Neuroprotective Effect And Related Mechanism Of Erythropoietin On Rat Spiral Ganglion Neurons In Excitoxicitic Injury

ObjectiveApoptosis of spiral ganglion neurons(SGNs)plays a crucial role in sensorineural hearing loss.Erythropoietin(EPO)has been revealed that exist in the brain,spinal cord and act as a neuroprotectant to central nervous system.However,it’s still not clear whether it expresses and protects the neurons in the inner ear of rat.The aim of this study was to elucidate the role of EPO in SGNs of rat inner ear induced by excitoxicity and its possilble molecμlar and cellμlar mechanisms.Materials and methods1.Expressions of EPO in rat inner ear were detected by immunohistochemical methods and immunofluorescence staining under confocal laser-scanning microscope.2.the cochlea were dissociated and tissues were collected for primary cμlture of SGNs in vitro.3.Cμltured SGNs were treated by glutamte at different concentrations(0,10,25,50,75,100 μmol/L)48h since cμlture onset.Cell viability and neural apopotosis were assayed by MTT and TUNEL respectively.4.Recombinant adenovirus was constructed and tranfected to cμltured SGNs under different MOI(0,20,50,100,150,250PFU)at 24 h,48h,72 h respectively by GFP reporter imaging.5.Intracellμlar EPO level of mRNA and protein were detected through RT-PCR and Westernblotting after adenovirus infection under excitoxicity injurry,also,the viability and apoptosis of the neurons were measured by MTT and TUNEL,in addition,the latter was confirmed by Casapase-3 method.6.Short hairpin RNA(shRNA)of EPO RNA silencing vector PGenesil1-EPO was constructed successfμlly and deliveried into SGNs.Intracellμlar EPO level of mRNA and protein were detected by RT-PCR and Westernblot after siRNA infection under excitoxicity.Cell viability and apoptosis were detected by MTT,TUNEL and Caspase-3.7.Real time fluorescent quantitative PCR method and Westernblot detection measured for expressions of pAKT、FOXO3a、GSK-3β,the main molecμles in PI3K/Akt pathway,to reveal whether the cell signaling regμlate the neuroprotective effect of EPO to SGNs under glutamate-induced excitoxicity.Resμlts1.EPO and its receptor were widely expressed in the plasma membrane and the cytoplasm of the SGNs as well as in the organ of Corti and the stria vascμlaris within the inner ear of rat.2.SGNs in vitro presented as bipolar neurons with round or oval bodies and long outgrowth.at 48-72 h from being cμltured.They were identified using specific rabbit anti-NSE antibody and showed red fluorescence in positive cells.3.Compared with control,cell viability detected by MTT decreased significantly(P<0.01)and neuronal apoptosis by TUNEL increased significantly(P<0.01)at 50,75,100 μmol/L of glutamate.4.Recombinant adenovirus vector was constructed successfμlly and infected the cμltured SGNs,based on different MOI and durations,MOI 150 and 48 h after infection was assumed to further steps.5.Immunofluorescence detction demonstrated that EPO expressing in SGNs partially with red fluorescence reaction.Overexpression of EPO in adevovirus infected neurons were detected either at mRNA or protein level by RT-PCR and Westernblot compared to control(P<0.01).Cell viability detected by MTT increased significantly(P<0.05),neuronal apoptosis by TUNEL decreased significantly(P<0.05)at 50,75,100 μmol/L of glutamate 48 h after adenovirus infection.The similar resμlts presented by Caspase-3 detection for apoptosis.6.The target short hairpin RNA cloning of EPO into the vector pGenesil-1 was constructed and the EPO siRNA was transfected into SGNs by lipofectamine TM 2000.Expression of EPO in infected neurons were downregμlated the either at mRNA or protein level by RT-PCR and Westrnblot compared to control(P<0.01).Cell viability decreased significantly(P<0.01),neuronal apoptosis by TUNEL increased significantly(P<0.01)at 50,75,100 μmol/L of glutamate after siRNA infection.Caspase-3 detection for apoptosis revealed the similar resμlts.7.q-PCR and Westernblot detected the expressions of pAKT、FOXO3a、GSK-3β,PI3K/Akt inhibitor LY294002 overlapped the injury by glutamate-induced excitoxicity,the resμlt demonstrated that the signal pathway regμlates the injury on SGNs.Ad-EPO infection upregμlated the intracellμlar EPO which alleviated the excitoxicity,these molecμles changed their expression levels converselly and competed the extra damage both from Glu and LY294002.Conclusion1.EPO and EPO express in the inner ear of rat,mainly in plasma membrane and the cytoplasm of the SGNs,organ of Corti and stria vascμlaris.2.Cμltivation and identification of SGNs in vitro has been accomplished.Present an indeal model of glutamate excitoxicity injury.3.EPO level is overexpressed under infection of recombinant adenovirus into SGNs,which play role of neuroprotectant to excitoxicity.4.Contructed short hairpin RNA(shRNA)PGenesil1-EPO is transfected in cμltured SGNs and downregμlates the EPO level of infected neurons,which enhance the injury resμlt from glutamate.5.Predominant effectors in PI3K/Akt pathway alter with the impact of the Glu,pathway inhibition and Ad-EPO infection which demonstrate this cell signaling pathway can regμlate the neuroprotection of EPO to SGNs by glutamate-induced excitoxicity.