Department of Anesthesiology, The Eye, Ear, Nose and Throat Hospital, Fudan University, Fenyang Road #83, Shanghai, 200031, People's Republic of China.
Department of Oro-Maxillofacial Head and Neck Oncology, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai Key Laboratory of Stomatology, Shanghai, China.
Cell Mol Neurobiol. 2022 Oct;42(7):2235-2244. doi: 10.1007/s10571-021-01098-z. Epub 2021 May 15.
Recent studies have shown that exposure to sevoflurane in developing brains causes neuronal apoptosis and cognitive dysfunction. "Necroptosis" is a novel pathway of necrosis. We introduced the caspase-specific inhibitor Z-VAD in addition to the receptor-interacting protein kinase 1 (RIPK1) inhibitor Nec-1, to ascertain the existence and importance of necroptosis. Sprague-Dawley rat pups postnatal day 7 were randomly assigned into one of five groups: control, sevoflurane + Z-VAD, sevoflurane + Nec-1, sevoflurane + Z-VAD + Nec-1 and 3% sevoflurane group. Neuronal apoptosis was evaluated by hematoxylin and eosin staining. The MTT assay was performed to evaluate cell viability. Immunofluorescence was employed to measure expression of RIPK1 and RIPK3. Western blots showing expression of RIPK1, RIPK3 and phosphorylation of mixed lineage kinase domain-like (p-MLKL) were used to explore the role of necroptosis. Binding of RIPK1/RIPK3 was detected via co-immunoprecipitation. Finally, the Morris water maze test was used to determine cognitive function. Exposure to 3% sevoflurane for 6 h induced neurotoxicity and inhibited cell viability. Neuron viability was low in the SEV, SEV + Z-VAD and SEV + Nec-1 groups. The study revealed that RIPK1 and RIPK3 protein expression increased significantly, but there was no significant differences between the SEV and SEV + Z-VAD groups. The expression of p-MLKL significantly increased in the SEV and SEV + Z-VAD groups, but not in the SEV + Nec-1 group or SEV + Z-VAD + Nec-1 group compared to the control group. Co-immunoprecipitation results showed that sevoflurane exposure enhanced binding of RIPK1/RIPK3 protein significantly. Blockade of apoptosis and necroptosis alleviated sevoflurane-induced cognitive impairment. Sevoflurane exposure elicited neurotoxicity within neonatal hippocampal neurons and tissues. Blockade of apoptosis or necroptosis alone did not attenuate sevoflurane-induced neurotoxicity (SIN). RIPK1/RIPK3-mediated necroptosis was involved in SIN in hippocampal neurons. SIN could be attenuated only by inhibiting both apoptosis and necroptosis.
最近的研究表明，七氟醚暴露于发育中的大脑会导致神经元凋亡和认知功能障碍。“坏死性凋亡”是一种新的坏死途径。我们引入了半胱天冬酶特异性抑制剂 Z-VAD 以及受体相互作用蛋白激酶 1（RIPK1）抑制剂 Nec-1，以确定坏死性凋亡的存在和重要性。新生第 7 天的 Sprague-Dawley 幼鼠被随机分为五组之一：对照组、七氟醚+Z-VAD、七氟醚+Nec-1、七氟醚+Z-VAD+Nec-1 和 3%七氟醚组。通过苏木精和伊红染色评估神经元凋亡。通过 MTT 测定评估细胞活力。免疫荧光用于测量 RIPK1 和 RIPK3 的表达。Western blot 显示 RIPK1、RIPK3 和混合谱系激酶结构域样（p-MLKL）磷酸化的表达，用于探索坏死性凋亡的作用。通过共免疫沉淀检测 RIPK1/RIPK3 的结合。最后，使用 Morris 水迷宫测试确定认知功能。暴露于 3%七氟醚 6 小时会引起神经毒性并抑制细胞活力。SEV、SEV+Z-VAD 和 SEV+Nec-1 组神经元活力较低。研究表明，RIPK1 和 RIPK3 蛋白表达显著增加，但 SEV 和 SEV+Z-VAD 组之间没有显著差异。SEV 和 SEV+Z-VAD 组 p-MLKL 的表达显著增加，但 SEV+Nec-1 组或 SEV+Z-VAD+Nec-1 组与对照组相比没有增加。共免疫沉淀结果表明，七氟醚暴露显著增强了 RIPK1/RIPK3 蛋白的结合。阻断凋亡和坏死性凋亡可减轻七氟醚引起的认知障碍。七氟醚暴露引起新生海马神经元和组织的神经毒性。单独阻断凋亡或坏死性凋亡并不能减轻七氟醚引起的神经毒性（SIN）。RIPK1/RIPK3 介导的坏死性凋亡参与了海马神经元中的 SIN。只有同时抑制凋亡和坏死性凋亡才能减轻 SIN。
