Background: Subarachnoid hemorrhage (SAH) is a cerebral hemorrhagic disease with a high disability and fatality rate. Cell pyroptosis is involved in the brain injury following SAH. Here, we explored the effect of HDAC inhibitor SAHA against cell pyroptosis after SAH. Methods: The rat SAH model was established by endovascular perforation and the rat microglia were treated with 25 μm oxyhemoglobin (OxyHb) for 24 h to mimic SAH model in vitro. Neurological score and brain edema were assessed in rat SAH model. TUNEL staining detected apoptosis. qRT-PCR and western blotting were employed to detect expression levels of miR-340, NEK7 and inflammatory cytokines. ELISA assay determined the secretion of IL-1β and IL-18 in rat serum and cell supernatant. A lactate dehydrogenase (LDH) kit measured the LDH activity in rat primary microglia. Microglia pyroptosis was detected by flow cytometry. RIP and dual luciferase reporter assays confirmed the binding relationship between miR-340 and NEK7. Results: SAHA alleviated neurological dysfunction, inflammatory injury and microglia pyroptosis in SAH rats. SAHA suppressed LDH release, inflammatory factor expression and pyroptosis in microglia treated with OxyHb. Meanwhile, SAHA increased miR-340 expression and inhibited NEK7 level in vivo and in vitro SAH models. Further, miR-340 directly targeted NEK7 to inhibit the NLRP3 signaling pathway. Knockdown of miR-340 or overexpression of NEK7 reversed the suppressive effects of SAHA on microglia inflammation and pyroptosis. Additionally, knockdown of NEK7 impaired microglia inflammation and pyroptosis induced by miR-340 inhibitor. Conclusion: HDAC inhibitor SAHA ameliorates microglia pyroptosis in SAH through triggering miR-340 expression to suppress NEK7 signaling. This novel mechanism provides promise for SAHA in SAH treatment.

【摘要】：背景蛛网膜下腔出血(SAH)是一种高致残率和致死率的脑出血性疾病。SAH 后的脑损伤涉及细胞焦亡。在这里，我们探讨了 HDAC 抑制剂 SAHA 对 SAH 后细胞焦亡的影响。方法: 通过血管内穿孔建立大鼠SAH模型，大鼠小胶质细胞用25 μm氧合血红蛋白（OxyHb）处理24小时以在体外模拟SAH模型。在大鼠 SAH 模型中评估神经学评分和脑水肿。TUNEL染色检测细胞凋亡。采用 qRT-PCR 和蛋白质印迹法检测 miR-340、NEK7 和炎性细胞因子的表达水平。ELISA测定法测定大鼠血清和细胞上清液中IL-1β和IL-18的分泌。乳酸脱氢酶 (LDH) 试剂盒测量了大鼠原代小胶质细胞中的 LDH 活性。通过流式细胞术检测小胶质细胞焦亡。RIP 和双荧光素酶报告基因检测证实了 miR-340 和 NEK7 之间的结合关系。结果: SAHA 减轻 SAH 大鼠的神经功能障碍、炎症损伤和小胶质细胞焦亡。SAHA 抑制了用 OxyHb 处理的小胶质细胞中的 LDH 释放、炎症因子表达和细胞焦亡。同时，SAHA 在体内和体外 SAH 模型中增加 miR-340 表达并抑制 NEK7 水平。此外，miR-340 直接靶向 NEK7 以抑制 NLRP3 信号通路。miR-340 的敲低或 NEK7 的过表达逆转了 SAHA 对小胶质细胞炎症和细胞焦亡的抑制作用。此外，敲低 NEK7 会损害 miR-340 抑制剂诱导的小胶质细胞炎症和细胞焦亡。结论: HDAC 抑制剂 SAHA 通过触发 miR-340 表达抑制 NEK7 信号传导来改善 SAH 中的小胶质细胞焦亡。这种新机制为 SAHA 治疗 SAH 提供了希望。