Department of Forensic Medicine, Medical College of Soochow University, Suzhou, Jiangsu 215123, P.R. China.
Mol Med Rep. 2019 Mar;19(3):2211-2219. doi: 10.3892/mmr.2019.9853. Epub 2019 Jan 14.
Sodium azide (NaN3), an inhibitor of cytochrome oxidase, induces the release of excitotoxins via an energy impairment and this, in turn, results in neurodegeneration. The present study aimed to investigate the toxic effects NaN3 on apoptosis of PC12 cells and its mechanism of action in peroxisome proliferator‑activated receptor γ co‑activator 1‑α (Pgc‑1α)‑associated signaling pathways. To induce apoptosis, PC12 cells were exposed to NaN3 (0, 5, 10, 20, 40 and 80 mM) for 12, 24, 48 and 72 h. Cell viability was determined by CCK‑8 assay. DAPI staining was employed to additionally examine apoptotic cells and their nuclear changes. Production of reactive oxygen species (ROS), mitochondrial membrane potential (ΔΨm) and apoptotic rate were also assessed by flow cytometry. Cellular ATP content was estimated by firefly luciferase assay. In addition, the expression levels of B‑cell lymphoma 2 (Bcl‑2), Bcl‑2‑associated X protein (Bax), phosphorylated (p)‑Ca2+/calmodulin‑dependent protein kinase (CaMK), p‑p38 mitogen‑activated protein kinase (p38 MAPK), Pgc‑1α, nuclear respiratory factor (Nrf)‑1, mitochondrial transcription factor A (Tfam), p‑extracellular signal‑regulated kinase (Erk)1/2, Nrf‑2 and complex IV (Cox IV) were determined by western blot analysis. The data suggested that NaN3 may induce PC12 cell injury and dose‑dependently decrease the cell viability. The expression levels of pro‑apoptotic proteins Bax and cytochrome c were upregulated, while the expression levels of anti‑apoptotic proteins procaspase‑3 and Bcl‑2 were downregulated. In addition, the phosphorylation of MAPK and Ca2+/calmodulin‑dependent protein kinase II (CaMKⅡ) family members including pan‑calcineurin A was increased, in particular the ratios of p‑p38/p38 and p‑CaMKⅡ/CaMKⅡ. However, the expression levels of Pgc‑1α and its associated proteins, including Nrf‑1/2, Tfam and p‑Erk1/2 were decreased. In addition, mitochondria were the target organelles of NaN3‑induced toxicity in PC12 cells, which moderated the dissipation of ΔΨm, preserved the cellular ATP content, promoted the production of ROS and increased the apoptotic rate. These results suggested that NaN3 induced cell death in PC12 cells via Pgc‑1α‑associated signaling pathways and provided a theoretical basis for additional investigation of the neurotoxic mechanism of NaN3, with applications in neurodegenerative disorders.
叠氮化钠（NaN3）是细胞色素氧化酶的抑制剂，通过能量损伤诱导神经递质释放，进而导致神经退行性变。本研究旨在探讨NaN3 对 PC12 细胞凋亡的毒性作用及其在过氧化物酶体增殖物激活受体γ共激活物 1-α（PGC-1α）相关信号通路中的作用机制。为了诱导细胞凋亡，将 PC12 细胞暴露于 0、5、10、20、40 和 80mM 的 NaN3 中 12、24、48 和 72 小时。通过 CCK-8 测定法测定细胞活力。使用 DAPI 染色进一步检查凋亡细胞及其核变化。通过流式细胞术评估活性氧（ROS）、线粒体膜电位（ΔΨm）和凋亡率。通过萤火虫荧光素酶测定法测定细胞内 ATP 含量。此外，通过 Western blot 分析测定 B 细胞淋巴瘤 2（Bcl-2）、Bcl-2 相关 X 蛋白（Bax）、磷酸化（p）钙/钙调蛋白依赖性蛋白激酶（CaMK）、p-丝裂原活化蛋白激酶 p38（p38 MAPK）、PGC-1α、核呼吸因子（Nrf）-1、线粒体转录因子 A（Tfam）、p-细胞外信号调节激酶（Erk）1/2、Nrf-2 和复合物 IV（Cox IV）的表达水平。数据表明，NaN3 可能诱导 PC12 细胞损伤，并呈剂量依赖性降低细胞活力。促凋亡蛋白 Bax 和细胞色素 c 的表达上调，而抗凋亡蛋白 procaspase-3 和 Bcl-2 的表达下调。此外，包括全钙调神经磷酸酶 A 在内的 MAPK 和钙/钙调蛋白依赖性蛋白激酶 II（CaMKⅡ）家族成员的磷酸化增加，特别是 p-p38/p38 和 p-CaMKⅡ/CaMKⅡ 的比值。然而，PGC-1α 及其相关蛋白（包括 Nrf-1/2、Tfam 和 p-Erk1/2）的表达水平下降。此外，线粒体是 NaN3 诱导的 PC12 细胞毒性的靶细胞器，其调节了ΔΨm 的耗散，保持了细胞内 ATP 含量，促进了 ROS 的产生并增加了凋亡率。这些结果表明，NaN3 通过 PGC-1α 相关信号通路诱导 PC12 细胞死亡，并为进一步研究 NaN3 的神经毒性机制提供了理论依据，可应用于神经退行性疾病。
