Secretory Protein And Exosome Mediated Molecular Defense Mechanism After Hydrogen Peroxide-induced Injury In HT22 Cells

Introduction:With the arrival of the aging process of the worldwide,the incidence of neurodegenerative diseases is increasing and has been identified as one of the chief causes of mortality especially in the elderly.Oxidative stress is considered to be the main cause of neurodegeneration,especially Alzheimer’s disease（AD）,and neurons in this population are characterized by higher susceptibility of oxidative stress.A plenty of studies have revealed that secretory proteins and exosomes acting as important communication forms of cells under the condition of H₂O₂ injury can enhance the capacity of recipient cells defending oxidative stress and apoptosis.However,whether secretory proteins and exosomes of neurons under oxidative stress are involved in mediating molecular defense mechanism remains unknown.Objective:We used a mouse hippocampal neurons HT22 cell line exposed to H₂O₂,as a model of oxidative stress,to investigate the changes of secretomics of HT22 cells after H₂O₂ injury.And clarifying the effects of secretory proteins and exosomes released after H₂O₂ stimulation on normal neurons to find potential molecular targets in corresponding signaling cascade of oxidative stress.Methods:HT22 cells were exposed to 100μM H₂O₂ for 24 hours.Observing the morphological characteristics of cells by inverted microscope,measuring the cell proliferation rate by CCK-8 and detecting apoptosis by Western Blot.Secretory proteins were extracted by ultrafiltration tube from conditioned medium,while exosomes were extracted by the SBI kit.After exosomes identification（the expression of CD81,Transmission electron microscopy and Nonparametric trajectory analysis）,secretory proteins and exosomes were respectively co-cultured with normal neurons for 24 hours.And then measuring the cell viability by CCK-8,and detecting the expression of apoptosis proteins Bcl2/Bax and Voltage-dependent anion channel 1（VDAC1）by Western blot.Using 2D-PAGE analyzed the changes of secretory proteins and LC-MS/MS mass spectrometry identified the differential proteins of HT22 cells exposed to H₂O₂,and finally analyzing GO and KEGG functional enrichment by bioinformatics.Results:The experimental results showed that the viability of HT22 cells gradually decreased with the increasing of H₂O₂ concentration（P<0.01）.When HT22 cells were treated with 100μM H₂O₂ for 24 hours,significant changes in cell morphology（contraction and condensation）could be observed and the expression of apoptosis protein Bcl-2/Bax decreased（P<0.01）.H₂O₂could induce HT22 cells apoptosis and the oxidative stress injury model was established by treating with 100μM H₂O₂ for 24 hours.Secretory proteins were extracted from conditioned medium,and heat shock protein 90β（HSP90β）expression was found to be up-regulated,while proteasome（Psma6）and vimentin（VIM）expression were down-regulated based on the technology of 2D-PAGE combined with mass spectrometry after HT22 cells exposed to H₂O₂.All differential proteins were also integrated and GO functional annotation revealed that secreted proteins were mainly derived from axons（GO:0150034）,growth cones（GO:0030426）as well as nucleosomes（GO:0000786）,and KEGG pathways were mainly enriched in protein processing （mmu04141）,Parkinson’s disease（mmu05012）as well as programmed cell death（mmu04217）pathways in the endoplasmic reticulum.Exosomes were successfully obtained by using the SBI kit.Western blot results detected that HSP90βprotein was expressed in the exosomes,and the expression level was higher after H₂O₂ stimulation（P<0.05）,and which was similar to the results of secretory proteins.After co-culture of conditioned secretory protein and exosome with HT22 cells,showed that the cell viability was increased,the expression of Bcl-2/Bax was increased（P<0.01）,and the expression of VDAC1 was decreased by exosomes intervention.（P<0.05）Conclusion:The expression of secretomics by HT22 cells exposed to H₂O₂ was different.The majority of secretory proteins are derived from distal axon,growth cones,as well as site of polarized growth;most of these proteins are involved in the endoplasmic reticulum,cellular responses to stimuli,and necroptosis.HSP90βis up-regulated in both secretory proteins and exosomes after H₂O₂ stimulation,and it may play a neuroprotective role by increasing cell viability and inhibiting apoptotic pathway in recipient cells.