Zheng Yonghe, Tang Wenwen, Zeng Hanhai, Peng Yucong, Yu Xiaobo, Yan Feng, Cao Shenglong
Department of Neurosurgery, The Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou, China.
Zhejiang University School of Medicine, Hangzhou, China.
Front Neurol. 2022 Mar 23;13:854671. doi: 10.3389/fneur.2022.854671. eCollection 2022.
Previous studies have proved that inhibiting inflammasome activation provides neuroprotection against early brain injury (EBI) after subarachnoid hemorrhage (SAH), which is mainly focused on the microglial inflammatory response, but the potential role of neuronal inflammasome activation in EBI has not been clearly identified. This study examined whether the pannexin-1 channel inhibitor probenecid could reduce EBI after SAH by inhibiting neuronal AIM2 inflammasome activation.
There are and parts in this study. First, adult male SD rats were subjected to the endovascular perforation mode of SAH. The time course of pannexin-1 and AIM2 expressions were determined after SAH in 72 h. Brain water content, neurological function, AIM2 inflammasome activation, and inflammatory response were evaluated at 24 h after SAH in sham, SAH, and SAH + probenecid groups. In the part, HT22 cell treated with hemin was applied to mimic SAH. The expression of AIM2 inflammasome was detected by immunofluorescence staining. Neuronal death and mitochondrial dysfunction were determined by the LDH assay kit and JC-1 staining.
The pannexin-1 and AIM2 protein levels were upregulated after SAH. Pannexin-1 channel inhibitor probenecid attenuated brain edema and improved neurological dysfunction by reducing AIM2 inflammasome activation and reactive oxygen species (ROS) generation after SAH in rats. Treating HT22 cells with hemin for 12 h resulted in AIM2 and caspase-1 upregulation and increased mitochondrial dysfunction and neuronal cell death. Probenecid significantly attenuated the hemin-induced AIM2 inflammasome activation and neuronal death.
AIM2 inflammasome is activated in neurons after SAH. Pharmacological inhibition of the pannexin-1 channel by probenecid attenuated SAH-induced AIM2 inflammasome activation and EBI and hemin-induced AIM2 inflammasome activation and neuronal death .
以往研究已证明，抑制炎性小体激活可为蛛网膜下腔出血（SAH）后的早期脑损伤（EBI）提供神经保护作用，其主要集中于小胶质细胞炎症反应，但神经元炎性小体激活在EBI中的潜在作用尚未明确。本研究检测泛连接蛋白1通道抑制剂丙磺舒是否可通过抑制神经元AIM2炎性小体激活来减轻SAH后的EBI。
本研究分为两部分。首先，成年雄性SD大鼠采用血管内穿刺法制备SAH模型。于SAH后72小时内测定泛连接蛋白1和AIM2的表达时程。在假手术组、SAH组和SAH+丙磺舒组中，于SAH后24小时评估脑含水量、神经功能、AIM2炎性小体激活及炎症反应。在第二部分中，应用经血红素处理的HT22细胞模拟SAH。通过免疫荧光染色检测AIM2炎性小体的表达。采用乳酸脱氢酶（LDH）检测试剂盒和JC-1染色法测定神经元死亡和线粒体功能障碍。
SAH后泛连接蛋白1和AIM2蛋白水平上调。泛连接蛋白1通道抑制剂丙磺舒可减轻大鼠SAH后的脑水肿，并通过降低AIM2炎性小体激活和活性氧（ROS）生成来改善神经功能障碍。用血红素处理HT22细胞12小时导致AIM2和半胱天冬酶-1上调，并增加线粒体功能障碍和神经元细胞死亡。丙磺舒可显著减轻血红素诱导的AIM2炎性小体激活和神经元死亡。
SAH后神经元中的AIM2炎性小体被激活。丙磺舒对泛连接蛋白1通道的药理学抑制作用减轻了SAH诱导的AIM2炎性小体激活和EBI，以及血红素诱导的AIM2炎性小体激活和神经元死亡。
