目的:先前报道醛脱氢酶2家族成员(ALDH2)在脑缺血/再灌注损伤中具有神经保护作用。然而,保护作用是否通过介导程序性细胞死亡尚未完全阐明。
方法:在HT22细胞和小鼠皮质神经元中建立体外氧糖剥夺/复氧(OGD/R)模型。随后,通过qRT-PCR和蛋白质印迹评估ALDH2表达。通过甲基化特异性PCR(MS-PCR)检查甲基化状态。然后,ALDH2表达被促进和抑制以探讨ALDH2在OGD/R处理的细胞中的作用。CCK-8法用于检测细胞活力,并应用流式细胞术评价细胞凋亡。Westernblot检测细胞凋亡相关蛋白(Caspase3、Bcl-2和Bax),坏死相关蛋白(RIP3和MLKL),焦亡相关蛋白(NLRP3和GSDMD),铁凋亡相关蛋白(ACSL4和GPX4),和自噬相关蛋白(LC3B,andp62).通过ELISA测定评估IL-1β和IL-18的产生。通过相应的检测试剂盒评估活性氧的产生和Fe2含量。
结果:在OGD/R处理的细胞中,ALDH2表达降低,这是由于ALDH2在启动子区域的过度甲基化。在OGD/R处理的细胞中,ALDH2过表达改善细胞活力,并且ALDH2敲低抑制细胞活力。我们还发现ALDH2过表达减弱了OGD/R诱导的细胞凋亡,焦亡,铁凋亡和自噬,而ALDH2敲低促进OGD/R诱导的细胞凋亡,焦亡,铁凋亡和自噬。
结论:总的来说,我们的结果表明ALDH2减弱了OGD/R诱导的细胞凋亡,焦亡,铁凋亡和自噬促进HT22细胞和小鼠皮质神经元的细胞活力。
OBJECTIVE: It is previously reported that aldehyde dehydrogenase 2 family member (ALDH2) shows neuroprotective effects in cerebral ischemia/reperfusion injury. However, whether the protective effects are through mediating the programmed cell death is yet to be fully elucidated.
METHODS: In vitro oxygen-glucose deprivation/reoxygenation (OGD/R) model was established in HT22 cells and mouse cortical neurons. Subsequently, ALDH2 expression were assessed by qRT-PCR and western blot. The methylation status was examined by methylation-specific PCR (MS-PCR). Then, ALDH2 expression was promoted and suppressed to explore the role of ALDH2 in OGD/R-treated cells. CCK-8 assay was applied to detect cell viability, and flow cytometry was applied to evaluate cell apoptosis. Western blot was applied to detect the apoptosis-related proteins (Caspase 3, Bcl-2 and Bax), necroptosis-related proteins (RIP3 and MLKL), pyroptosis-related proteins (NLRP3 and GSDMD), ferroptosis-related protein (ACSL4 and GPX4), and autophagy-related proteins (LC3B, and p62). IL-1β and IL-18 production was evaluated by ELISA assay. Reactive oxygen species production and Fe2+ content were evaluated by the corresponding detection kit.
RESULTS: In OGD/R-treated cells, ALDH2 expression was decreased, which was due to the hypermethylation of ALDH2 in the promoter region. ALDH2 overexpression improved cell viability and ALDH2 knockdown suppressed cell viability in OGD/R-treated cells. We also found that ALDH2 overexpression attenuated OGD/R-induced cell apoptosis, pyroptosis, ferroptosis and autophagy, while ALDH2 knockdown facilitated the OGD/R-induced cell apoptosis, pyroptosis, ferroptosis and autophagy.
CONCLUSIONS: Collectively, our results implied that ALDH2 attenuated OGD/R-induced cell apoptosis, pyroptosis, ferroptosis and autophagy to promote cell viability in HT22 cells and mouse cortical neurons.