College of Forensic Medicine, Hebei Medical University, Hebei Key Laboratory of Forensic Medicine, Collaborative Innovation Center of Forensic Medical Molecular Identification, Shijiazhuang, PR China.
College of Forensic Medicine, Hebei Medical University, Hebei Key Laboratory of Forensic Medicine, Collaborative Innovation Center of Forensic Medical Molecular Identification, Shijiazhuang, PR China.
Brain Res Bull. 2020 Sep;162:73-83. doi: 10.1016/j.brainresbull.2020.06.005. Epub 2020 Jun 13.
Methamphetamine (METH) is an illegal amphetamine-typed psychostimulant that is abused worldwide and causes serious public health problems. METH exposure induces apoptosis and autophagy in neuronal cells. However, the role of pyroptosis in METH-induced neurotoxicity is still unclear. Here, we investigate whether pyroptosis is involved in METH-induced hippocampal neurotoxicity and the potential mechanisms of Endoplasmic reticulum (ER) stress in hippocampal neuronal cells. For this purpose, the expression levels of pyroptosis-related proteins, GSDMD and GSDME, were analyzed by immunoblotting and immunohistochemistry in the hippocampal neuron cell line HT-22. Next, we explored METH-induced pyroptosis in HT-22 using immunoblotting, LDH assays and SYTOX green acid staining. Further, the relationship between pyroptosis and ER stress in METH-induced hippocampal neuron damage was studied in HT-22 cells using inhibitors including TUDCA, a specific inhibitor of ER stress, GSK-2656157, a PERK pathway inhibitor and STF-0803010, an inhibitor of IRE1α endoribonuclease activity. This relationship was also studied using siRNAs, including siTRAF2, an siRNA against IRE1α kinase activity and siATF6 against the ATF6 pathway, which were analyzed by immunoblotting, LDH assays and SYTOX green acid staining. GSDME but not GSDMD was found to be expressed in HT-22 cells. METH treatment induced the upregulation of cleaved GSDME-NT and LDH release, as well as the increase of SYTOX green positive cells in HT-22 cells, which was partly reversed by inhibitors and siRNAs, indicating that the ER stress signaling pathway was involved in GSDME-dependent cell death induced by METH. In summary, these results revealed that METH induced ER stress that mediated GSDME-dependent cell death in hippocampal neuronal cells. These findings provide novel insight into the mechanisms of METH-induced neurotoxicity.
甲基苯丙胺（METH）是一种在全球范围内被滥用的非法安非他命类精神兴奋剂，会导致严重的公共健康问题。METH 暴露会诱导神经元细胞凋亡和自噬。然而，细胞焦亡在 METH 诱导的神经毒性中的作用尚不清楚。在这里，我们研究了细胞焦亡是否参与 METH 诱导的海马神经毒性以及内质网（ER）应激在海马神经元细胞中的潜在机制。为此，通过免疫印迹和免疫组织化学分析 HT-22 海马神经元细胞系中细胞焦亡相关蛋白 GSDMD 和 GSDME 的表达水平。接下来，我们使用免疫印迹、LDH 测定和 SYTOX 绿色酸染色研究 METH 诱导的 HT-22 细胞焦亡。此外，在 HT-22 细胞中，使用包括 TUDCA（一种 ER 应激的特异性抑制剂）、GSK-2656157（一种 PERK 途径抑制剂）和 STF-0803010（一种 IRE1α 内切核酸酶活性抑制剂）在内的抑制剂研究了 METH 诱导的海马神经元损伤中细胞焦亡与 ER 应激之间的关系。还使用包括针对 IRE1α 激酶活性的 siTRAF2 和针对 ATF6 途径的 siATF6 在内的 siRNA 研究了这种关系，通过免疫印迹、LDH 测定和 SYTOX 绿色酸染色进行分析。发现 GSDME 而不是 GSDMD 在 HT-22 细胞中表达。METH 处理诱导 cleaved GSDME-NT 的上调和 LDH 释放，以及 HT-22 细胞中 SYTOX 绿色阳性细胞的增加，这部分被抑制剂和 siRNA 逆转，表明 ER 应激信号通路参与了 METH 诱导的依赖 GSDME 的细胞死亡。总之，这些结果表明 METH 诱导的 ER 应激介导了海马神经元细胞中 GSDME 依赖性细胞死亡。这些发现为 METH 诱导的神经毒性机制提供了新的见解。
