Neuroprotective Effect Of ALOXE3 Overexpression On H₂O₂-induced Oxidative Stress In Hippocampal HT22 Cells

IntroductionNeurodegenerative diseases are a type of disease characterized by progressive loss of neuronal structure and function which leads to cell death.Oxidative stress（OS）,a key early event in neurodegenerative diseases,results in lipid peroxidation,DNA damage,metabolic disorders and mitochondrial dysfunction,which initiate apoptosis-associated signaling pathways and then lead to neuronal death.Arachidonic acid（AA）,a polyunsaturated fatty acid in cells,plays an important role in the regulation of brain function.Recent evidences have suggested that rapid increase in intracellular AA is involved in the OS-induced cell damage and neurodegeneration with regard to activation of p38,MAPK,and JNK pathways.Therefore,we propose that AA and its metabolic pathways should play important roles in OS-induced cell damage and neurodegeneration.Arachidonic acid lipoxygenase-3（ALOXE3）is a key enzyme in the AA metabolic pathway.Our previous studies have identified that ALOXE3 was highly expressed in mouse hippocampus,and overexpression of ALOXE3 could significantly reduce the intracellular AA levels.However,whether ALOXE3 can alleviate the OS-induced neuronal damage by catalyzing the intracellular AA.ObjectiveThis study aims to investigate the effect of ALOXE3 overexpression on H₂O₂-induced oxidative stress and cell damages in hippocampal neurons,which will reveal the potential pathways for ALOXE3’s neuroprotective effect and provide a new cue for future diagnosis and treatment of neurodegenerative diseases.Methods1.Hippocampal HT22 cells were treated with a titrated dose of H₂O₂.Cell viability was determined by a CCK-8 kit,and intracellular ROS were measured by a ROS detection kit.Intracellular AA level was detected using an Elisa kit.2.HT22 cells were treated with a titrated dose of AA.The cell viability and intracellular ROS levels were determined according to above descriptions.3.Western blot and RT-q PCR experiments were performed to detect protein and m RNA levels of ALOXE3,SOD1,and SOD2 in the cells treated with H₂O₂ or AA.4.A recombinant vector containing an Aloxe3 coding region was constructed,and the recombinant vector was transfected into the HT22 cells by liposome-mediated transfection to overexpress ALOXE3.Simultaneously,the cells were treated with H₂O₂or AA,and then the cell viability,intracellular ROS levels and the ALOXE3,SOD1 and SOD2 levels were analyzed according to above descriptions.5.Western blots were used to detect the changes in phosphorylation modifications of some key MAPK-pathway proteins（such as ERK1/2,p38 and JNK）in the HT22 cells upon the above-described treatments.Results1.Oxidative stress and cell damage in the HT22 cells treated with H2O2 or AAData from the HT22 cells treated with a titrated（0-500μM）H₂O₂ showed that the cell viability gradually decreased with an increasing usage of H₂O₂,while under the same condition,the intracellular ROS and AA levels gradually increased.Compared with the control group,the changes of cell viability,ROS and AA levels were significant when the H₂O₂ dose increased to 200μM,which indicated that this dose is reasonable for H₂O₂-induced oxidative stress and cell damage in this cell line.In addition,the cell viability gradually decreased with an increased usage of exogenous AA（0-50μM）,and the intracellular ROS level increased in a dose-dependent pattern.Compared with the control group,the cell viability was significantly decreased when the AA dose increased to 20μM,and the ROS levels were significantly increased when the AA dose increased to 10μM.Together,the above results indicate that H₂O₂ or AA treatment could lead to oxidative stress and cell damage in HT22 cells.2.Decreases of SOD2 and ALOXE3 expression in H2O2-or AA-treated cellsTo investigate the relationship between oxidative stress and AA metabolism,this study detected the expression of oxidative stress-related enzymes SOD and AA metabolic enzyme ALOXE3 in the HT22 cells upon H₂O₂ or AA treatment.The data showed that the protein and m RNA levels of SOD2,instead of SOD1,were significantly decreased the cells treated with H₂O₂（200μM,500μM）or AA（20μM,50μM）in a dose-dependent pattern.In addition,the ALOXE3 protein and m RNA levels were also significantly downregulated upon H₂O₂ or AA treatment.Therefore,these results suggest that exogenous H₂O₂ or AA treatment can lead to downregulation of SOD2 and ALOXE3expression.3.ALOXE3 overexpression alleviates the H2O2-or AA-induced oxidative stress and cell damageTo determine whether ALOXE3 plays a neuroprotective role in H₂O₂-or AA-induced oxidative stress and cell damage,we then overexpressed ALOXE3 in the H₂O₂-or AA-treated HT22 cells.Compared with the control group,the cell viability of the cells treated with H₂O₂（200μM）or AA（20μM）significantly decreased,and the intracellular ROS levels significantly increased upon H₂O₂ or AA treatment.Compared with the H₂O₂or AA treatment,overexpression of ALOXE3 significantly enhanced cell viability and downregulated ROS levels.Thus,these data indicate that ALOXE3 overexpression could alleviate the H₂O₂-or AA-induced oxidative stress and decrease in cell viability.4.ALOXE3 overexpression reverses the H2O2-or AA-induced downregulation of SOD2 expressionTo investigate the potential factors for the neuroprotective effect of ALOXE3overexpression,this study analyzed the expression of antioxidant stress kinases SOD1and SOD2 upon both overexpression of ALOXE3 and H₂O₂ or AA treatment.Our data showed that,the expression of SOD2,instead of SOD1,significantly decreased upon H₂O₂ or AA treatment compared to that in the control group.Compared with the H₂O₂or AA treatment,SOD2 expression significantly increased upon overexpression of ALOXE3,and no significant change in SOD1 expression was observed between the two group.Therefore,it could be speculated that ALOXE3 overexpression should play a protective effect on oxidative stress-induced neuronal damage by promoting upregulation of SOD2 expression.5.ALOXE3 overexpression impedes the H2O2-or AA-induced activation of MAPK pathwayPrevious evidences have suggested some essential roles of MAPK activation in the oxidative stress-induced neuronal damage,we propose that that overexpression of ALOXE3 may inhibit the oxidative stress-induced activation of MAPK pathway.Our Western blot data indicated that the relative ratios of three key MAPK components,including p-ERK/ERK,p-p38/p38,and p-JNK/JNK,significantly increased upon H₂O₂or AA treatment.Compared with the H₂O₂ or AA treated group,the relative ratios of p-ERK/ERK,p-38/38,and p-JNK/JNK significantly decreased upon overexpression of ALOXE3.These results suggest that ALOXE3 overexpression could inhibit the H₂O₂-or AA-induced activation of MAPK pathway,which might alleviate the oxidative stress-induced neuronal damage.ConclusionThis study suggests that H₂O₂-or AA-induced oxidative stress downregulate ALOXE3 expression,and overexpression of ALOXE3 could restore the H₂O₂-or AA-induced decline in cell viability and decrease in SOD2 expression.Restoration of SOD2expression by ALOXE3 overexpression alleviates oxidative stress,and then inhibit MAPK pathway,which ultimately alleviate the oxidative stress-induced neuronal damages.Therefore,our study suggests that ALOXE3 might be a potential intervention target for oxidative stress-related neurodegenerative disorders.