Abstract TP26: Hypothermic and Non-Hypothermic Neuroprotection by Phenothiazine in Acute Ischemic Stroke: Modulation of NLRP3 Inflammasome-Related-Inflammation via Different Pathways of RIPK1/RIPK3-DRP1 or HIF-1α

Abstract

Background: Inflammation after successful revascularization worsened outcomes in ischemic stroke. NLRPyrin domain containing 3 (NLRP3) inflammasome mediated inflammatory responses, in which receptor-interacting protein kinase (RIPK1) activated dynamin-related protein 1 (DRP1) in a RIPK3-dependent fashion. Hypoxia-inducible factor 1α (HIF-1α) regulated ischemic inflammation through NLRP3 inflammasome complex, thus apoptosis after stroke. Phenothiazine, including chlorpromazine and promethazine (C+P), induced hypothermic and non-hypothermic neuroprotection. We determined effects of RIPK1/RIPK3-DRP1 or HIF-1α pathways underlying the dual neuroprotection by C+P. Methods: 144 adult male Sprague-Dawley rats were subjected to 2 h of middle cerebral artery occlusion (MCAO), followed by 24 h reperfusion. 8mg/kg of C+P was injected at the onset of reperfusion. Infarct volumes, mRNA and protein expressions of HIF-1α, RIPK1, RIPK3, DRP-1, NLRP3-inflammation (NLRP3, ASC, Caspase-1, IL-1β and IL-18) and cytochrome C-apoptosis (cytochrome C, APAF-1, Caspase-9 and Caspase-3) were assessed. Apoptotic cell death, and the infiltration of neutrophils and macrophages were evaluated. Co-localization of RIPK1/RIPK3 and HIF-1α/NLRP3 were determined. Results: C+P significantly reduced brain inflammation, neuronal apoptosis and immune cell infiltration, with or without hypothermia. C+P with induced-hypothermia and RIPK1 inhibitor significantly reduced infarct volumes, the expression of RIPK1, RIPK3, DRP-1, NLRP3-inflammation process and cytochrome C-apoptosis, as well as the co-localization of RIPK1/RIPK3, suggesting the hypothermic effect underlying the RIPK1/RIPK3-DRP1 pathway. In non-hypothermia effect, HIF-1α expression was reduced by C+P or HIF-1α inhibitor, in concomitant with reduced NLRP3 inflammasome, cytochrome C-apoptosis, and decreased interaction of HIF-1α and NLRP3. Conclusion: Hypothermic and non-hypothermic neuroprotection of C+P involve different pathways. The hypothermic effect was via RIPK1/RIPK3-DRP1 signaling, while non-hypothermic effect was mediated by HIF-1α. This provides a theoretical basis for future precise exploration of hypothermic and non-hypothermic neuroprotection.