Study On Role And Mechanism Of Autophagy Flux Regulated By Homer1a In Neuronal Oxidative Injury

?Background?Neurological diseases,such as neurotrauma,stroke,Alzheimer's disease and Parkinson's disease and so on,have become significant ones which affect the global disease burden.Oxidative stress-induced neuronal injury is the common pathogenesis of all kinds of acute brain injury and neurodegenerative diseases.Oxidative stress-induced damage refers to the imbalance between intracellular antioxidant system and oxidation system,resulting from the production of considerable reactive oxygen species?ROS?.If excessive ROS cannot be cleared in time,it will lead to intracellular metabolic disorder and structural damage,and ultimately lead to cell death.Therefore,it is of great significance to find potential targets to reduce damage caused by oxidative stress.Homer1 is an important component of neuronal postsynaptic density.Our previous study found that Homer1 subtype Homer1a plays a neuroprotective role in traumatic brain injury and ischemic brain injury,and confirmed that Homer1a can protect mitochondrial function and reduce apoptosis.However,the role and mechanism of Homer1a in oxidative stress-induced neuronal injury is not clear.Autophagy is a metabolic process in which cells degrade intracellular organelles and longevity proteins through autophagosomes and lysosomes.Autophagy flux refers to the whole dynamic process including autophagy production,binding between autophagosomes and lysosomes,and substrate degradation in lysosomes.Previous study reported that enhanced autophagy remove organelles damaged by oxidative stress and reduce cell injury in neurons,suggesting that autophagy may be an intervention target to alleviate oxidative stress injury.However,the relationship between autophagy flux and oxidative stress-induced neuronal damage has not been elucidated and needs to be further clarified.In addition,it has been reported that Homer might interacts with some molecules related to the regulation of autophagy flux,but the relationship between Homer1a and neuronal autophagy flux is not clear.?Methods?1.HT-22 cell line and primary hippocampal neurons were used to induce oxidative stress injury by hydrogen peroxide?H₂O₂?and the neuronal oxidative stress-induced injury models were established respectively.2.Lactate dehydrogenase?LDH?release rate,cell viability and propidium iodide?PI?staining were used to evaluate the cell injury.3.The expression of related molecules was detected by fluorescence real-time quantitative PCR,Western blot?WB?and immunofluorescence.4.Neuronal apoptosis was evaluated by Terminal dUTP Nick end labeling?TUNEL?and Caspase-3 activation rate.5.The level of reactive oxygen species?ROS?in was detected by 2,7-dichlorodihydrofluorescein diacetate?H₂DCFDA?assay.6.The lipid peroxidation was evaluated via malondialdehyde?MDA?assay and 4-hydroxynonenal?4-HNE?assay.7.Mitochondrial membrane potential was evaluated by rhodamine 123 staining and mitochondrial energy metabolism was measured by adenosine triphosphate?ATP?assay.8.Up-regulation or down-regulation of Homer1a expression in neurons was by lentivirus transfection.9.The distribution of autophagosomes in neurons was observed by Transmission Electron Microscope.10.EGFP-RFP-LC3 lentivirus transfection and EGFP-RFP-LC3 transgenic mice were used to visualize autophagy flux in primary hippocampal neurons,and autophagy flow was regulated by autophagic agonists and inhibitors.?Result?Section one:After the oxidative stress injury model was established by HT-22 cell line and primary hippocampal neurons,the expression of Homer1a increased gradually after H₂O₂ treatment.Up-regulation of Homer1a alleviated oxidative stress-induced neuronal damage;down-regulation of Homer1a aggravated oxidative stress-induced neuronal damage.Further studies found that up-regulation of Homer1a reduced apoptosis and oxidative stress and protected mitochondrial function.Section two:Oxidative stress damage mainly led to impaired autophagy flux.Enhancing autophagy flux alleviated oxidative stress injury.Section three:Up-regulation of Homer1a prompted autophagic flux,and blocking autophagy flow weakened the neuroprotective effect of Homer1a.Further study found that up-regulation of Homer1a could increase the phosphorylation of AMPK.When AMPK pathway was inhibited,the regulation of autophagic flux by Homer1a disappeared,and the neuroprotective effect of Homer1a was weakened.?Conclusion?These studies first found that Homer1a,as an endogenous regulator of autophagy flux,played a protective role in neuronal injury induced by oxidative stress.In addition,it was also suggested that impaired autophagy flux was involved in mechanism of neuronal injury induced by oxidative stress.These results provided potential intervention targets and new entry points for the diagnosis and treatment of nervous system diseases and oxidative stress-induced neuronal injury.