The Molecular Mechanism Of Iron Metabolism In Parkinson Disease Model Intervened By α-Lipoic Acid Through FBXL5-IRP2 Pathway

Objective: To observe the effect of α-lipoic acid(α-LA)on the iron content,oxidative stress level,and the changes in expressions of FBXL5,iron regulatory protein 2(IRP2),and ferroportin 1(FP1)in the substantia nigra(SN)of PD model rats and PD cell models,and explore the mechanism by which α-LA regulates iron metabolism in PD model.Methods:(1)Animal experiment: Healthy male Sprague-Dawley(SD)rats were randomly divided into sham group(n=15),PD groups(n=15)and α-LA treatment group(n=15).Evaluated the effect of α-LA on behavioral changes of PD rats by APO-induced rotation test and cylinder test.The midbrain right SN was taken in each group,the expression of enzyme tyrosine hydroxylase(TH)was detected by immunohistochemical staining,the iron content was detected by Prussian blue iron staining and spectrophotometer,the levels of FBXL5,IRP2,FP1 were detected by Western Blot,the glutathione(GSH)content and superoxide dismutase(SOD)activity were by spectrophotometry and WST-1 method respectively.(2)Cell experiment:PC12 cells were induced by 6-OHDA to establish the cellular models of PD,which was treated with α-LA.Cell viability was detected using MTT assay.It was divided into 3 groups: the normal control group,the model group(200μmol/L 6-OHDA)and the α-LA treat group(10μmol/L α-LA+200μmol/L 6-OHDA).The content of intracellular iron was detected spectrophotometer.The levels of FBXL5,IRP2,FP1 were detected by Western Blot.The level of intracellular reactive oxygen species(ROS)was detected by 2,7-dichlorodihydrofluorescein diacetate(DCFH-DA)staining.The content of intracellular malondialdehyde(MDA)was detected by colorimetry.Results:(1)Animal experiment: compared with the sham group rats,the APO-induced rotational speed of the PD group rats was significantly increased(P<0.01),the left forelimb use rate of PD model group was significantly reduced(P<0.01),The TH expression of midbrain right SN of PD group rats was significantly decreased(P<0.01),the number of iron staining positive cells and total iron content were significantly increased(P<0.01,P<0.01),the expression of FBXL5 was significantly reduced(P<0.01),the expression of IRP2 was significantly increased(P<0.01),the expression of FP1 was significantly reduced(P<0.01),and the GSH content and SOD activity were significantly decreased(P<0.01,P<0.01);compared with the PD group rats,the APO-induced rotational speed of the α-LA treatment group rats was significantly decreased(P<0.01),the left forelimb use rate of α-LA treatment group was significantly increased(P<0.01),the TH expression of midbrain right SN of α-LA treatment group rats was significantly increased(P<0.01),the number of iron staining positive cells and total iron content were significantly reduced(P<0.01,P<0.01),the expression of FBXL5 was significantly increased(P<0.05),the expression of IRP2 was significantly reduced(P<0.01),the expression of FP1 was significantly increased(P<0.05),and the GSH content and SOD activity were significantly increased(P<0.05,P<0.01).(2)Cell experiment: after treated with 200 μmol/L 6-OHDA for 24 h,the cell viability decreased to 65.05±5.61%,which was treated as PD model cells.After pretreatment with 10 μmol/L α-LA for 1 h and then adding 200 μmol/L 6-OHDA for 24 h,the cell viability of PC12 cells rising to 87.79 ± 3.76%,which was treated as α-LA treat group.Compared with the control group,the cells viability of the PD model group was significantly decreased(P<0.01),the contents of intracellular iron was significantly increased(P<0.01),the expression of FBXL5 was significantly reduced(P<0.01),the expression of IRP2 was significantly increased(P<0.01),the expression of FP1 was significantly reduced(P<0.01),the contents of intracellular MDA and ROS were significantly increased(P<0.01,P<0.01);Compared with the PD modle group,the cells viability of the PD model group was significantly increased(P<0.01),the contents of intracellular iron was significantly reduced(P<0.01),the expression of FBXL5 was significantly increased(P<0.01),the expression of IRP2 was significantly reduced(P<0.05),the expression of FP1 was significantly increased(P<0.05),the contents of intracellular MDA and ROS were significantly decreased(P<0.01,P<0.01).Conclusion:(1)α-LA inhibit the abnormal deposition of iron may by up-regulate FP1 through the FBXL5-IRP2 pathway to increase iron turnover in the PD model.(2)α-LA up-regulate the expression of FBXL5 protein by decreasing the level of oxidative stress in the PD model.