Wei Ruili, Bao Wangxiao, He Fangping, Meng Fangxia, Liang Hui, Luo Benyan
Department of Neurology, Brain Medical Centre, First Affiliated Hospital, Zhejiang University School of Medicine, 79 Qingchun Road, Hangzhou 310003, China.
Department of Neurology, Brain Medical Centre, First Affiliated Hospital, Zhejiang University School of Medicine, 79 Qingchun Road, Hangzhou 310003, China.
Neuroscience. 2020 Jun 15;437:23-33. doi: 10.1016/j.neuroscience.2020.02.042. Epub 2020 Mar 12.
Recent studies have demonstrated that programmed necrosis (necroptosis) is a delayed component of ischemic neuronal injury and our previous study has shown that pannexin 1 channel is involved in cerebral ischemic injury and cellular inflammatory response. Here, we examined whether the pannexin 1 channel inhibitor, panx, could reduce focal ischemic brain injury in rats by inhibiting cellular necroptosis and the associated inflammation. Male Sprague-Dawley rats were randomly divided into sham-operated, MCAO (transient middle cerebral artery occlusion) group, and panx-treated groups. We investigated the effect of panx by assessing infarct volume and neurological deficit. Further, we determined the potential mechanism using immunofluorescent staining, Western blotting, enzyme-linked immunosorbent assay (ELISA) and TUNEL assay. We demonstrated that panx reduced infarct volume and alleviated neurological deficit in the MCAO injury model. panx ameliorated post-ischemic neuronal death, but it did not reduce the TUNEL positive neurons and expression of cleaved-caspase3. In contrast, expression of necroptosis related protein receptor-interacting protein 3 (RIP3) was significantly decreased. Furthermore, panx reduced the release of high mobility group box 1 (HMGB1) from neurons and inhibited microglial activation and secretion of pro-inflammatory factors. Immunent co-labeling of RIP3 with HMGB1 showed that RIP3 protein was closely related with the release of HMGB1 from nucleus to cytoplasm. Our data suggested that panx treatment may ameliorate MCAO injury by reducing RIP3-mediated necroptosis, HMGB1 release and associated inflammatory response. RIP3 may play an important role in the release of HMGB1 and inflammation after stroke.
近期研究表明，程序性坏死（坏死性凋亡）是缺血性神经元损伤的一个延迟组成部分，并且我们之前的研究已表明，泛连接蛋白1通道参与脑缺血损伤及细胞炎症反应。在此，我们研究了泛连接蛋白1通道抑制剂panx是否可通过抑制细胞坏死性凋亡及相关炎症来减轻大鼠局灶性缺血性脑损伤。雄性Sprague-Dawley大鼠被随机分为假手术组、大脑中动脉闭塞（短暂性大脑中动脉闭塞）组和panx治疗组。我们通过评估梗死体积和神经功能缺损来研究panx的作用。此外，我们使用免疫荧光染色、蛋白质印迹法、酶联免疫吸附测定（ELISA）和TUNEL测定来确定潜在机制。我们证明，panx可减小大脑中动脉闭塞损伤模型中的梗死体积并减轻神经功能缺损。panx改善了缺血后神经元死亡，但并未减少TUNEL阳性神经元及裂解型半胱天冬酶3的表达。相反，坏死性凋亡相关蛋白受体相互作用蛋白3（RIP3）的表达显著降低。此外，panx减少了神经元中高迁移率族蛋白B1（HMGB1）的释放，并抑制了小胶质细胞的激活及促炎因子的分泌。RIP3与HMGB1的免疫共标记显示，RIP3蛋白与HMGB1从细胞核向细胞质的释放密切相关。我们的数据表明，panx治疗可能通过减少RIP3介导的坏死性凋亡、HMGB1释放及相关炎症反应来改善大脑中动脉闭塞损伤。RIP3可能在中风后HMGB1的释放及炎症中起重要作用。
