Study On The Mechanisms And Protective Effect Of Erythropoietin On β-amyloid-induced PC12 Cells Death

Part 1 Protective effect of Erythropoietin onβ-amyloid-inducedPC12 cells death through antioxidant mechanismsObjective To investigate the neuroprotective effect of EPO against Aβ-inducedapoptosis in cultured PC12 cells.In addition,we have explored possible mechanisms forthese effects.Methods Cell viability was determined using the conventional MTT reduction assay.The degrees of DNA nick formation Genomic DNA fragmentation was determined by theterminal deoxynucleotidyl transferase nick end labeling (TUNEL) assay and Hoechst 33342staining.Flow cytometry assay was used to analyze the apoptosis ratio of PC12 cells.Intracellular reactive species was monitored by using the fluorescent DCF-DA,Themitochondrial membrane potential was measured by using rhodamine 123 fluorescent dye,Western blot analysis for Bcl-2,Bax,and pl 7 subunit of caspase-3.Results EPO exerted a concentration dependent neuroprotective effect against the lossof cell viability induced by Aβ_25-35 in PC12 cells.The maximum neuroprotection occurredwhen 2U/ml EPO concomitant with 20μM Aβ_25-35.MTT assay and apoptotic staining(TUNEL and Hoechst 33342) provided the first evidence that EPO could protect PC12 cellsagainst Aβ_25-35 induced PC12 cells apoptosis.Furthermore,EPO inhibited theAβ_25-35-induced ROS increase in DCF fluorescence,and prevented the decrease in theretention of rhodamine 123 induced by Aβ_25-35.EPO treatment (2U/ml) could alsoprevented the Aβ_25-35-induced decrease of the Bcl-2/Bax ratio,and activation of Caspase -3.Conclusion EPO also possesses the ability to protect neurons from Aβ_25-35-induceddamage,possibly by inhibiting oxidative stress and apoptosis. Part 2 STAT5 signaling is essential for erythropoietin-mediatedprotection against PC12 cells apoptosis induced by Aβ_25-35Objective To investigate the role of JAK2/STAT5 signaling pathway in the protectiveeffect of EPO onβ-amyloid₂₅₃₅-induced apoptosis.Methods Cell viability was determined using the conventional MTT reduction assay.The apoptotic cells were observed by Hoechst 33342 staining.Western blot analysis forJAK2,p-JAK2,STAT5 and p-STAT5.Results (1) There was a significant increase in the phosphorylation of JAK2 inAβ_25-35-treated cells by EPO.Treatment of PC12 cells with JAK2 inhibitors AG490decreased the level of p-JAK2 induced by EPO.(2)EPO induced the phosphorylation ofSTAT5 in Aβ_25-35-treated cells.AG490 pretreatment abolished phosphorylation of STAT5.(3) JAK2 inhibitors AG490 significantly decreased the PC12 cells viability and increasedthe ratio of apoptosis.Conclusion JAK2/STAT5 singaling pathway contribute to the protective effect of EPOagainst Aβ_25-35-induced apoptosis.JAK2 inhibitor AG490 blocked EPO-induced activationof JAK2/STAT5 signaling pathway,proliferation,and antiapoptotic effect. Part3 Erythropoietin Protects PC12 cells fromβ-amyloid2s₃s-induced apoptosis via PI3K/Akt signaling pathwayObjective To investigate the role of PI3K/Akt signaling pathway in the protective effectof EPO on Aβ₂₅₃₅-induced apoptosis.Methods Cell viability was determined using the conventional MTT reduction assay.The degrees of DNA nick formation Genomic DNA fragmentation was determined by theterminal deoxynucleotidyl transferase nick end labeling (TUNEL) assay and Hoechst 33342staining.Western blot analysis forAkt,p-Akt,GSK-3β,p- GSK-3βand Bcl-2.Results (1)EPO induced time-dependent phosphorylation of phosphatidylinositol 3-kinasesubstrate Akt.There was also a significant increase in the phosphorylation of Akt inAβ₂₅₃₅-treated cells by EPO.Treatment of PC12 cells with PI3K inhibitors LY294002decreased the level of p-Akt induced by EPO.(2)EPO induced the phosphorylation ofglycogen synthase kinase-3β(GSK-3β) in Aβ₂₅₃₅-treated cells.LY294002 pretreatmentabolished phosphorylation of GSK-3β.(3) the expression of anti-apoptotic protein Bcl-2was increased by EPO,which could be inhibited by PI3K inhibitors LY294002.(4) PI3Kinhibitors LY294002 significantly decreased the PC12 cells viability and increased the ratioof apoptosis.Conversely,GSK-3βinhibitors lithium chloride blocked Aβ_25-35-induced cellapoptosis in a manner similar to EPO.Conclusion the treatment of EPO resulted in an enhanced phosphorylation of moleculesthat were associated with neuroprotection in PC12 cells,including Akt and GSK-3β,as wellas increased expression of anti-apoptotic protein Bcl-2.