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) 是一种非法的苯丙胺类精神兴奋剂，在世界范围内被滥用并导致严重的公共健康问题。METH 暴露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仍不清楚。在这里，我们研究细胞焦亡是否涉及 METH 诱导的海马神经毒性以及海马神经元细胞中内质网 (ER) 应激的潜在机制。为此，通过免疫印迹和免疫组织化学在海马神经元细胞系 HT-22 中分析细胞焦亡相关蛋白 GSDMD 和 GSDME 的表达水平。接下来，我们使用免疫印迹、LDH 测定和 SYTOX 绿酸染色研究了 HT-22 中 METH 诱导的细胞焦亡。更远，在 HT-22 细胞中使用抑制剂研究了 METH 诱导的海马神经元损伤中细胞焦亡和 ER 应激之间的关系，这些抑制剂包括 TUDCA，一种 ER 应激的特异性抑制剂，GSK-2656157，一种 PERK 通路抑制剂和 STF-0803010，一种 IRE1α 的抑制剂内切核糖核酸酶活性。还使用 siRNA 研究了这种关系，包括 siTRAF2、一种针对 IRE1α 激酶活性的 siRNA 和针对 ATF6 途径的 siATF6，通过免疫印迹、LDH 测定和 SYTOX 绿酸染色进行分析。发现 GSDME 而不是 GSDMD 在 HT-22 细胞中表达。METH 处理诱导裂解的 GSDME-NT 和 LDH 释放的上调，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，表明内质网应激信号通路参与了 METH 诱导的 GSDME 依赖性细胞死亡。总之，这些结果表明 METH 诱导内质网应激，介导海马神经元细胞中 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。