Study On The Regulatory Mechanism Of Oxidative Stress On Iron Regulatory Protein IRP2 In SH-SY5Y Cells

Parkinson's disease?PD?is a common neurodegenerative disorder of the central nervous system that occurred in the elderly.The main pathological changes of PD are degeneration of dopaminergic neurons in the substantia nigra and the formation of Lewy body in the remaining neurons.At present,the pathogenesis of PD remains unclear,oxidative stress,as a negative effect of free radicals produced in the body,is considered to be an important cause of PD.It caused the death of dopaminergic neurons through a variety of pathways that has gained widespread attentions by researchers.Neuroimaging and postmortem studies reported that iron selective aggregation in the substantia nigra led to the increased iron content in residual dopaminergic neurons,suggesting that the misregulation of iron metabolism played a key factor in neuronal injury in PD.The cellular iron homeostasis is dependent on the functions of iron regulatory protein?IRPs?in the cytosol.They could regulate the expression of some iron metabolism proteins through bindinge with their iron regulatory elements on the 3?or 5?untranslated region of their m RNA.IRPs could be divided into two types: IRP1 and IRP2.Although both are widely expressed in the body,IRP1 is mainly expressed in peripheral tissues,while IRP2 is expressed in the central nervous system.Six-month-old IRP2-/-mice showed neural degeneration,and iron deposited in the substantia nigra,cerebellum,hippocampus and caudate nuclei.In comparison,IRP1-/-mice at the same age did not show any neurological manifestations,indicating that IRP2 is a key protein regulating brain iron homeostasis.However,there are few studies on the regulation of IRP2 expression by oxidative stress.The elucidation of this question will provide a new evidence to understand the mechanism of iron aggregation in the substantia nigra.The expression of IRP2 is regulated by multiple pathways,including which are related in transcription,post-transcription,translation and post-translational modifications.Among these,post-translational modifications are the main ways to maintain IRP2 protein stability,and ubiquitination plays an important role in protein degradation process.Ubiquitin ligase?E3?is prerequisite to ubiquitination for target protein.E3 recognizes and binds to the specific sequence of a protein,which is subsequently degraded by 26 S proteasome.It is found that the F-box of leucine rich repeat protein 5?FBXL5?is a kind of iron and oxygen dependent E3 ubiquitin ligase.Under iron and oxygen sufficient conditions,the activity and stability of FBXL5 is enhanced and mediates the degradation of IRP2.However,it is not clear whether the expression of IRP2 is regulated by FBXL5 in PD.To evaluate the effect of different oxidative stress factors including H₂O₂,ferric ammonium citrate?FAC?and sodium nitroprusside?SNP?on the regulation of IRP2 expression and the underlying mechanism,SH-SY5 Y cells was used in this study.Methylthiazolyldiphenyl-tetrazolium bromide?MTT?assay was used to observe the changes of cell viability.Flow cytometry was used to test the intracellular reactive oxygen species?ROS?contents and the mitochondrial membrane potential???m?.Real-time quantitative of PCR was performed to detect the m RNA levels of FBXL5 and IRP2.Western blots was used to measure the protein levels of FBXL5 and IRP2;confocal microscopy detection was used to observe the cell iron uptake functions.The results were as follows:1.SH-SY5 Y cells were treated with different concentrations of H₂O₂ for 24 hours,MTT method was performed to detect the cell viability.The results showed that the cell viability was decreased with the increased concentration of H₂O₂.In cooperation with the control group,the cell survival rate in 20,30,50,200,300,500 and 1000 mol/L H₂O₂ group was decreased by 10.2% 12.9%,13.7%,15.3%,21.2%,33.6% and 38.6%,respectively,which displayed significant differences?P<0.05?.2.After SH-SY5 Y cells were treated with 200 ?mol/L and 300 ?mol/L of H₂O₂ for 24 hours,the intracellular ROS levels were increased by 52.2% and 87.3%,respectively,and the difference was statistically significant?P<0.01?when compared with that of the control