Innate immunity can facilitate early brain injury (EBI) following subarachnoid hemorrhage (SAH). Numerous studies suggest that pyroptosis could exacerbate extracellular immune responses by promoting secretion of inflammatory cytokines. Transforming growth factor-β-activated kinase 1 (TAK1) is a quintessential kinase that positively regulates inflammation through NF-κB and MAPK signaling cascades. However, the effects of TAK1 on neuroinflammation in EBI following SAH are largely unknown. Two hundred and forty-six male C57BL/6J mice were subjected to the endovascular perforation model of SAH. A selective TAK1 inhibitor, 5Z-7-oxozeaenol (OZ) was administered by intracerebroventricular (i.c.v) injection at 30 min after SAH induction. To genetic knockdown of TAK1, small interfering RNA (siRNA) was i.c.v injected at 48 h before SAH induction. SAH grade, brain water content, BBB permeability, neurological score, western blot, real-time PCR, ELISA, transmission electron microscope, and immunofluorescence staining were performed. Long-term behavioral sequelae were evaluated by the rotarod and Morris water maze tests. Furthermore, OZ was added to the culture medium with oxyhemoglobin (OxyHb) to mimic SAH in vitro. The reactive oxygen species level was detected by DCFH-DA staining. Lysosomal integrity was assessed by Lyso-Tracker Red staining and Acridine Orange staining. The neuronal phosphorylated TAK1 expression was upregulated following SAH. Pharmacologic inhibition of TAK1 with OZ could alleviate neurological deficits, brain edema, and brain-blood barrier (BBB) disruption at 24 h after SAH. In addition, OZ administration restored long-term neurobehavioral function. Furthermore, blockade of TAK1 dampened neuronal pyroptosis by downregulating the N-terminal fragment of GSDMD (GSDMD-N) expression and IL-1β/IL-18 production. Mechanistically, both in vivo and in vitro, we demonstrated that TAK1 can induce neuronal pyroptosis through promoting nuclear translocation of NF-κB p65 and activating nucleotide-binding oligomerization domain (NOD)-like receptor pyrin domain containing 3 (NLRP3) inflammasome. TAK1 siRNA treatment mitigated SAH-induced neurobehavioral deficits and restrained phosphorylated NF-κB p65 expression and NLRP3 inflammasome activation. TAK1 blockade also ameliorated reactive oxygen species (ROS) production and prevented lysosomal cathepsin B releasing into the cytoplasm. Our findings demonstrate that TAK1 modulates NLRP3-mediated neuronal pyroptosis in EBI following SAH. Inhibition of TAK1 may serve as a potential candidate to relieve neuroinflammatory responses triggered by SAH.

中文翻译：

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TAK1介导蛛网膜下腔出血后早期脑损伤的神经元焦亡

先天免疫可以促进蛛网膜下腔出血 (SAH) 后的早期脑损伤 (EBI)。大量研究表明，细胞焦亡可能通过促进炎性细胞因子的分泌而加剧细胞外免疫反应。转化生长因子-β-活化激酶 1 (TAK1) 是一种典型的激酶，可通过 NF-κB 和 MAPK 信号级联来正向调节炎症。然而，TAK1 对 SAH 后 EBI 中神经炎症的影响在很大程度上是未知的。对246只雄性C57BL/6J小鼠进行SAH血管内穿孔模型。在 SAH 诱导后 30 分钟，通过脑室内 (icv) 注射给予选择性 TAK1 抑制剂 5Z-7-oxozaeenol (OZ)。对于 TAK1 的基因敲低，在 SAH 诱导前 48 小时注射小干扰 RNA (siRNA)。SAH级，进行脑含水量、BBB通透性、神经学评分、蛋白质印迹、实时PCR、ELISA、透射电子显微镜和免疫荧光染色。通过旋转棒和莫里斯水迷宫测试评估长期行为后遗症。此外，将 OZ 添加到含氧合血红蛋白 (OxyHb) 的培养基中以在体外模拟 SAH。DCFH-DA染色检测活性氧水平。通过 Lyso-Tracker Red 染色和吖啶橙染色评估溶酶体完整性。SAH 后神经元磷酸化 TAK1 表达上调。用 OZ 对 TAK1 进行药理学抑制可以减轻 SAH 后 24 小时的神经功能缺损、脑水肿和脑血屏障 (BBB) 破坏。此外，OZ 管理恢复了长期的神经行为功能。此外，TAK1 的阻断通过下调 GSDMD 的 N 末端片段 (GSDMD-N) 表达和 IL-1β/IL-18 的产生来抑制神经元焦亡。在体内和体外的机制上，我们证明 TAK1 可以通过促进 NF-κB p65 的核转位和激活含有 3 个 (NLRP3) 炎性体的核苷酸结合寡聚化结构域 (NOD) 样受体 pyrin 结构域来诱导神经元细胞焦亡。TAK1 siRNA 治疗减轻了 SAH 诱导的神经行为缺陷并抑制了磷酸化的 NF-κB p65 表达和 NLRP3 炎性体激活。TAK1 阻断还改善了活性氧 (ROS) 的产生并阻止溶酶体组织蛋白酶 B 释放到细胞质中。我们的研究结果表明 TAK1 调节 SAH 后 EBI 中 NLRP3 介导的神经元细胞焦亡。