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, 10panx, 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 10panx-treated groups. We investigated the effect of 10panx 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 10panx reduced infarct volume and alleviated neurological deficit in the MCAO injury model. 10panx 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, 10panx 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 10panx 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.

中文翻译：

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Pannexin1通道抑制剂（10panx）通过抑制大鼠中RIP3的表达和炎症反应，保护其免受短暂性局灶性脑缺血性损伤。

最近的研究表明，程序性坏死（坏死病）是缺血性神经元损伤的延迟成分，而我们先前的研究表明，pannexin 1通道参与了脑缺血性损伤和细胞炎症反应。在这里，我们检查了pannexin 1通道抑制剂10panx是否可以通过抑制细胞坏死病和相关的炎症减轻大鼠局灶性缺血性脑损伤。将雄性Sprague-Dawley大鼠随机分为假手术组，MCAO（短暂性中脑动脉阻塞）组和10panx治疗组。我们通过评估梗死体积和神经功能缺损来研究10panx的影响。此外，我们使用免疫荧光染色，蛋白质印迹，酶联免疫吸附测定（ELISA）和TUNEL测定确定了潜在的机制。我们证明了10panx减少了MCAO损伤模型中的梗塞体积并减轻了神经功能缺损。10panx改善了缺血后神经元的死亡，但并没有减少TUNEL阳性神经元和裂解的caspase3的表达。相反，坏死病相关蛋白受体相互作用蛋白3（RIP3）的表达明显降低。此外，10panx减少了神经元高迁移率族1（HMGB1）的释放，并抑制了小胶质细胞的活化和促炎因子的分泌。RIP3与HMGB1的免疫共标记显示RIP3蛋白与HMGB1从细胞核到细胞质的释放密切相关。我们的数据表明10panx治疗可通过减少RIP3介导的坏死病，HMGB1释放和相关的炎症反应来减轻MCAO损伤。