine the optimal concentration of MPP+ and Ferrostatin-1(Fer).The Ferro Orange fluorescence(Ferro Orange),DCFH-DA(DCFH-DA),and Liperfluo probes(Liperfluo)were used to measure the cellular iron,ROS,and lipid peroxidation levels.Western blotting was used to analyze the levels of GAPDH,ACSL4,GPX4 and α-Syn.GSH and MDA kits were used to measure the GSH and MDA levels.Therefore,whether iron deposition could induce the DA neuron damage through the metabolic regulatory mechanisms of lipid peroxidation in ferroptosis was confirmed at the cellular level.(3)MN9D cell model: The cells were divided into control group(Control),MPP+control group(MPP+Control),p62 overexpression group(MPP+OV-p62),and p62 overexpression empty group(MPP+OV-p62-NC).Inhibitors Brusatol and Zn PP inhibited the activation of Nrf2 and HO-1 and the inhibitors were divided into Brusatol group(MPP+OV-p62+Brusatol)and Zn PP group(MPP+OV-p62+Zn PP).RT-q PCR was used to detect transfection efficiency of cells in each group,and CCK8 was used to detect cell activity.Ferro Orange,DCFH-DA,and Liperfluo probes were used to detect intracellular iron,reactive oxygen species,and lipid peroxidation levels.Western blotting was used to detect the levels of Nrf2,HO-1,Keap1 and their downstream GPX4,ACSL4.GSH and MDA kits were used to measure the GSH and MDA levels.Cell level validation demonstrated that activation of the Keap1-Nrf2-ARE pathway has antioxidant protection against ferroptosis in DA neurons.Results:(1)(1)Locomotor behavioral tests of PD zebrafish showed that compared to the control group,larval fish in the MPTP group had reduced movement ability,velocity,and distance.However,Nomi treatment of MPTP-treated fish could restore the movement ability and velocity(P<0.05).Immunofluorescence assay showed that compared to the control group,substantial DA neuron damages were detected in the MPTP group,while Nomi treatment could partially rescue the MPTP-induced DA neuron damages.No signal of(α-Syn)was detected in the zebrafish,which could be associated with the non-specificity of the antibody in zebrafish.(2)The detection results of ferroptosis related indicators in zebrafish showed that compared with the normal control group,the content of GSH and GPX4 were decreased in the MPTP group,while compared with the MPTP group,the content of GSH and GPX4 were increased in the MPTP+Nomi group,with a statistical difference among the three groups(P<0.05).Compared with the normal control group,the content of iron and LPO werer increased in the MPTP group,while compared with the MPTP group,the content of iron and LPO weredecreased in the MPTP+Nomi group,with a statistical difference among the three groups(P<0.05).Compared with the normal control group,the MDA content was increased in the MPTP group,while compared with the MPTP group,the MDA content was decreased in the MPTP+Nomi group(P>0.05).There was no significant change in CAT content in the normal control group,MPTP group,and MPTP+Nomi group(P>0.05).In the normal control,MPTP,and MPTP+Nomi groups,the proportion of ROS positive cells was 31.6%,46.2%,and 31.3%,respectively.(2)(1)MN9D cells were activated by MPP+,and then the viability of cells at the concentration of MPP+ of 2.5,5,and 10 mmol/L was evaluated by CCK-8 assay.The findings showed that cell viability was approximately 50% when the concentration of MPP+ was 5 mmol/L,which was selected as the concentration for the subsequent experiments.Fer,a ferroptosis inhibitor,was used to treat the cells at the concentrations of 1,2,4,6,8,and 10μm,which showed that 10μm Fer inhibits cell death and increases cell viability.Therefore,this concentration was selected for the subsequent experiments.(2)The immunofluorescence probe flow cytometry results showed that compared with the normal control group,the content of three probes in the MPP+model group were significantly increased(P<0.01),While compared with the MPP+group,the content of Ferro Orange and Liperfluo were decreased(P<0.001)and the content of DCFH-DAwere decreased(P>0.05)in the MPP+Fer group,indicating that the levels of intracellular iron,reactive oxygen species,and lipid peroxidation in the MPP+group were increased,while the levels of intracellular iron,reactive oxygen species,and peroxides in the Fer inhibitor group were decreased.(3)Western blotting results showed that compared with the normal group,α-Syn and ACSL4 proteins were upregulated in MPP+model group,while GPX4 protein was downregulated in MPP+model group(P<0.01);Compared to the MPP+model group,α-Syn and ACSL4 proteins in the MPP+Fer group were down regulated,while the expression of GPX4 protein in the MPP+Fer group was up regulated,with a statistically significant difference(P<0.01).(4)The results of GSH and MDA kit showed that compared with the normal control group,the content of GSH was significantly decreased(P<0.01)but the content of MDA was increased in the MPP+model group(P<0.001).Compared with MPP+model group,the content of GSH was increased(P>0.05)but the content of MDA was decreased in MPP+Fer group(P<0.01).(3)(1)The results of RT-q PCR showed that compared with the control group,the expression of p62 gene in the p62 overexpression group was significantly increased,and the expression of p62 gene in the Brusatol group and Zn PP group was significantly increased,indicating that the transfection was successful(P < 0.05).(2)The immunofluorescence probe flow cytometry results showed that compared with the normal control group,the content of the three probes were significantly increased in the MPP+model group(P<0.01),and compared with the MPP+model group,the content of the three probes were decreased in the P62 overexpression group(P<0.01),indicating that the levels of intracellular iron,reactive oxygen species,and lipid peroxidation were increased in the MPP+group,while the levels of intracellular iron,reactive oxygen species,and lipid peroxidation were decreased in the P62 overexpression group.(3)Compared with the control group,the expression of Nrf2,HO-1and GPX4 were increased in the MPP+p62 overexpression group(P<0.05),while the expression of Keap1 and ACSL4 were decreased in the MPP+p62 overexpression group(P<0.05).Compared with the MPP+control group,Nrf2 and GPX4 were increased in the MPP+p62 overexpression group,while ACSL4 was decreased in the MPP+p62 overexpression group(P<0.05).The GSH and MDA kit results showed that compared with the normal control group,the content of GSH was significantly lower,while the content of MDA was higher in the MPP+control group.Compared with MPP+model group,the content of GSH was higher,while the content of MDA was lower in MPP+p62 overexpression group(P<0.01).Compared with the MPP+p62 overexpression group,The content of Nrf2,HO-1,and GPX4 were decreased in the Brusatol and Zn PP groups(P<0.001).Conclusion: On the basis of MPTP induced zebrafish and MPP+induced MN9 D cells,it was found that there was ferroptosis in PD zebrafish and dopaminergic MN9 D cells,which was related to lipid peroxidation accumulation.It has been verified from animal and cellular levels that the damage of DA neurons caused by iron deposition which is related to the lipid peroxidation metabolic mechanism of ferroptosis.Based on the transfection of P62 plasmid,it was found that P62 plasmid can inhibit the lipid peroxidation of ferroptosis in dopaminergic nerve cells by activating the Nrf2 signaling pathway,thereby exerting a protective effect on DA neurons. |
PDF Full Text Request