China-America Institute of Neuroscience, Beijing Luhe Hospital, Capital Medical University, China; Department of Neurology, Beijing Luhe Hospital, Capital Medical University, China.
Department of Neurosurgery, Wayne State University School of Medicine, Detroit, MI, USA.
Redox Biol. 2024 Jul;73:103169. doi: 10.1016/j.redox.2024.103169. Epub 2024 Apr 26.
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 c-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 c-apoptosis, as well as the interaction of HIF-1α and NLRP3. These were also evidenced in vitro, 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.
在缺血性中风再灌注后，炎症和随后的线粒体功能障碍和细胞死亡会使转归恶化。受体相互作用蛋白激酶 1（RIPK1）以含 NOD、LRR 和 pyrin 结构域蛋白 3（NLRP3）炎性小体依赖性的方式激活动力相关蛋白 1（DRP1），而缺氧诱导因子（HIF）-1α在该过程中起关键作用。本研究旨在确定具有低温和常温模式的吩噻嗪类药物（氯丙嗪和丙嗪（C+P））如何在提供神经保护方面影响 RIPK1/RIPK3-DRP1 和 HIF-1α 通路。
总共将 150 只成年雄性 Sprague-Dawley 大鼠进行 2 小时大脑中动脉闭塞（MCAO），随后进行 24 小时再灌注。在再灌注开始时给予 8mg/kg 的 C+P。评估 HIF-1α、RIPK1、RIPK3、DRP-1、NLRP3-炎症和细胞色素 c-凋亡的 mRNA 和蛋白表达。评估细胞凋亡、中性粒细胞和巨噬细胞浸润以及线粒体功能。评估 RIPK1/RIPK3 与 HIF-1α/NLRP3 之间的相互作用。在经历氧/葡萄糖剥夺（OGD）的 SH-SY5Y 细胞中，研究 C+P 的常温作用对炎症和凋亡的影响。
C+P 显著降低了梗死体积、线粒体功能障碍（ATP 和 ROS 浓度、柠檬酸合酶和 ATP 酶活性）、炎症和凋亡，无论是否诱导低温。在 33°C 时，C+P 显著降低了 RIPK1、RIPK3、DRP-1、NLRP3-炎性小体和细胞色素 c-凋亡的过表达，以及 RIPK1 抑制剂（Nec1s），表明 C+P 通过 RIPK1/RIPK3-DRP1 通路具有低温作用。当体温保持在 37°C 时，C+P 和 HIF-1α 抑制剂（YC-1）降低了 HIF-1α 的表达，导致线粒体功能障碍、NLRP3 炎性小体和细胞色素 c-凋亡减少，以及 HIF-1α 和 NLRP3 的相互作用。这在体外也得到了证实，表明 C+P 通过 HIF-1α 具有常温作用。
C+P 的低温和常温神经保护作用涉及不同的途径。常温作用是通过 HIF-1α 介导的，而低温作用是通过 RIPK1/RIPK3-DRP1 信号转导介导的。这为未来对低温和常温神经保护的精确探索提供了理论基础。
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