The Protective Effect Of GPX4 On Dopaminergic Neurons By Suppressing Ferroptosis And Pyroptosis

Backgroud:Parkinson’s disease(PD)is a common age-related neurodegenerative disease,the prevalence of which increases with the aging of the population,accounting for about1%of the population over the age of 60.Gpx4 is a selenium-containing antioxidant in the glutathione peroxidases(GPXs)family.Previous studies have shown that Gpx4plays an important role in the occurrence and development of Parkinson’s disease.However,there is no clear study on whether Gpx4 protect dopaminergic neurons by inhibiting ferroptosis and pyroptosis through a common pathway?Therefore,this study will firstly detect the expression of ferroptosis and pyroptosis-related factors in early Parkinson’s disease patients.Then,experiments in vitro and in vivo was used to explore whether GPX4 is involved in inhibiting the ferroptosis and pyroptosis pathways in Parkinson’s disease.The protective effect is expected to provide new targets and follow-up experimental basis for the treatment of Parkinson’s disease.Method:In this study,firstly,peripheral blood samples from healthy controls(n=8 cases)and early PD patients(n=17 cases)were collected,and q PCR,ELISA and Western blot(WB)were used to detect the changes of gene transcription level and protein expression level of GPX4 and pyroptosis-related factors.And statistical comparisons were performed.Using the GSE7621 database,the expression differences in the substantia nigra tissue of normal and postmortem patients with Parkinson’s disease were analyzed for clinical validation.Then,by constructing plasmids to overexpress and inhibit the expression of Gpx4,MPP~+induced SH-SY5Y into PD cells in vitro Model(MPP~++SH-SY5Y),and the PD cell model was treated with Fer-1 inhibiting ferroptosis and TETD inhibiting pyroptosis detected by cell apoptosis,cell activity,cell mitochondrial membrane status,lipid peroxidation degree,reactive oxygen species(ROS)level.Finally,the PD subacute mouse model established by MPTP induction was treated with Fer-1 and TETD to detect changes in the Behavior Experiment(Open Filed Test,Rotarod Test,Pole test),Nissl staining,immunohistochemistry,related protein expression levels and other indicators,to clarify the effect of inhibiting ferroptosis and pyroptosis on PD dopaminergic neurons.Results:The results of the study found thatα-syn,NLRP3,GSDMD,and IL-1βwere increased at the gene level of leukocytes in the peripheral blood of early Parkinson’s disease,and GPX4 and caspase-1 were decreased.Compared to control group,NLRP3,IL-1βgene expression was statistically significant(p<0.05)in the detection of gene levels in early Parkinson’s disease patients.In the detection of protein levels,it was found thatα-syn,NLRP3,IL-1βincreased,and GPX4 decreased in early PD patients.Compared with the control group,the protein expressions of GPX4,NLRP3,and IL-1βwere statistically significant(p<0.05)in early PD group.Through re-clustering mapping and heat map difference analysis,it was found that ferroptosis and pyroptosis-related genes have significant expression differences between HC and PD group(p<0.05).On the basis of PD cell model,overexpressed and knocked down of GPX4 were treated by the inhibitors Fer-1 and TETD,respectively.It was found that GPX4overexpression,Fer-1,and TETD all could reduce the apoptosis and enhance the Cell viability,improved mitochondrial membrane potential,reduced lipid peroxidation,and suppress ROS overproduction(p<0.05)in PD cell model.WB analysis showed that the change of GPX4 level could affect the level of NLRP1/NLRP3-caspase-1 and GSDMD,as well as the level of intracellularα-synuclein.Correspondingly,the apparent cell function changes of PD cell model which were treated by ferroptosis inhibitor Fer-1and pyroptosis inhibitor TETD were basically consistent with those of previous PD cell model in order to maintain cell viability.In MPTP-induced PD mouse model,both Fer-1 and TETD can improve motor dysfunction,depression and anxiety symptoms of PD.Fer-1 and TETD can significantly increase Nissl-positive neurons(p<0.001)and TH-positive neurons(p<0.001)in substantia nigra,striatum,hippocampal DG and hippocampal CA regions area,decreaseα-syn-positive neurons(p<0.001)in hippocampal DG and CA regions.Finally,Fer-1 and TETD also ameliorated neuroinflammation,decreased the protein expression of GSDMD-Caspase-1(p<0.001),and increased the antioxidant enzyme GPX4 expression(p<0.01)in MPTP-induced PD mouse model.Thereby,Fer-1 and TETD could attenuated MPTP-induced cytotoxicity and protected the cellular activity of dopaminergic neurons in the brains of PD animal models.Conclusion:The results of this study suggest that:1)there are significant differences in the expression of genes related to ferroptosis and pyroptosis by Transcriptomic analysis of the substantia nigra of normal and Parkinson’s disease patients.There are significant differences in the transcriptional level and protein levels of molecules related to cell pyroptosis and ferroptosis pathway in peripheral blood of patients with early PD.There are high expression of pyroptosis related factors(such as NLRP3,IL-1β)and low expression of ferroptosis key factor GPX4 in peripheral blood of patients with early PD.2)GPX4、fer-1 and TETD show protective effects on dopaminergic neurons through affecting ferroptosis-related phenotypes,such as lipid peroxidation,mitochondrial membrane potential,MDA and GSH.GPX4 regulates the process of ferroptosis induced by MPP+through NLRP1/NLRP3-caspase-1 signal pathway.3)GPX4 may exert a protective effect on dopaminergic neurons for improving motor and non-motor symptoms in animal models of PD by affecting the inflammasome-caspase-1 signaling pathway,inhibitingα-syn production and loss of TH.