Istradefylline Upregulated HIF1α Inhibits Ferroptosis In Models Of Parkinson’s Disease Via MAPK Family Proteins

Background:Parkinson’s disease（PD）is the second most common neurodegenerative disease in the world.Currently,yet no disease-modifying therapy is available.Therefore,it is urgent to explore new targets in the study of PD.PD is characterized by the loss of dopaminergic neurons in the substantia nigra and striatum.The pathogenesis of PD involves ferroptosis,α-alpha-synuclein（α-syn）aggregation,oxidative stress,mitochondrial dysfunction,neuroinflammation and other aspects.Previous studies have suggested that ferroptosis is both a cause and a result of the development and progression of PD.The process of ferroptosis is very similar to the signature process of PD neuropathology because both involve iron deposition,lipid peroxidation,glutathione depletion,α-syn aggregation,mitochondrial dysfunction,oxidative stress,and inflammation.The aggregation ofα-syn can induce ferroptosis by stimulating the production of reactive oxygen species and accelerating lipid peroxidation through the interaction with the cell membrane.The loss of dopamine may increase the susceptibility of PD to ferroptosis.Taken together,regulation of ferroptosis pathway may be a potential therapeutic strategy for PD.Studies have found that mitogen-activated protein kinase（MAPK）signaling pathway is involved in regulating the expression of hypoxia inducible factor 1 subunit alpha（Hif1α）,which plays a protective role in PD mice and PD cell models.After neurotoxic insults,Adenosine A_2Areceptors can activate several intracellular cascades,including downstream signaling via p38,ERK and JNK MAP kinases.Activation of these kinases contributes to neuronal death with a variety of lethal triggers.At present,a new selective adenosine A_2Areceptor antagonist,istradefylline,has been approved for the treatment of PD in Japan and the United States.To clarify whether istradefylline regulates ferroptosis in PD by inhibiting MAPK family proteins and stabilizing HIF1α,we used bioinformatics methods to screen differentially expressed genes（DEGs）in SNpc of PD patients and healthy people.We intersected DEGs with a ferroptosis database（Ferr Db,http://www.zhounan.org/ferrdb/current/）to obtain ferroptosis-related DEGs in PD.HIF1αwas the most significantly up-regulated gene.The aim of this study is to investigate the mechanism of istradefylline alleviating ferroptosis in PD via upregulated the hub target gene HIF1α,which is expected to find a new therapeutic target for PD treatment.Purpose:To demonstrate that the expression of HIF1αwas down-regulated during ferroptosis in dopaminergic neurons of PD mice model.It was clarified that istradefylline could alleviate ferroptosis in dopaminergic neurons of PD model.To investigate the molecular mechanism of itracylline inhibiting ferroptosis in dopaminergic neurons of PD model by inhibiting the MAPK pathway and up-regulating the expression of HIF1α.Method:1.To identify ferroptosis-related DEGs,the m RNA expression profile dataset（GSE114918）was downloaded from GEO（http://www.ncbi.nlm.nih.gov/geo/）.The R software was used to screene the differentially expressed genes（DEGs）between 5 PD patients and 16 healthy individuals by bioinformatics.The genes in the ferroptosis dataset（Ferr Db,http://www.zhounan.org/ferrdb/current/）were intersected with the DEGs to identify ferroptosis-related DEGs.GO and KEGG enrichment analysis were used to annotate their functions,and the protein-protein interaction network of PD ferroptosis-related DEGs was constructed,hub genes were identified using Cytohubba.2.The PD mouse model was constructed by intraperitoneal injection of1-methyl-4 phenyl-1,2,3,6-tetrahydropyridine（MPTP）.The behavioral changes of the mice were observed by open-field,pole,traction,and forced swimming tests,hematoxylin eosin（HE）staining,immunohistochemical method（IHC）in the substantia nigra and striatum of PD mouse model were performed,which were used to confirme the success of PD mouse model.Transmission electron microscope,iron Assay Kit,MDA Assay Kit,GSH and GSSG Assay Kit were used to detect the relevant markers of ferroptosis.Western blot was used to detect the expression levels of TH,α-syn,HIF1α,vascular endothelial growth factor A（VEGFA）,transferrin（TF）,transferrin receptor（TFR）,ferroportin 1（FPN1）,and glutathione peroxidase 4（GPX4）,so as to confirm the presence of ferroptosis in the PD mouse model.The m RNA expression levels of hub genes in the substantia nigra were verified by real-time quantitative PCR（RT-PCR）.3.To verify that itracylline inhibits ferroptosis and regulates MAPK pathway and HIF1αin mouse model of PD,the experimental animals were randomly divided into control group,model group,istradefylline treatment group.The model group and istradefylline treatment group received intraperitoneal MPTP（30mg/kg）,the control group received intraperitoneal saline,qd,for 8 consecutive days.On the 9th day,no drug treatment or other intervention was given in each group,and on the 10th day,behavioral tests were performed.Compared with the control group,those with behavioral differences entered the subsequent experiment.The control model group and itracylline treatment group were given