Inflammation and subsequent mitochondrial dysfunction and cell death worsen outcomes after revascularization in ischemic stroke. Receptor-interacting protein kinase 1 (RIPK1) activated dynamin-related protein 1 (DRP1) in a NLRPyrin domain containing 3 (NLRP3) inflammasome-dependent fashion and Hypoxia-Inducible Factor (HIF)-1α play key roles in the process. This study determined how phenothiazine drugs (chlorpromazine and promethazine (C + P)) with the hypothermic and normothermic modality impacts the RIPK1/RIPK3-DRP1 and HIF-1α pathways in providing neuroprotection. A total of 150 adult male Sprague-Dawley rats were subjected to 2 h middle cerebral artery occlusion (MCAO) followed by 24 h reperfusion. 8 mg/kg of C + P was administered at onset of reperfusion. Infarct volumes, mRNA and protein expressions of HIF-1α, RIPK1, RIPK3, DRP-1, NLRP3-inflammation and cytochrome c-apoptosis were assessed. Apoptotic cell death, infiltration of neutrophils and macrophages, and mitochondrial function were evaluated. Interaction between RIPK1/RIPK3 and HIF-1α/NLRP3 were determined. In SH-SY5Y cells subjected to oxygen/glucose deprivation (OGD), the normothermic effect of C + P on inflammation and apoptosis were examined. C + P significantly reduced infarct volumes, mitochondrial dysfunction (ATP and ROS concentration, citrate synthase and ATPase activity), inflammation and apoptosis with and without induced hypothermia. Overexpression of RIPK1, RIPK3, DRP-1, NLRP3-inflammasome and cytochrome -apoptosis were all significantly reduced by C + P at 33 °C and the RIPK1 inhibitor (Nec1s), suggesting hypothermic effect of C + P via RIPK1/RIPK3-DRP1pathway. When body temperature was maintained at 37 °C, C + P and HIF-1α inhibitor (YC-1) reduced HIF-1α expression, leading to reduction in mitochondrial dysfunction, NLRP3 inflammasome and cytochrome -apoptosis, as well as the interaction of HIF-1α and NLRP3. These were also evidenced , indicating a normothermic effect of C + P via HIF-1α. Hypothermic and normothermic neuroprotection of C + P involve different pathways. The normothermic effect was mediated by HIF-1α, while hypothermic effect was via RIPK1/RIPK3-DRP1 signaling. This provides a theoretical basis for future precise exploration of hypothermic and normothermic neuroprotection.

中文翻译：

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吩噻嗪通过 RIPK1/RIPK3-DRP1 或 HIF-1α 信号调节 NLRP3 炎症小体相关炎症在急性缺血性中风后低温和常温神经保护中的作用

炎症和随后的线粒体功能障碍和细胞死亡使缺血性中风血运重建后的结果恶化。受体相互作用蛋白激酶 1 (RIPK1) 激活包含 NLRPyrin 结构域 3 (NLRP3) 炎症小体依赖性方式的动力相关蛋白 1 (DRP1)，缺氧诱导因子 (HIF)-1α 在此过程中发挥关键作用。本研究确定了吩噻嗪药物（氯丙嗪和异丙嗪 (C + P)）在低温和常温模式下如何影响 RIPK1/RIPK3-DRP1 和 HIF-1α 通路以提供神经保护。总共 150 只成年雄性 Sprague-Dawley 大鼠接受 2 小时大脑中动脉闭塞 (MCAO)，然后再灌注 24 小时。再灌注开始时施用8mg/kg的C+P。评估梗塞体积、HIF-1α、RIPK1、RIPK3、DRP-1、NLRP3 炎症和细胞色素 c 凋亡的 mRNA 和蛋白表达。评估了细胞凋亡、中性粒细胞和巨噬细胞的浸润以及线粒体功能。确定了 RIPK1/RIPK3 和 HIF-1α/NLRP3 之间的相互作用。在进行缺氧/葡萄糖剥夺 (OGD) 的 SH-SY5Y 细胞中，检查了 C + P 对炎症和细胞凋亡的常温作用。无论有或没有诱导低温，C + P 均显着减少梗塞体积、线粒体功能障碍（ATP 和 ROS 浓度、柠檬酸合酶和 ATP 酶活性）、炎症和细胞凋亡。 33°C 下的 C + P 和 RIPK1 抑制剂 (Nec1s) 均显着降低 RIPK1、RIPK3、DRP-1、NLRP3 炎症小体和细胞色素凋亡的过表达，表明 C + P 通过 RIPK1/RIPK3-DRP1 途径产生低温作用。当体温维持在37℃时，C+P和HIF-1α抑制剂（YC-1）降低HIF-1α表达，导致线粒体功能障碍、NLRP3炎症小体和细胞色素凋亡以及HIF的相互作用减少-1α和NLRP3。这些也得到了证实，表明 C + P 通过 HIF-1α 发挥常温作用。 C+P 的低温和常温神经保护作用涉及不同的途径。正常体温作用是由 HIF-1α 介导的，而低温作用是通过 RIPK1/RIPK3-DRP1 信号传导介导的。这为今后精准探索低温和常温神经保护提供了理论基础。