The Protective Effects Of Cordycepin On Dopaminergic Neurons In PD Models And Its Effects On MAPK And PI3K/Akt-Nrf2 Signaling Pathways

Objective:To explore the protective effects of cordycepin(Cor)on dopaminergic neurons in PD models and to investigate the potential mechanisms involved through the MAPK,Nrf2,and PI3K/Akt signaling pathways.Methods:1.In vivo:C57BL/6 mice were randomly divided into the control group,the model group(30mg/kg MPTP)and the Cor groups(2.5,5.0 and 10.0 mg/kg Cor).Motor performance of mice was evaluated by open field,rotarod and pole tests.Contents of dopamine(DA),dihydroxyphenylacetic acid(DOPAC)and homovanillic acid(HVA)in striatum(Str)were determined by HPLC-ECD.The numbers of TH-immunoreactive cells in substantia nigra pars compacta(SNpc)and apoptotic cells in substantia nigra(SN)were measured by immunohistochemistry staining and TUNEL staining,respectively.The content of MDA and the activities of SOD and GSH-Px were assayed by spectrophotometry.Expressions of Cleaved Caspase-3,Bax,Bcl-2,p38,p-p38,ERK1/2,p-ERK1/2,JNK1/2,and p-JNK1/2 proteins in SN were determined by Western blotting.2.In vitro:PC12 cells were divided into the control group,the model group(500 μM MPP+)and the Cor groups(0.5×10-6,2.0×10-6 and 8.0×10-6 μM Cor).Cell viability was measured by MTT assay.Apoptosis of PC 12 cells was detected by Hoechst 33258 nuclear staining and the apoptotic rate was analyzed by flow cytometry.The intracellular levels of reactive oxygen species(ROS)were measured by using the DCFH-DA fluorescent probe.Expressions of intracellular Cleaved Caspase-3.Bax,Bcl-2,p53,HO-1,Akt,and p-Akt proteins as well as that of Nrf2 in the cytoplasm and nucleus were determined by Western blotting.To further explore the relationship between the PI3K/Akt and the Nrf2 signaling pathways,cell viability.ROS content and the expressions of HO-1,Akt,p-Akt,and the cytoplasmic and nuclear Nrf2 proteins were determined after adding PI3K inhibitor(LY294002)using the methods described above.respectively.Results:1.Protective effects of Cor on apoptosis of DA neurons and its effects on the MAPK signaling pathway in MPTP-PD mice.(1)The protective effects of Cor on DA neurons in PI)mice:After Cor pretreatment,the spontaneous activity,muscle tension,and body coordination of’ mice induced by MPTP were significantly improved,the contents of DA.DOPAC and I IVA in Str were increased,and the loss of TH-immunoreactive neurons in SNpc was reduced.(2)Cor inhibited the apoptosis of DA neurons in PD mice:Cor markedly reduced the number of apoptotic cells in SN of mice induced by MPTP.Meanwhile,the expressions of apoptotic proteins Bax and Ceaved Caspase-3 were significantly down-regulated and that of anti-apoptotic protein Bcl-2 as well as the ratio of Bcl-2/Bax were dramatically up-regulated in SN of PD mice pretreated with Cor.(3)Cor improved the antioxidant capacity in SN of the PD mice:Cor decreased the content of MDA and increased the activities of antioxidant enzymes,such as SOD and GSH-Px.(4)Cor inhibited the activation of MAPK signaling pathway:The expressions of p-p38.p-ERK1/2 and p-JNK1/2 proteins were down-regulated in SN of PD mice treated with different doses of Cor.while no significant differences in the expressions of p38.ERK1/2 and JNK1/2 proteins were observed in each group.2.Cor inhibited MPP+-induced oxidative stress injury in PC12 cells by activating the PI3K/Akt-Nrf2 pathway(1)Cor inhibited MPP+-induced PC12 cell apoptosis:Cor improved the cell morphology of PC12 cells induced by MPP+ and prominently reduced the number of cell with apoptotic characteristics such as nuclear condensation and fragmentation.And hence the overall apoptosis rate was significantly decreased.The expressions of Bax and p53 as well as the activation of Caspase-3 were also significantly inhibited and the expression of Bcl-2 and the ratio of Bcl-2/Baxwere up-regulated by Cor pretreatment.(2)Effects of Cor on the oxidative stress-related Nrf2 pathway and the PI3K/Akt pathway:The cell survival rate was obviously increased and intracellular ROS accumulation was significantly reduced in MPP+-induced PC 12 cells treated with Cor.By inducing a translocation of Nrf2 from cytoplasm to nucleus,Cor activated the Nrf2 signaling pathway and dramatically increased the protein expression of HO-1.At the same time,Cor noticeably increased Akt phosphorylation to activate the PI3K/Akt signaling pathway.(3)The activation of the Nrf2 pathway induced by Cor was dependent on the activation of the PI3K/Akt pathway:The protective effects of Cor on MPP+-induced cell injury and its inhibitory effect on intracellular ROS accumulation were significantly diminished,respectively,after the addition of the PI3K inhibitor(LY294002).Moreover,the effects of Cor,such as nuclear translocation of Nrf2,and up-regulation of p-Akt and HO-1 protein expressions,were also evidently inhibited by LY294002.Conclusion:The findings from our investigation confirm that Cor has neuroprotective effects on the MPTP/MPP+-induced PD models.The underlying mechanisms may involve the activation of the PI3K/Akt-Nrf2 signaling pathway and the inhibition of the MAPK signaling pathway activation to reduce oxidative stress injury and thereby inhibit the apoptosis of DA neurons.