group.The ??m in cells was decreased by 1.7% and 8.2%,comparing with the control group,which was significant in 300 ?mol/L H₂O₂ treatment group?P<0.05?.3.After SH-SY5 Y cells were treated with 200 ?mol/L and 300 ?mol/L of H₂O₂ for 24 hours,the expression levels of FBXL5 were increased by 20% and 31%,whereas the expression levels of IRP2 were decreased by 44% and 75%.In comparison with the control group,the difference was statistically significant?P<0.05?.Moreover,the m RNA levels of FBXL5 and IRP2 showed no significant difference?P>0.05?.After incubation with the ubiquitin proteasome inhibitor MG132,the expression levels of FBXL5 in 200 ?mol/L and 300 ?mol/L H₂O₂ treated cells showed no significant difference,while IRP2 levels were increased by 49% and 28% in two groups,the difference was statistically significant?P<0.05?.After incubation with the si-FBXL5,FBXL5 levels were decreased by 86% and 101%,and IRP2 levels were increased by 39% and 26% in two groups,the difference was statistically significant?P<0.05?.4.After 200 ?mol/L and 300 ?mol/L of H₂O₂ treatment for 24 hours,the SH-SY5 Y cells were incubated with 100 ?mol/L Fe2+,The cell iron uptake ability was observed using laser confocal microscopy,and the results showed that the intracellular fluorescence intensity was increased with time,indicating a decreased Fe2+ uptake.The differences were statistically significant?P<0.05?.5.After SH-SY5 Y cells were treated with different concentrations of FAC for 24 hours,MTT results showed that the survival rate of cells in 10,30 and 50 ?g/m L groups had no obvious change;however,it was in 100,300,500 and 1000 ?g/m L groups decreased with the increased FAC levels,and the decreases were 14.3%,26%,29.4% and 38.5%,respectively,which the differences were statistically significant?P<0.05?.After 10 ?g/m L and 100 ?g/m L FAC treated SH-SY5 Y cells for 24 hours,the intracellular ROS contents were increased by 63.3% and 94.8%,respectively,and the difference was statistically significant?P<0.01?.The ??m in cells was decreased by 12% and 27%,which the differences were statistically significant?P<0.05?.The expression levels of FBXL5 were increased by 21% and 37%,whereas the expression levels of IRP2 were decreased by 28% and 45%.In comparison with the control group,the difference was statistically significant?P<0.05?.6.After SH-SY5 Y cells were treated with different concentrations of SNP for 24 hours,MTT results showed that the survival rate of cells in 10,30,50 and 100 ?mol/L groups had no obvious change;however,it was in 300 and 500 ?mol/L groups decreased with the increased FAC levels,and the decreases were 21.9% and 42% respectively,which the differences were statistically significant?P<0.05?.After 100 ?mol/L and 300 ?mol/L SNP treated SH-SY5 Y cells for 24 hours,the intracellular ROS content was increased by 58.6% and 82.3%,respectively,and the difference was statistically significant?P<0.01?.The ??m in cells was decreased by 43% and 60%,which the differences were statistically significant?P<0.05?.The expression levels of FBXL5 were increased by 29% and 40%,whereas the expression levels of IRP2 were decreased by 13% and 47%.In comparison with the control group,the difference was statistically significant?P<0.05?.In summary,the experimental results showed that the oxidative stress factors including H₂O₂,FAC and SNP treatment could increase ROS levels,decrease the mitochondrial membrane potential,cause oxidative stress in SH-SY5 Y cells,and further induce an increase in FBXL5 levels and a decrease in IRP2 levels,which resulted in a reduction of iron uptake ability by cells.The m RNA levels of FBXL5 and IRP2 did not change,which indicated that the decrease of IRP2 levels were not caused by the alteration in transcription.After the treatment using the ubiquitin proteasome pathway inhibitor MG132,FBXL5 expression was unchanged,while the expression of IRP2 was significantly increased,indicating that MG132 blocked IRP2 ubiquitin degradation process that may be mediated by FBXL5.This study implicates that oxidative stress factors could regulate IRP2 expression through ubiquitination pathway,and it could provide a new experimental basis to reveal the neuronal mechanism of IRP2 regulation in the pathogenesis of PD.