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.

Microglial phagocytosis benefits neurological recovery after stroke. Large-conductance Ca2+-activated K+ currents are expressed in activated microglia, and BK channel knockout aggravates cerebral ischemic injury. However, the effect of BK channels on microglial phagocytosis after ischemic stroke remains unknown. Here, we explored whether BK channel activation is beneficial for neurological outcomes through microglial phagocytosis after ischemic stroke. ICR mice after transient middle cerebral artery occlusion (tMCAO) were treated with dimethyl sulfoxide (DMSO), BK channel activator NS19504, and inhibitor Paxilline. The results showed a decrease in BK channel expression after tMCAO. BK channel activator NS19504 alleviates neurological deficit after experimental modeling of tMCAO in mice compared to the control. Furthermore, we treated primary microglia with DMSO, NS19504, and Paxilline after oxygen glucose deprivation (OGD). NS19504 promoted primary microglial phagocytosing fluorescent beads and neuronal debris, which reduced neuronal apoptosis after stroke. These effects could be reversed by BK channel inhibitor Paxilline. Finally, NS19504 increased relative phosphorylated extracellular signal-regulated kinase 1/2 expression compared to the Paxilline group at the third day after stroke. Our findings indicate that microglial BK channels are a potential target for acute stage of ischemic stroke therapy.

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