ACT001 attenuates microglia-mediated neuroinflammation after traumatic brain injury via inhibiting AKT/NFκB/NLRP3 pathway

Abstract

Background Microglia-mediated neuroinflammatory response following traumatic brain injury (TBI) is considered as a vital secondary injury factor, which drives trauma-induced neurodegeneration and is lack of efficient treatment. ACT001, a sesquiterpene lactone derivative, is reportedly involved in alleviation of inflammatory response. However, little is known regarding its function in regulating innate immune response of central nervous system (CNS) after TBI. This study aimed to investigate the role and underlying mechanism of ACT001 in TBI. Methods Controlled cortical impact (CCI) models were used to establish model of TBI. Cresyl violet staining, evans blue extravasation, neurobehavioral function assessments, immunofluorescence and transmission electron microscopy were used to evaluate therapeutic effects of ACT001 in vivo. Microglial depletion was induced by administering mice with colony stimulating factor 1 receptor (CSF1R) inhibitor, PLX5622. Cell-cell interaction models were established as co-culture system to simulate TBI conditions in vitro. Cytotoxic effect of ACT001 on cell viability was assessed by cell counting kit-8 and activation of microglia cells were induced by Lipopolysaccharides (LPS). Pro-inflammatory cytokines expression was determined by Real-time PCR and nitric oxide production. Apoptotic cells were detected by TUNEL and flow cytometry assays. Tube formation was performed to evaluate cellular angiogenic ability. ELISA and western blot experiments were used to determine proteins expression. Pull-down assay was used to analyze proteins that bound ACT001. Results ACT001 relieved the extent of blood-brain barrier integrity damage and alleviated motor function deficits after TBI via reducing trauma-induced activation of microglia cells. Delayed depletion of microglia with PLX5622 hindered therapeutic effect of ACT001. Furthermore, ACT001 alleviated LPS-induced activation in mouse and rat primary microglia cells. Besides, ACT001 was effective in suppressing LPS-induced pro-inflammatory cytokines production in BV2 cells, resulting in reduction of neuronal apoptosis in HT22 cells and improvement of tube formation in bEnd.3 cells. Mechanism by which ACT001 functioned was related to AKT/NFκB/NLRP3 pathway. ACT001 restrained NFκB nuclear translocation in microglia cells through inhibiting AKT phosphorylation, resulting in decrease of NLRP3 inflammasome activation, and finally down-regulated microglial neuroinflammatory response. Conclusions Our study indicated that ACT001 played critical role in microglia-mediated neuroinflammatory response and might be a novel potential chemotherapeutic drug for TBI.

Abrocitinib Attenuates Microglia-Mediated Neuroinflammation after Traumatic Brain Injury via Inhibiting the JAK1/STAT1/NF-κB Pathway

“…NF-κB signaling could be activated by hypoxia, reactive oxygen species, and several inflammatory mediators [ 51 ]. Once activated, NF-κB can cause the production of proinflammatory cytokines, such as IL-1β, IL-6, and TNF-α, as well as the activation of NLRP3 inflammasome [ 5 , 52 ]. Additionally, some inflammatory cytokines, such as IL-1 and TNF-α, can activate NF-κB further, creating a positive feedback loop that exacerbates the inflammatory response [ 19 , 53 ].…”

“…The pathology of TBI can be categorized as primary brain injury which is caused by mechanical damage during the initial impact, or secondary brain injury, caused by inflammation, oxidative stress, mitochondrial dysfunction, apoptosis, etc. [ 3 , 4 , 5 ]. Among these pathological events, neuroinflammation, which is the main pathological process, can lead to extensive tissue injury and neurological dysfunctions [ 6 , 7 ].…”

“…After the stimulation of a TBI, the inhibitor of NF-κB (IκB) is phosphorylated and degraded, which leads to the release of the NF-κB dimer. Then, the activated NF-κB translocates to the nucleus, resulting in the transcription and activation of pro-inflammatory factors, such as NLR family pyrin domain containing protein 3 (NLRP3) inflammasomes, which are key elements in the procession of pyroptosis and are composed of an inflammasome sensor molecule, an apoptosis-associated speck-like protein containing a CARD (ASC), and caspase-1 [ 5 , 21 ].…”

Novel recombinant protein flagellin A N/C attenuates experimental autoimmune encephalomyelitis by suppressing the ROS/NF-κB/NLRP3 signaling pathway

“…We weighed the mice daily and scored their clinical behavior from 0-5 according to the respective criteria for EAE: 0, no clinical symptoms; 1, the tail tension disappeared or the gait was awkward; 2, weakness in hind limbs; 3, paralysis of hind limbs; 4, paralysis of hind limbs and weakness in anterior limbs; 5, near-death state ( Yu et al, 2020 ; Zilkha-Falb et al, 2020 ; Kim et al, 2021 ; Cai et al, 2022 ).…”

“…For inflammation: 0 = non-inflammatory cells; 1 = a small amount of scattered inflammatory cells; 2 = organization of inflammatory infiltrates around blood vessels; 3 = extensive perivascular cuffing with extension into parenchyma. For demyelination: 0 = none; 1 = rare foci; 2 = several demyelination areas; 3 = large areas of demyelination ( Yu et al, 2020 ; Kim et al, 2021 ; Cai et al, 2022 ).…”