Resolvin D1 Attenuates Necroptosis-Associated Neuronal Injury After Intracerebral Hemorrhage Through ALX/FPR2-PI3K/AKT Signaling With ANXA1-Dependent Downstream Regulation

BACKGROUND: Intracerebral hemorrhage (ICH) triggers secondary brain injury characterized by inflammation and regulated cell death. Resolvin D1 (RvD1) has neuroprotective potential, but its effects on necroptosis-associated injury after ICH remain incompletely defined. METHODS: Collagenase-induced ICH was established in Sprague-Dawley rats. RvD1 (222 μg/kg) was administered intraperitoneally 3 h after ICH, and outcomes were assessed at 24 h. Neurological function, histopathology, plasma RvD1, inflammatory cytokines, and necroptosis-associated proteins were measured. HT22 cells were exposed to hemin (80 μM) with or without RvD1 (250 nM). Necrostatin-1, WRW4, LY294002, and si-ANXA1 were used to interrogate necroptosis-associated injury, ALX/FPR2, PI3K/AKT, and ANXA1-dependent responses; the combined RvD1/si-ANXA1 intervention was used to test the functional contribution of ANXA1 to the measured RvD1 responses. RESULTS: ICH reduced plasma RvD1 and neurological function, increased IL-1β, IL-6, TNF-α, RIPK1, RIPK3, p-MLKL, and MLKL, and produced histopathological injury; RvD1 attenuated these changes. In hemin-exposed HT22 cells, RvD1 improved viability and reduced representative PI staining, cytokine release, and necroptosis-associated proteins. Necrostatin-1 produced a parallel protective pattern. In the combined RvD1/si-ANXA1 intervention, ANXA1 knockdown was verified by western blotting and attenuated RvD1-mediated changes in viability, cytokine release, RIPK1-RIPK3-MLKL signaling, and PI3K/AKT phosphorylation. WRW4 and LY294002 each attenuated RvD1-associated protection and PI3K/AKT activation. CONCLUSION: RvD1 reduced acute ICH-associated injury, inflammatory output, and necroptosis-associated signaling under the tested conditions. The principal mechanistic evidence supports ALX/FPR2-dependent PI3K/AKT signaling. The combined RvD1/si-ANXA1 intervention supported a functional requirement for ANXA1 in the measured downstream molecular and cellular responses to RvD1, but its position relative to ALX/FPR2 remains unresolved.