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.

Increased NOX2 expression in astrocytes leads to eNOS uncoupling through dihydrofolate reductase in endothelial cells after subarachnoid hemorrhage

“…An experimental SAH model was established by injecting autologous arterial blood into the prechiasmatic cistern of the optic chiasm as described previously, with minor modifications ( Deng et al, 2021 ). The mice were anesthetized using 100% O 2 containing 2% isoflurane and fixed on a stereotaxic apparatus while maintaining anesthesia with 1% isoflurane.…”

Potential preventive markers in the intracerebral hemorrhage process are revealed by serum untargeted metabolomics in mice using hypertensive cerebral microbleeds

“…In addition, L-NAME, which was used to induce HCMB, was only used in the HCMB group from days 1 to 28 and was added to drinking water at a dose of 100 mg/kg/day ( 24 , 25 , 27 – 29 ). During the experiment, if we found that some mice showed signs of reduced neurological function, we performed MRI on the mice with neurological function scores below 16 (according to the modified Garcia score) ( 30 , 31 ) and excluded them if they were considered to have ICH. Prior to the formal blood pressure measurement, we anesthetized the mice using barbiturates, immobilized them on the MRI instrument after they were better anesthetized, and then performed MRI measurements.…”

Metformin Inhibits NLR Family Pyrin Domain Containing 3 (NLRP)-Relevant Neuroinflammation via an Adenosine-5′-Monophosphate-Activated Protein Kinase (AMPK)-Dependent Pathway to Alleviate Early Brain Injury After Subarachnoid Hemorrhage in Mice

“…Nissl staining was used to evaluate neuronal damage in the ipsilateral cortex, as described previously ( Deng et al, 2021 ). Briefly, at 24 h after SAH, the mice were deeply anesthetized and transcardially perfused with 50 ml of phosphate-buffered saline (PBS; 0.1 M) followed by 50 ml of 4% paraformaldehyde (PFA; pH 7.4).…”