Affiliation:
1. Anhui Medical College: Anhui Medical University
2. Anhui Medical University
Abstract
Abstract
Aims
The current study investigated differences in ethanol-induced apoptosis of spinal cord dorsal horn neurons at different developmental stages, and the molecular mechanisms involved.
Methods
A mouse ethanol intervention model was established at postnatal days 4, 7, and 12. Primary cells were derived from the spinal cord at postnatal day 4. Western blotting, immunofluorescence, and flow cytometry were used to detect apoptosis-related proteins in spinal cord and primary cells. Kyoto Encyclopedia of Genes and Genomes enrichment analysis of differentially expressed genes originating from the Gene Expression Omnibus dataset GSE184615 was conducted. Effects on Akt/GSK3β pathway proteins were investigated using the GSK3β inhibitor AR-A014418, and the Akt inhibitor DHA. Lentiviral knockdown and overexpression of intervening GSK3β were used in HT22 cell lines to investigate the effects of alcohol on GSK 3β and caspase proteins. J-aggregates, reactive oxygen species assays, and calcein-AM assays were used to investigate mitochondrial function and cell viability.
Results
Ethanol caused downregulation of Akt activity, and upregulation of GSK3β activity and apoptosis. DHA, AR-A014418, and knockdown of GSK3β effectively counteracted ethanol-induced apoptosis, whereas overexpression of GSK3β enhanced the injury process. PI3K activity was unchanged during these processes. Fluorescence colocalization analysis indicated that BAX was translocated to mitochondria during the apoptotic process. BAX was downregulated as the spinal cord developed, consistent with a reduced susceptibility to ethanolinduced apoptosis.
Conclusions
Akt/GSK3β signaling, and BAX together determine the direction of alcoholinduced apoptosis and its susceptibility to change during developmental stages in the spinal cord.
Publisher
Research Square Platform LLC
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