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A20 Establishes Negative Feedback With TRAF6/NF-κB and Attenuates Early Brain Injury After Experimental Subarachnoid Hemorrhage.

ABSTRACT: Nuclear factor (NF)-κB-ty -50mediated neuroinflammation plays a crucial role in early brain injury (EBI) after subarachnoid hemorrhage (SAH). As an important negative feedback regulator of NF-κB, A20 is essential for inflammatory homeostasis. Herein, we tested the hypothesis that A20 attenuates EBI by establishing NF-κB-associated negative feedback after experimental SAH. In vivo and in vitro models of SAH were established. TPCA-1 and lentivirus were used for NF-κB inhibition and A20 silencing/overexpression, respectively. Cellular localization of A20 in the brain was determined via immunofluorescence. Western blotting and enzyme-linked immunosorbent assays were applied to observe the expression of members of the A20/tumor necrosis factor receptor-associated factor 6 (TRAF6)/NF-κB pathway and inflammatory cytokines (IL-6, IL-1β, TNF-α). Evans blue staining, TUNEL staining, Nissl staining, brain water content, and modified Garcia score were performed to evaluate the neuroprotective effect of A20. A20 expression by astrocytes, microglia, and neurons was increased at 24 h after SAH. A20 and inflammatory cytokine levels were decreased while TRAF6 expression was elevated after NF-κB inhibition. TRAF6, NF-κB, and inflammatory cytokine levels were increased after A20 silencing but suppressed with A20 overexpression. Also, Bcl-2, Bax, MMP-9, ZO-1 protein levels; Evans blue, TUNEL, and Nissl staining; brain water content; and modified Garcia score showed that A20 exerted a neuroprotective effect after SAH. A20 expression was regulated by NF-κB. In turn, increased A20 expression inhibited TRAF6 and NF-κB to reduce the subsequent inflammatory response. Our data also suggest that negative feedback regulation mechanism of the A20/TRAF6/NF-κB pathway and the neuroprotective role of A20 to attenuate EBI after SAH.

SUBMITTER: Deng HJ

PROVIDER: S-EPMC8350325 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Background and purposeNeuroinflammation has been proven to play an important role in the pathogenesis of early brain injury after subarachnoid hemorrhage (SAH). EZH2 (enhancer of zeste homolog 2)-mediated H3K27Me3 (trimethylation of histone 3 lysine 27) has been recognized to play a critical role in multiple inflammatory diseases. However, there is still a lack of evidence to address the effect of EZH2 on the immune response of SAH. Therefore, the aim of this study was to determine the role of EZH2 in SAH-induced neuroinflammation and explore the effect of EZH2 inhibition with its specific inhibitor EPZ6438.MethodsThe endovascular perforation method was performed on rats to induce subarachnoid hemorrhage. EPZ6438, a specific EZH2 inhibitor, was administered intraperitoneally at 1 hour after SAH. SOCS3 (Suppressor of cytokine signaling 3) siRNA and H3K27me3 CRISPR were administered intracerebroventricularly at 48 hours before SAH to explore potential mechanisms. The SAH grade, short-term and long-term neurobehavioral tests, immunofluorescence staining, and western blots were performed after SAH.ResultsThe expression of EZH2 and H3K27me3 peaked at 24 hours after SAH. In addition, inhibition of EZH2 with EPZ6438 significantly improved neurological deficits both in short-term and long-term outcome studies. Moreover, EPZ6438 treatment significantly decreased the levels of EZH2, H3K27Me3, pathway-related proteins TRAF6 (TNF [tumor necrosis factor] receptor family 6), NF-κB (nuclear factor-κB) p65, proinflammatory cytokines TNF-α, IL (interleukin)-6, IL-1β, but increased the expression levels of SOCS3 and anti-inflammatory cytokine IL-10. Furthermore, administration of SOCS3 siRNA and H3k27me3-activating CRISPR partly abolished the neuroprotective effect of EPZ6438, which indicated that the neuroprotective effect of EPZ6438 acted, at least partly, through activation of SOCS3.ConclusionsIn summary, the inhibition of EZH2 by EPZ6438 attenuated neuroinflammation via H3K27me3/SOCS3/TRAF6/NF-κB signaling pathway after SAH in rats. By targeting EZH2, this study may provide an innovative method to ameliorate early brain injury after SAH.

Project description:BackgroundNeuroinflammation and blood-brain barrier (BBB) disruption are two critical mechanisms of subarachnoid hemorrhage (SAH)-induced brain injury, which are closely related to patient prognosis. Recently, angiogenic factor with G-patch and FHA domain 1 (Aggf1) was shown to inhibit inflammatory effect and preserve vascular integrity in non-nervous system diseases. This study aimed to determine whether Aggf1 could attenuate neuroinflammation and preserve BBB integrity after experimental SAH, as well as the underlying mechanisms of its protective roles.MethodsTwo hundred forty-nine male Sprague-Dawley rats were subjected to the endovascular perforation model of SAH. Recombinant human Aggf1 (rh-Aggf1) was administered intravenously via tail vein injection at 1 h after SAH induction. To investigate the underlying neuroprotection mechanism, Aggf1 small interfering RNA (Aggf1 siRNA) and PI3K-specific inhibitor LY294002 were administered through intracerebroventricular (i.c.v.) before SAH induction. SAH grade, neurological score, brain water content, BBB permeability, Western blot, and immunohistochemistry were performed.ResultsExpression of endogenous Aggf1 was markedly increased after SAH. Aggf1 was primarily expressed in endothelial cells and astrocytes, as well as microglia after SAH. Administration of rh-Aggf1 significantly reduced brain water content and BBB permeability, decreased the numbers of infiltrating neutrophils, and activated microglia in the ipsilateral cerebral cortex following SAH. Furthermore, rh-Aggf1 treatment improved both short- and long-term neurological functions after SAH. Meanwhile, exogenous rh-Aggf1 significantly increased the expression of PI3K, p-Akt, VE-cadherin, Occludin, and Claudin-5, as well as decreased the expression of p-NF-?B p65, albumin, myeloperoxidase (MPO), TNF-?, and IL-1?. Conversely, knockdown of endogenous Aggf1 aggravated BBB breakdown, inflammatory response and neurological impairments at 24 h after SAH. Additionally, the protective roles of rh-Aggf1 were abolished by LY294002.ConclusionsTaken together, exogenous Aggf1 treatment attenuated neuroinflammation and BBB disruption, improved neurological deficits after SAH in rats, at least in part through the PI3K/Akt/NF-?B pathway.

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A20 Establishes Negative Feedback With TRAF6/NF-κB and Attenuates Early Brain Injury After Experimental Subarachnoid Hemorrhage.

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