MPTP（30mg/kg）or MPTP（30mg/kg）intraperitoneal injection,respectively The behavioral differences between the two groups were compared.On the 11th day,the istradefylline treatment group was given intraperitoneal injection of istradefylline（10mg/kg）,qd for 10 consecutive days,and the other two groups were given saline.Behavioral tests were performed on the 22nd day.The mice were sacrificed on the 23rd day.HE staining and IHC staining of TH were performed on the midbrain substantia nigra and striatum.Western blot was used to detect the expression levels of ferroptosis-related proteins such as TF,TFR,FPN1,GPX4 in the substantia nigra and striatum of midbrain,as well as MAPK family proteins,TH,α-syn,HIF1αand VEGFA.Transmission electron microscopy,iron Assay Kit,MDA Assay Kit,GSH and GSSG Kit were used to detect ferroptosis indicators.4.To further confirm the mechanism of istradefylline inhibiting ferroptosis in PD cells by inhibiting MAPK pathway upregulation of HIF1α,Human neuroblastoma cell SH-SY5Y were treated with 1-methyl-4-phenyl-pyridine salt（MPP⁺）to construct the PD cell model.The concentrations of MPP⁺and istradefylline used were verified by measuring cell viability by CCK-8 assay.The test cells were subsequently divided into control group,model group,and istradefylline treatment group.Mito Sox DCFH-DA kit was used to detect the levels of superoxide in mitochondria and reactive oxygen species in cytoplasm.Immunofluorescence staining（IF）was used to detect the expression of TH.Western blot was used to detect the expression of MAPK family proteins,TH,α-Syn,HIF1α,VEGFA,TF,TFR,FPN1 and GPX4.Iron Assay Kit,MDA Assay Kit,GSH and GSSG Kit were used to detect ferroptosis indicators.In the istradefylline treatment group,the ERK agonist LY-2828360 was given,and the protein expression levels of ERK,HIF1α,VEGFA,TH,α-syn,TF,TFR,FPN1and GPX4 were detected by Western blot.The HIF1αgene silencing cell model was constructed by lentivirus transfection of sh RNA interference fragments.The m RNA of HIF1αand VEGFA genes was detected by RT-PCR.After verified the successful construction of the cell model,the HIF1αgene silencing cell model was treated with MPP⁺,and then treated with istradefylline.The protein expression of ERK,TH,α-syn,HIF1α,VEGFA,TF,TFR,FPN1 and GPX4 was detected by Western blot.Results:1.PCA was used to evaluate the repeatability of intragroup data,and the results showed that the GSE114918 data had good repeatability.A total of 2811 DEGs were identified according to the preset criteria（log2|FC|>1,adjusted P<0.05）,When the Ferr Db data were intersected with the GSE114918 data,we found 67ferroptosis-related DEGs,including the 28 up-regulated genes and 39 down-regulated genes.The GO enrichment analysis showed that the most significant GO enrichment terms were as follows:cellular iron ion homeostasis（biological process）;autolysosome（cellular component）;and antioxidant activity（molecular function）.The KEGG enrichment analysis revealed that the ferroptosis-related DEGs were mainly related to the following pathways:ferroptosis,peroxisome,fatty acid metabolism.There were 57 nodes and 142 edges in the PPI network.The top 10 genes were identified as hub genes by"Cytohubba".2.The PD mouse model was successfully constructed by intraperitoneal injection of MPTP.Statistically significant differences were observed in the results of the open-field test,traction test,pole test,and forced swimming test in PD mouse model.Immunohistochemical staining revealed an approximately 40%reduction in the number of TH-positive neurons in the SNpc and an approximately 40%reduction in TH immunoreactivity in the striatum of PD model mice compared with control mice.The results of transmission electron microscope showed that mitochondrial shrinkage,increased membrane density and outer membrane disruption in the substantia nigra and striatum in mouse model of PD;the myelin morphology changed significantly in striatum,and the morphology became irregular,myelin separation,disordered arrangement and uneven staining.The Fe²⁺deposition,increased MDA,and decreased GSH in the substantia nigra and striatum in the PD mouse model group.Western bolt results showed that increased TF and TFR in the substantia nigra and striatal regions,proving that more iron ions turned into the cells.Decreased FPN1showed that iron ions are less expelled from the cell.Decreased GPX4 suggested that the ability of glutathione to reduce lipid peroxide is decreased.Decreased TH,and increased a-syn confirmed severe damage to dopaminergic neurons.In conclusion,ferroptosis is present in the PD mouse model.RT-PCR results showed that the m RNA transcript levels of GSK3B,HIF1A,VEGFA,STK11 and PPARA in substantia nigra of PD mouse model were consistent with the bioinformatics results.Western bolt results showed that the expression of HIF1αand VEGFA protein was decreased,indicating that the adaptation ability of PD mouse model to hypoxia was decreased3.Behavioral tests showed that compared with the model group,istradefylline had a significant protective effect on the motor dysfunction of PD mouse model.The results of transmission electron microscope suggested that the mitochondrial contraction was significantly improved,the membrane rupture was