| Neurodegenerative diseases are a group of diseases in which there is a gradual loss of neuronal structure or function until the neurons die,resulting in neurological dysfunction,which is a serious threat to human health and quality of life.The causative factors and pathogenesis of neurodegenerative diseases vary,but cellular damage caused by oxidative stress is evident in all diseased neural tissues,which is an important cause of neurodegenerative lesions.Recent studies have found that oxidative stress contributes directly to the dysregulation of mitochondrial energy metabolism and further damages mitochondria,thereby promoting the development of neurodegenerative diseases.Although more studies have been reported on the relevance of mitochondrial oxidative damage to neurodegenerative diseases,the molecular mechanism of mitochondrial oxidative damage is not yet clear.Prx Ⅱ is a member of the peroxidase family,which relies on structurally conserved cysteine residues to react rapidly with ROS and reduce intracellular levels of oxidative stress,thereby participating in a variety of cellular functions.We found that Prx Ⅱ protects against alcohol-induced mitochondria-dependent apoptosis in hippocampal neurons via oxidative stress.This suggests that Prx Ⅱ is a key gene that regulates oxidative stress-induced mitochondrial damage in hippocampal neurons.Therefore,in this study,to further investigate the molecular mechanism of Prx Ⅱ regulation of oxidative stress-induced mitochondrial damage,we used mouse hippocampal neuronal cells as the research object to elucidate the mechanism of oxidative stress-induced mitochondrial damage,and clarify the regulatory role of Prx Ⅱ in it by RNA sequencing combined with cell Biology experimental methods to provide basic theory for the prevention and treatment of oxidative stress-induced neurodegenerative disease development.Alcohol treatment of hippocampal neuronal cell line HT22,1.Using Transmission Electron Microscopy,Flow Cytometry,and Western Blotting in combination with the Ca2+chelator BAPTA,AM,we examined the role of MAM formation in mock and shPrx Ⅱ HT22 after alcohol treatment in regulating mitochondrial damage and mitochondria-dependent apoptosis through modulation of mitochondrial Ca2+levels.2.RNA sequencing of alcohol-treated mock and shPrx Ⅱ HT22 was performed to analyze the mechanism of MAM formation and validated at the mRNA and protein levels using qRT-PCR and protein immunoblotting,respectively.3.Based on the RNA sequencing results combined with Bioinformatics analysis,the expression of miRNAs that play a role in regulating mitochondrial transport was detected and validated at the RNA level.4.Based on the miRNAs obtained from the screening,the predicted transcription factors that can regulate miRNAs were combined with the Jaspar database,and the predicted transcription factors were validated,and the signaling pathways that Prx Ⅱ may regulate transcription factors were analyzed and detected.Results:1.In the observation of alcohol treated HT22 using Transmission Electron Microscopy,there was a significant increase in MAM formation and elevated mitochondrial Ca2+levels within shPrx Ⅱ HT22.After retransfer to Prx Ⅱ,mitochondrial Ca2+levels were downregulated within shPrx Ⅱ HT22 after alcohol treatment.After addition of the Ca2+chelator BAPTA,mitochondrial Ca2+levels within shPrx Ⅱ HT22 were downregulated after alcohol treatment,mitochondrial membrane potential was restored,and mitochondria-dependent apoptosis was inhibited.2.RNA sequencing of alcohol-treated mock and shPrx Ⅱ HT22 and examination of mitochondrial subcellular localization using confocal microscopy showed that mitochondria were significantly clustered around the nucleus within shPrx Ⅱ HT22 after alcohol treatment.RNA sequencing of mock and shPrx Ⅱ HT22 after alcohol treatment revealed a significant down-regulation of the expression level of Armcx3,a key protein that regulates mitochondrial transport,and was verified at both the RNA and protein levels.3.To investigate how Prx Ⅱ regulates the downregulation of Armcx3 mRNA level,miRWalk,miRDB,and starBase Bioinformatics software were used to analyze the miRNAs that can regulate Armcx3 mRNA expression,and a total of 18 intersections were taken from each other,and only miR-18 1b-5p expression level was up-regulated by qRT-PCR.4.combined with Jaspar database predicted transcription factors that can regulate miR-181b-5p and found that transcription factor ATF3 can bind to miR-181b-5p mutual precursors,and in the follow-up demonstrated that PrxⅡ regulates transcription factor ATF3 expression by scavenging ROS,regulating ERS,and activating PERK signaling pathway.These results lead to the following conclusions:1.Alcohol inhibits Armcx3 expression in hippocampal neurons through oxidative stress,inhibits mitochondrial transport and promotes mitochondria-associated endoplasmic reticulum membrane formation,resulting in elevated mitochondrial Ca2+ levels,which induces mitochondrial damage and thus mitochondria-dependent apoptosis.2.Prx Ⅱ gene inhibits oxidative stress through ERS activation of PERK signaling pathway,inhibits transcription of transcription factor ATF3,suppresses miR-181b-5p expression,and thus regulates Armcx3 expression levels,thereby protecting against alcohol-induced mitochondrial damage and mitochondria-dependent apoptosis in hippocampal neurons.In conclusion,we elucidated the molecular mechanism of Prx Ⅱ protection against alcohol-induced mitochondrial oxidative damage,providing a fundamental theoretical and experimental basis for targeting mitochondria-associated endoplasmic reticulum membranes to regulate mitochondrial oxidative damage,and offering new ideas and perspectives for the treatment of neurodegenerative diseases. |
