Modulation of NLRP3 inflammasome-related-inflammation via RIPK1/RIPK3-DRP1 or HIF-1α Signaling by Phenothiazine in Hypothermic and Normothermic Neuroprotection after Acute Ischemic Stroke

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. 8mg/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.