reduced,the morphological arrangement of myelin sheath began to become regular,the staining tended to be uniform,the myelin sheath became closely arranged,and the structural abnormalities were obviously recovered in striatum.IHC staining suggested that compared with the model group,TH staining positive cells in substantia nigra and striatal areas increased significantly after istradefylline treatment,which confirmed that istradefylline exerts a protective effect on dopaminergic neurons in PD.Fe²⁺decreased significantly,MDA decreased significantly,and reduced GSH increased significantly after istradefylline treatment.The results of Western bolt showed that the expression of TF,TFR decreased;FPN1,GPX4 increased;HIF1α,VEGFA and TH increased;andα-syn decreased in the SN and striatum of the istradefylline treatment group,what suggested that istradefylline has an inhibitory effect on ferroptosis.Istradefylline up-regulated the expression of HIF1αand protected dopaminergic neurons in substantia nigra and striatum in PD.The expression of P-P38、P-JNK and P-ERK was significantly increased in the model group compared with the normal group,and the expression of P-P38,P-JNK and P-ERK were significantly decreased in the istradefylline treated group compared with the model group in the result of Western bolt.The results showed that istradefylline may inhibit the ferroptosis of dopaminergic neurons in substantia nigra and striatum by inhibiting P-P38,P-JNK and P-ERK and up-regulating HIF1αto improve the ability of cells to resist hypoxia.4.We used CCK-8 detection kit to detect the cell viability of SH-SY5Y cells treated with different concentrations of MPP for 24h.SH-SY5Y cell viability decreased to about 69%when MPP was 2m M.We treated SH-SY5Y cells with MPP2m M for 24h to build PD cell model.The cell viability of SH-SY5Y cells pretreated with 3n M istradefylline for 2 hours and then co-cultured with 2m M MPP for 24 hours was about 80%,so 3n M was selected as the therapeutic concentration of istradefylline.Both the IF and Western blot results showed a decreased protein expression of TH in the PD cell model,confirming the success of the PD cell model construction.Compared with the model group,the concentration of Fe²⁺and MDA decreased,the level of reduced GSH increased,and the level of superoxide in mitochondria and reactive oxygen species in cytoplasm decreased in the istradefylline treatment group,in addition,the results of Western blot showed that the expression of TF,TFR was decreased,and the expression of FPN1,GPX4 was increased,suggesting that ferroptosis was inhibited.The results of Western blot showed that compared with the control group,the expression of P-P38,P-JNK and P-ERK was significantly increased,and the expression of HIF1αand VEGFA was decreased in the model group;compared with the model group,the expression of P-P38,P-JNK and P-ERK was significantly decreased,and the expression of HIF1αand VEGFA was increased in the istradefylline treatment group,suggesting that the treatment with istradefylline may inhibit P-P38,P-JNK and P-ERK and up-regulate the expression of HIF1αprotein,improve the ability of cells to resist hypoxia,and inhibit the occurrence of ferroptosis in PD cell model.In order to further explore the upstream mechanism,we chose to administer ERK agonist LY-2828360 to activate ERK while treating PD cell models with istradefylline,and 20μM LY-2828360 ERK agonist could significantly activate SH-SY5Y p-ERK protein expression（P<0.05）.After the combined use of ERK agonist LY-2828360,ferroptosis related protein changed significantly contrast with the istradefylline treatment group.Compared with istradefylline treatment group,After LY-2828360 treatment,TF and TFR expressions increased,FPN1 and GPX4expressions decreased,and ferroptosis increased.The decreased expression level of HIF1αand VEGFA protein indicates that HIF1αconsumption increases after P-ERK activation,which is not conducive to the protective effect of PD cell model.After successful transfection of lentiviral sh RNA interference fragments to construct HIF1αgene silencing cell model,Western blot showed that the therapeutic effect of istradefylline on ferroptosis in PD cell model was lost after silencing HIF1α,which indicates that silencing HIF1αattenuated the protective effect of istradefylline on ferroptosis in PD cell model,and that the protective effect of ferroptosis in PD cell model was exerted by improving the expression of HIF1αprotein.In conclusion,istradefylline exerts a protective effect on ferroptosis in PD cell model by upregulating HIF1αexpression by inhibiting the ERK pathway.Conclusion:1.HIF1αwas the most significant DEG associated with PD ferroptosis in bioinformatics analysis;2.Ferroptosis of dopaminergic neurons was observed in MPP⁺-treated SH-SY5Y PD cell model and in the substantia nigra and striatum in MPTP mouse model of PD.Reduced expression of HIF1αpromotes ferroptosis of dopaminergic neurons.HIF1αmay be a key molecule regulating ferroptosis of dopaminergic neurons in PD model;3.Istradefylline can upregulate HIF1αby inhibiting ERK signaling pathway and inhibits ferroptosis of MPP⁺-treated SH-SY5Y PD cell line model,HIF1αmolecule may be a target for the treatment of ferroptosis in PD.