Department of Emergency, Yuhuangding Hospital Affiliated to Qingdao University, Yantai, 264000, Shandong, China.
Department of Neurology, Yuhuangding Hospital Affiliated to Qingdao University, No. 20 Yudong Road, Zhifu District, Yantai, 264000, Shandong, China.
Clin Transl Oncol. 2023 Nov;25(11):3203-3216. doi: 10.1007/s12094-023-03190-w. Epub 2023 Apr 27.
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.
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 Fe content were evaluated by the corresponding detection kit.
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.
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.
先前有报道称，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，其保护作用是否通过调节程序性细胞死亡还尚未完全阐明。
在 HT22 细胞和小鼠皮质神经元中建立体外氧葡萄糖剥夺/再氧合（OGD/R）模型。随后，通过 qRT-PCR 和 Western blot 检测 ALDH2 的表达。通过甲基化特异性 PCR（MS-PCR）检测甲基化状态。然后，促进和抑制 ALDH2 的表达，以探讨 ALDH2 在 OGD/R 处理的细胞中的作用。CCK-8 法检测细胞活力，流式细胞术检测细胞凋亡。Western blot 检测凋亡相关蛋白（Caspase 3、Bcl-2 和 Bax）、坏死性凋亡相关蛋白（RIP3 和 MLKL）、焦亡相关蛋白（NLRP3 和 GSDMD）、铁死亡相关蛋白（ACSL4 和 GPX4）和自噬相关蛋白（LC3B 和 p62）。通过 ELISA 法检测 IL-1β 和 IL-18 的产生。通过相应的检测试剂盒评估活性氧（ROS）的产生和铁含量。
在 OGD/R 处理的细胞中，ALDH2 的表达降低，这是由于启动子区域的 ALDH2 过度甲基化所致。ALDH2 过表达可提高细胞活力，而 ALDH2 敲低则抑制 OGD/R 处理的细胞活力。我们还发现，ALDH2 过表达可减轻 OGD/R 诱导的细胞凋亡、焦亡、铁死亡和自噬，而 ALDH2 敲低则促进 OGD/R 诱导的细胞凋亡、焦亡、铁死亡和自噬。
综上所述，我们的结果表明，ALDH2 通过减轻 OGD/R 诱导的细胞凋亡、焦亡、铁死亡和自噬来促进 HT22 细胞和小鼠皮质神经元的细胞活力。
