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Activation of Galanin Receptor 1 with M617 Attenuates Neuronal Apoptosis via ERK/GSK-3β/TIP60 Pathway After Subarachnoid Hemorrhage in Rats.

ABSTRACT: Subarachnoid hemorrhage (SAH) is a devastating cerebrovascular disease. Neuronal apoptosis plays an important pathological role in early brain injury after SAH. Galanin receptor 1 (GalR1) activation was recently shown to be anti-apoptotic in the setting of ischemic stroke. This study aimed to explore the anti-neuronal apoptosis effect of GalR1 activation after SAH, as well as the underlying mechanisms. GalR1 CRISPR and GalR1 selective agonist, M617, was administered, respectively. Extracellular-signal-regulated kinase (ERK) inhibitor (U0126) and glycogen synthase kinase 3-beta (GSK3-β) CRISPR were administered to investigate the involvement of the ERK/GSK3-β pathway in GalR1-mediated neuroprotection after SAH. Outcome assessments included neurobehavioral tests, western blot, and immunohistochemistry. The results showed that endogenous ligand galanin (Gal) and GalR1 were markedly increased in the ipsilateral brain hemisphere at 12 h and 24 h after SAH. GalR1 were expressed mainly in neurons, but expression was also observed in some astrocytes and microglia. GalR1 CRISPR knockdown exacerbated neurological deficits and neuronal apoptosis 24 h after SAH. Moreover, activation of GalR1 with M617 significantly improved short- and long-term neurological deficits but decreased neuronal apoptosis after SAH. Furthermore, GalR1 activation dysregulated the protein levels of phosphorylated ERK and GSK-3β, but downregulated the phosphorylated Tat-interactive protein 60 (TIP60) and cleaved caspase-3 at 24 h after SAH. GalR1 CRISPR, U0126, and GSK-3β CRISPR abolished the beneficial effects of GalR1 activation at 24 h after SAH in rats. Collectively, the present study demonstrated that activation of GalR1 using M617 attenuated neuronal apoptosis through the ERK/GSK-3β/TIP60 pathway after SAH in rats. GalR1 may serve as a promising therapeutic target for SAH patients.

SUBMITTER: Shi H

PROVIDER: S-EPMC8609084 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND:Oxidative stress and neuronal apoptosis play important roles in the pathogenesis of early brain injury (EBI) after subarachnoid hemorrhage (SAH). The activation of TGR5, a novel membrane-bound bile acid receptor, possesses anti-oxidative stress and anti-apoptotic effects in hepatobiliary disease and kidney disease. The present study aimed to explore the neuroprotective effect of TGR5 activation against EBI after SAH and the potential underlying mechanisms. METHODS:The endovascular perforation model of SAH was performed on 199 Sprague Dawley rats to investigate the beneficial effects of TGR5 activation after SAH. INT-777, a specific synthetic TGR5 agonist, was administered intranasally at 1?h after SAH induction. TGR5 CRISPR and ALDH2 CRISPR were administered intracerebroventricularly at 48?h before SAH to illuminate potential mechanisms. The SAH grade, short-term and long-term neurobehavioral tests, TUNEL staining, Fluoro-Jade C staining, Nissl staining, immunofluorescence staining, and western blots were performed at 24?h after SAH. RESULTS:The expressions of endogenous TGR5 and ALDH2 gradually increased and peaked at 24?h after SAH. TGR5 was expressed primarily in neurons, as well as in astrocytes and microglia. The activation of TGR5 with INT-777 significantly improved the short-term and long-term neurological deficits, accompanied by reduced the oxidative stress and neuronal apoptosis at 24?h after SAH. Moreover, INT-777 treatment significantly increased the expressions of TGR5, cAMP, phosphorylated PKC?, ALDH2, HO-1, and Bcl-2, while downregulated the expressions of 4-HNE, Bax, and Cleaved Caspase-3. TGR5 CRISPR and ALDH2 CRISPR abolished the neuroprotective effects of TGR5 activation after SAH. CONCLUSIONS:In summary, the activation of TGR5 with INT-777 attenuated oxidative stress and neuronal apoptosis via the cAMP/PKC?/ALDH2 signaling pathway after SAH in rats. Furthermore, TGR5 may serve as a novel therapeutic target to ameliorate EBI after SAH.

Project description:BackgroundNeuroinflammation and neuronal apoptosis are closely associated with a poor prognosis in patients with subarachnoid hemorrhage (SAH). We investigated the role of C-C motif chemokine receptor 2 (CCR2) in SAH.MethodsPre-processed RNA-seq transcriptome datasets GSE167110 and GSE79416 from the Gene Expression Omnibus (GEO) database were screened for genes differentially expressed between mice with SAH and control mice, using bioinformatics analysis. The endovascular perforation model was performed to establish SAH. RS504393 (a CCR2 antagonist) and LY294002 (PI3K inhibitor) were administered to explore the mechanism of neuroinflammation after SAH. SAH grading, neurological scoring, brain water content and blood-brain barrier (BBB) permeability determination, enzyme-linked immunosorbent assay (ELISA), western blotting, and immunofluorescence were performed. An in vitro model of SAH was induced in H22 cells by hemin treatment. The protective mechanism of CCR2 inhibition was studied by adding RS504393 and LY294002. Clinical cerebrospinal fluid (CST) samples were detected by ELISA.ResultsExpression of CCR2 was upregulated in both datasets and was identified as a hub gene. CCR2 expression was significantly upregulated in the cytoplasm of neurons after SAH, both in vitro and in vivo. RS significantly reduced the brain water content and blood-brain barrier permeability, alleviated neuroinflammation, and reduced neuronal apoptosis after SAH. Additionally, the protective effects of CCR2 inhibition were abolished by LY treatment. Finally, the levels of CCR2, inflammatory factors, and apoptotic factors were elevated in the CSF of patients with SAH. CCR2 levels were associated with patient outcomes at the 6-month follow-up.ConclusionCCR2 expression was upregulated in both in vitro and in vivo SAH models. Additionally, inhibition of CCR2, at least partly through the PI3K/AKT pathway, alleviated neuroinflammation and neuronal apoptosis in vivo and in vitro. CCR2 levels in the CSF have a moderate diagnostic value for 6-month outcome prediction in patients with SAH.

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Activation of Galanin Receptor 1 with M617 Attenuates Neuronal Apoptosis via ERK/GSK-3β/TIP60 Pathway After Subarachnoid Hemorrhage in Rats.

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