Sun Shuaichen, Wang Zizhuo, Xu Xiaoxiang, Ding Xihui, Xu Jianguang, Nan Xiang, Li Xiaohui, Xu Jinyong, Ren Zhenhua
Department of Anatomy, Anhui Medical University, 81 Meishan Road, Hefei, 230032, Anhui, China.
College and Hospital of Stomatology, Key Lab. of Oral Diseases Research of Anhui Privine, Anhui Medical University, Hefei, 230032, Anhui, China.
Mol Neurobiol. 2025 Apr;62(4):4301-4318. doi: 10.1007/s12035-024-04510-z. Epub 2024 Oct 23.
The current study investigated differences in ethanol-induced apoptosis of spinal cord dorsal horn neurons at different developmental stages and the molecular mechanisms involved. A mouse ethanol intervention model was established on 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 the 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. 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 ethanol-induced apoptosis. Akt/GSK3β signaling and BAX together determine the direction of alcohol-induced apoptosis and its susceptibility to change during developmental stages in the spinal cord.
本研究调查了不同发育阶段乙醇诱导的脊髓背角神经元凋亡差异及其相关分子机制。在出生后第4、7和12天建立小鼠乙醇干预模型。原代细胞来源于出生后第4天的脊髓。采用蛋白质免疫印迹法、免疫荧光法和流式细胞术检测脊髓和原代细胞中凋亡相关蛋白。对来自基因表达综合数据库GSE184615的差异表达基因进行京都基因与基因组百科全书富集分析。使用GSK3β抑制剂AR-A014418和Akt抑制剂DHA研究对Akt/GSK3β信号通路蛋白的影响。在HT22细胞系中使用慢病毒敲低和过表达干预性GSK3β，以研究酒精对GSK 3β和半胱天冬酶蛋白的影响。采用J-聚集体、活性氧检测和钙黄绿素-AM检测来研究线粒体功能和细胞活力。乙醇导致Akt活性下调、GSK3β活性上调和细胞凋亡。DHA、AR-A014418和GSK3β敲低有效对抗乙醇诱导的细胞凋亡，而GSK3β过表达则增强损伤过程。在这些过程中PI3K活性未发生变化。荧光共定位分析表明，在凋亡过程中BAX转位至线粒体。随着脊髓发育，BAX表达下调，这与乙醇诱导凋亡的易感性降低一致。Akt/GSK3β信号通路和BAX共同决定酒精诱导凋亡的方向及其在脊髓发育阶段易感性的变化。
