TTP/NOX2/ROS Protects Dopaminergic Neurons From Oxidative Damage In MPP+/MPTP Model Of Parkinson's Disease

Background:Parkinson's Disease(PD)is one of the common neurodegenerative diseases,which generally affects patients' motor function and quality of life worldwide.Parkinson's disease is currently associated with oxidative stress,mitochondrial dysfunction,and abnormal aggregation of misfoldingalpha-synuclein.Under normal physiological conditions,reactive oxygen species(ROS)are involved in cell signal transmission and affect cell functions,including gene expression,proliferation,differentiation,and migration.However,excess ROS in PD can negatively affect cell function and survival by destroying intracellular proteins,lipids and nucleic acids.Therefore,elucidating the molecular mechanism of oxidative stress of DA neurons in PD is of important significance for the early detection,early diagnosis and early treatment ofPD.Purposes:Gene expression regulation is the key to maintaining cell survival and death,and the role of post-transcriptional regulation in the pathogenesis of Parkinson's disease is still unclear.Tristetraprolin(TTP)is an RNA-binding protein encoded by the ZFP36 gene.It plays an important role in post-transcriptional regulation of genes.Previous studies have shown that TTP is essential for neural differentiation and plays a protective role in the subarachnoid hemorrhage model.TTP plays a tumor suppressive role in gliomas;however,its relationship with neurodegenerative diseases such as Parkinson's disease is unclear.This study intends to explore the effects and mechanisms of TTP on dopaminergic neuron damage in Parkinson's disease models through the NOX2/ROS signaling pathway at the molecular,cellular,and animal levels,and to provide new targets for subsequent TTP-based PD therapy.Methods:Cell levelMN9D mouse dopaminergic(DA)neuron cells or human neuroblastoma SH-SY5Y cells were treated with 1-methyl-4-phenylpyridine salt(MPP+),and then Western blot,quantify reverse transcription polymerase chain reaction(RT-qPCR)and immunofluorescence technologywere used to to detect the expression of TTP in vitro;After down-regulating TTP's expression by small interfering RNA(si-TTP)or up-regulating by OE-TTP lentivirus,oxidative stress indicator NOX2 expression and reactive oxygen species(ROS)production were detected,Glutathione(GSH)and superoxide dismutase(SOD)content,flow cytometry was used to detect the apoptosis rate and survival rate of dopaminergic neurons,Western blot was used to detect apoptosis-related protein Bcl-2 and cleaved-caspase-3.By down-regulating NOX2 or taking ROS inhibitor N-acetyl cysteine(NAC),we investigate whether TTP regulates the oxidative damage of dopaminergic neurons through the NOX2/ROS axis.Molecular levelBy down-regulating or up-regulating TTP,Western blot,rt-qPCR,and other technologies were used to detect the effects of TTP on the NOX2 pathway.Database prediction,luciferase assay,RNA immunoprecipitation(RIP)and mRNA stability analysis were used to further detect whether TTP directly binds to NOX2.Animal levelA sub-acute animal model of MPTP was constructed,and the expression levels of TTP and NOX2 were detected in vivo using techniques such as western blot,RTq-PCR,and tissue immunofluorescence.After Stereotactic injection of TTP interfered or over-expressed lentivirus,Western blot,RTq-PCR and tissue immunofluorescence were used to detect the effects of TTP on NOX2 and oxidative damage of dopaminergic neurons in vivo.Research result1.TTP's expression was increased in MPP+ cell model of Parkinson's disease,TTP could reduce apoptosis of DA neurons caused by MPP+2.TTP reduced DA neuron oxidative stress in PD model through ROS to regulate DA neuron apoptosis3.TTP binds with NOX2 directly,TTP regulates oxidative stress and apoptosis of DA neurons through NOX24.TTP's expression were increased in DA neurons of MPTP subacute model,TTP regulates NOX2 expression and DA neuron oxidative damage in vivoin conclusionThe TTP/NOX2/ROS axis reduces oxidative damage of dopaminergic neurons in the MPP+/MPTP model of Parkinson's disease.