Department of Forensic Pathology, School of Forensic Medicine, China Medical University, Shenyang, Liaoning 110122, PR China; Department of General Surgery, Shengjing Hospital of China Medical University, No. 36, San Hao Street, Shenyang, Liaoning 110004, PR China.
Department of Forensic Pathology, School of Forensic Medicine, China Medical University, Shenyang, Liaoning 110122, PR China; The People's Procuratorate of Liaoning Province Judicial Authentication Center, Shenyang, Liaoning 110032, PR China.
Toxicol In Vitro. 2021 Apr;72:105104. doi: 10.1016/j.tiv.2021.105104. Epub 2021 Jan 28.
Long-term alcohol intake may cause nerve cell apoptosis and induce various encephalopathies. Previously, we have shown that the expression of Na/Ca exchanger 3 (NCX3) was associated with the intracellular calcium concentration ([Ca2+]i) and apoptosis, involved in the spatial memory impairment in male C57BL/6 mice with chronic ethanol (EtOH) exposure. However, the mechanism involved is unclear. Here, we investigated the expression of NCX3 and its protective effect on SK-N-SH cells (a nerve cell line) after EtOH exposure. [Ca]i was measured using Fluo-3 AM reagent. Cell viability and the apoptotic rate were assayed using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) and flow cytometry, respectively. The expression of p-cAMP-responsive element binding protein1(p-CREB 1), NCX3 protein, and mRNA were observed using Western blotting and quantitative real-time polymerase chain reaction (qRT-PCR), respectively. Cleaved-caspase-3, caspase-3, rabbit anti- poly (ADP-ribose) polymerase-1 (PARP-1) and calpain-1 proteins were used to assess the degree of apoptosis. Our results showed that EtOH increased [Ca]i and apoptosis of SK-N-SH cells in a concentration- and time-dependent manner. The expression of NCX3 protein and mRNA was up regulated obviously after SK-N-SH cells were treated with EtOH. The phosphorylation levels of Akt and CREB 1 were up regulated in cells treated with EtOH. The expression of NCX3 protein was reduced in the SK-N-SH cells treated with Akt phosphorylation inhibitor (LY294002). The [Ca]i and apoptosis rate of SK-N-SH cells increased 1.31-fold and 1.52-fold after silencing NCX3 compared with those treated with 200 mM EtOH alone for 2 d. In contrast, the [Ca]i and apoptosis rate of SK-N-SH cells decreased 0.26-fold and 0.35-fold after overexpression of NCX3 in the 2 d-200 mM EtOH treatment group. These results suggest that NCX3 plays a critical role in neuronal protection via the elimination of intracellular Ca, which may be a promising target for the prevention and treatment of encephalopathy after ethanol exposure.
长期饮酒可能导致神经细胞凋亡，并引发各种脑病。此前，我们已经表明，钠/钙交换器 3（NCX3）的表达与细胞内钙离子浓度([Ca2+]i)和细胞凋亡有关，参与了慢性乙醇（EtOH）暴露的雄性 C57BL/6 小鼠的空间记忆障碍。然而，其涉及的机制尚不清楚。在这里，我们研究了 NCX3 的表达及其对 EtOH 暴露后的 SK-N-SH 细胞（一种神经细胞系）的保护作用。使用 Fluo-3 AM 试剂测量 [Ca]i。使用 3-(4,5-二甲基噻唑-2-基)-5-(3-羧基甲氧基苯基)-2-(4-磺苯基)-2H-四唑鎓，内盐（MTS）和流式细胞术分别测定细胞活力和凋亡率。使用 Western blot 和实时定量聚合酶链反应（qRT-PCR）分别观察 p-CAMP 反应元件结合蛋白 1（p-CREB 1）、NCX3 蛋白和 mRNA 的表达。使用裂解型半胱天冬酶-3、半胱天冬酶-3、兔抗多聚（ADP-核糖）聚合酶 1（PARP-1）和钙蛋白酶-1 蛋白评估细胞凋亡程度。我们的结果表明，EtOH 以浓度和时间依赖的方式增加 SK-N-SH 细胞的 [Ca]i 和凋亡。EtOH 处理后 SK-N-SH 细胞中 NCX3 蛋白和 mRNA 的表达明显上调。EtOH 处理的细胞中 Akt 和 CREB 1 的磷酸化水平上调。用 Akt 磷酸化抑制剂（LY294002）处理 SK-N-SH 细胞后，NCX3 蛋白的表达减少。与单独用 200 mM EtOH 处理 2 天相比，沉默 NCX3 后 SK-N-SH 细胞的 [Ca]i 和凋亡率增加了 1.31 倍和 1.52 倍。相反，在 2 天-200 mM EtOH 处理组中过表达 NCX3 后，SK-N-SH 细胞的 [Ca]i 和凋亡率降低了 0.26 倍和 0.35 倍。这些结果表明，NCX3 通过消除细胞内 Ca 发挥关键的神经元保护作用，这可能是预防和治疗乙醇暴露后脑病的有希望的靶点。
