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Background: Neuroinflammation is a vital pathophysiological process during ischemic stroke. Activated astrocytes play a major role in inflammation. Lipocalin-2 (LCN2), secreted by activated astrocytes, promotes neuroinflammation. Pyroptosis is a pro-inflammatory form of programmed cell death that has emerged as a new area of research in stroke. Nevertheless, the potential role of LCN2 in astrocyte pyroptosis remains unclear. Methods: An ischemic stroke model was established by middle cerebral artery occlusion (MCAO) in vivo. In this study, in vitro, oxygen–glucose deprivation and reoxygenation (O/R) were applied to cultured astrocytes. 24p3R (the LCN2 receptor) was inhibited by astrocyte-specific adeno-associated virus (AAV-GFAP-24p3Ri). MCC950 and Nigericin sodium salt (Nig) were used to inhibit or promote the activation of NLRP3 inflammasome pharmacologically, respectively. Histological and biochemical analyses were performed to assess astrocyte and neuron death. Additionally, the neurological deficits of mice were evaluated. Results: LCN2 expression was significantly induced in astrocytes 24 h after stroke onset in the mouse MCAO model. Lcn2 knockout (Lcn2−/−) mice exhibited reduced infarct volume and improved neurological and cognitive functions after MCAO. LCN2 and its receptor 24p3R were colocalized in astrocytes. Mechanistically, suppression of 24p3R by AAV-GFAP-24p3Ri alleviated pyroptosis-related pore formation and the secretion of pro-inflammatory cytokines via LCN2, which was then reversed by Nig-induced NLRP3 inflammasome activation. Astrocyte pyroptosis was exacerbated in Lcn2−/− mice by intracerebroventricular administration of recombinant LCN2 (rLCN2), while this aggravation was restricted by blocking 24p3R or inhibiting NLRP3 inflammasome activation with MCC950. Conclusion: LCN2/24p3R mediates astrocyte pyroptosis via NLRP3 inflammasome activation following cerebral ischemia/reperfusion injury. [ABSTRACT FROM AUTHOR]

Bradykinin receptors play important roles in cerebral ischaemia-reperfusion (I/R) injury of non-diabetics. Their functions in diabetics, however, have not been studied. In this study, we hypothesized that bradykinin 1 receptor (B1R) and bradykinin 2 receptor (B2R) would be upregulated and participate in the regulation of diabetic ischaemic stroke. To investigate this, we first evaluated B1R and B2R expression at different time points after I/R in non-diabetic and diabetic rats (Sprague-Dawley) by using real-time quantitative reverse transcription polymerase chain reaction, western blotting, and immunofluorescence. Then, pharmacological inhibitors were separately administered via the tail vein to analyse their effects on cerebral ischaemia in diabetics. Both receptors were significantly upregulated after cerebral I/R in non-diabetic and diabetic rats. B1R expression in diabetic rats increased in a sharper manner than in non-diabetic rats, whereas B2R expression increased to the same level during the early stage of reperfusion but later became lower. Interestingly, the upregulated B1R was expressed in astrocytes, whereas B2R was mainly located in neurons in the ischaemic penumbra. Functional studies showed that inhibition of B1R significantly reduced infarct volume, neurological deficits, cell apoptosis, and neuron degeneration, probably by attenuating blood-brain barrier (BBB) disruption and postischaemic inflammation, at 24 h after reperfusion. In contrast, B2R antagonist had opposite effects, and exacerbated BBB penetrability and tissue inflammation. These findings suggest that B1R and B2R have detrimental and beneficial effects, respectively in diabetic cerebral ischaemia, which might open new avenues for the treatment of ischaemic stroke in diabetic patients through selective pharmacological blockade or activation. [ABSTRACT FROM AUTHOR]

Ferroptosis is a form of non-apoptotic cell death caused by iron-dependent peroxidation of lipids. It contributes to ischemic stroke-induced neuronal damage. Elabela (ELA), a novel endogenous ligand for Apelin receptor (APJ), regulates oxidative stress and exerts a protective role in cardiovascular disease. However, the effect of ELA-APJ axis on cellular ferroptosis in cerebral ischemia/reperfusion (I/R) remains elusive. The present study showed that ELA and APJ were expressed on neurons and increased after cerebral I/R injury. The I/R insult triggered typical molecular and morphological features of neuronal ferroptosis, including iron and MDA accumulation, mitochondrial shrink and membrane rupture, upregulation of positive ferroptosis regulators and downregulation of negative regulators. ELA-32 treatment reduced brain infarction and ameliorated neurobehavioral deficits and cognitive dysfunction. Moreover, ELA-32 administration alleviated neuronal ferroptosis, accompanied by reduced iron deposition, decreased mitochondrial damage, relived lipid peroxidation and glutathione reduction. Such effects of ELA-32 were abolished by AAV-APJ-RNAi or nuclear factor erythroid 2-related factor 2 (NRF2) inhibitor ML385. Mechanistically, ELA was shown to bind to APJ and activate NRF2/ARE anti-oxidative signaling pathway via Gα13. Together, these findings suggested that ELA-APJ axis mitigates neuronal ferroptosis after ischemic stroke and that the ELA-32 peptide may be a putative therapeutic avenue for ischemic stroke. [Display omitted] • Ischemic stroke induces Elabela and APJ expression and neuronal ferroptosis. • Activation of APJ with ELA-32 ameliorates ischemia/reperfusion injury and cognitive dysfunction. • Treatment with ELA-32 reduces neuronal lipid peroxidation and ferroptosis via NRF2/ARE signaling pathway. • Elabela binds to APJ and subsequently triggers NRF2-mediated anti-oxidative response through Gα13. [ABSTRACT FROM AUTHOR]