BRD4 suppression alleviates cerebral ischemia-induced brain injury by blocking glial activation via the inhibition of inflammatory response and pyroptosis

https://doi.org/10.1016/j.bbrc.2019.07.097Get rights and content

Highlights

  • BRD4 inhibition alleviates ischemic injury and glial activation in MCAO mice.

  • BRD4 inhibition inhibits inflammation and pyroptosis in MCAO mice.

  • Suppressing BRD4 attenuates inflammatory response and pyroptosis in OGDR-treated glial cells.

Abstract

Ischemic stroke is a major cause of death and disability worldwide. Hyperneuroinflammation significantly contributes to ischemic stroke. Bromodomain-containing protein 4 (BRD4) is a member of the Bromo and Extra-Terminal (BET) family, and promotes inflammatory response in various types of tissue and cells. Thereby, we examined the contribution of BRD4 after cerebral ischemic/reperfusion (I/R) injury in a mouse middle cerebral artery occlusion (MCAO) model. Here, we showed that BRD4 expression was correlated with glial activation and cerebral I/R injury after MCAO in mice. Intriguingly, we found that BRD4 inhibition using its selective inhibitor, JQ1, showed a protective role in cerebral I/R injury in mice. Suppressing BRD4 by JQ1 reduced the infarction volume, brain water contents and neurological deficit score of MCAO mice. In addition, MCAO-induced glial activation was also blunted by JQ1, as proved by the significantly reduced expression of glial fibrillary acidic protein (GFAP) and Iba-1. Consistently, JQ1 treatment decreased the expression of pro-inflammatory factors by blocking nuclear factor kappa B (NF-κB) signaling. Furthermore, inflammasome activation and pyroptosis found in MCAO mice were markedly attenuated by JQ1, which were through suppressing the expression of NLRP3 (nucleotide-binding domain, leucine-rich repeat containing protein 3), ASC (apoptosis-associated speck-like protein containing a CARD), Caspase-1 and GSDMD (gasdermin D). The protective effects of BRD4 inhibition on cerebral ischemia-induced brain injury were verified in astrocytes and microglial cells via the inhibition of inflammation and pyroptosis. In summary, blocking BRD4 expression might serve as a potential therapeutic strategy for stroke therapy.

Introduction

Stroke is a major cause of mortality and disability in the world, and the ischemic stroke accounts for >80% of total stroke [1]. Astrocytes and microglia, the major cellular contributors to post-injury inflammation, have the potential to function as markers of disease onset and progression and to contribute to neurological outcome of acute brain injury [2,3]. After acute brain injury, these resident cells are rapidly and significantly activated, which then undergo dramatic morphological and phenotypic alterations [4]. The inflammatory microenvironment generated by glial cells has been considered to participate in blood-brain barrier (BBB) after ischemic stroke [5]. However, the molecular mechanism of activated glia on cerebral I/R injury remains to be further explored.

Pyroptosis is reported as a pro-inflammatory form of cell death and a regulated form of necrosis, which is distinct from other forms of cell death related to inflammasome activation [6,7]. The inflammasome acts as a sensor to detect cellular damage danger signals, and results in the excessive release of potent pro-inflammatory cytokines, contributing to inflammation-regulated organ injury [6,8]. Accumulating studies have indicated that this novel type of cell death plays a pivotal role in the pathogenesis of central nervous system diseases, including cerebral I/R injury, which is characterized by neuroinflammation [9,10]. Moreover, a range of cell types in central nervous system might undergo inflammasome-triggered pyroptosis, including astrocytes and microglia [11,12]. Bromodomain-containing protein 4 (BRD4), as a member of the Bromo and Extra-Terminal (BET) family, may bind to acetylated histones and transcription factors through bromodomains and modulate various pathophysiological activities such as inflammation [13]. Suppressing BRD4 by its selective inhibitor of JQ1 was suggested to alleviate lipopolysaccharides-triggered expression of pro-inflammatory cytokines [14]. Recently, growing evidence suggest that JQ1 treatment blunt osteoarthritis and rheumatoid arthritis progression partly through inflammation inhibition [15,16]. Also, spinal cord injury was also found to be repressed by JQ1 mainly via blocking microglial inflammatory response [17]. Herein, we hypothesized that BRD4 suppression might be a promising therapeutic target against ischemic stroke.

In this study, we found that BRD4 expression was associated with cerebral I/R injury in MCAO mice. Blunting BRD4 by JQ1 alleviated ischemic stroke in mice following MCAO by reducing glial activation, inflammation and pyroptosis, which indicated that BRD4 might serve as a promising therapeutic target for stroke treatment.

Section snippets

Animals and MCAO model

Male C57BL/6 mice (6–8 weeks old) were purchased from the Academy of Military Medical Science (Beijing, China). These mice were maintained in a specific pathogen-free (SPF) animal facility. The animal experiments were approved by the Animal Ethics Committee of Changhai Hospital, Naval Medical University (Shanghai, China) and were performed in accordance with animal care guidelines. Mice were randomly divided into 4 groups: 1) Sham without JQ1 treatment; 2) Sham with JQ1 treatment; 3) MCAO

BRD4 expression is induced following reperfusion in a mouse MCAO model

In ischemic cortex at 6 h, 12 h, 24 h, 72 h and 7 d after MCAO, the number of PI-positive cells was gradually increased, and peaked at 72 h (3 d) in the peri-infarct area after MCAO, indicating the cell death and brain injury triggered by MCAO (Fig. 1A and B). Then, BRD4 expression from mRNA and protein levels in the lateral cortex tissues of peri-infarct area were progressively up-regulated and reached to the highest levels at 3 d following MCAO (Fig. 1C and D). Immunofluorescent results

Discussion

Deleterious glia activation is crucially involved in the pathophysiology of cerebral ischemia. Activated glial cells have been reported in peri-infarct areas in clinical stroke and are considered to orchestrate neuronal damage in the penumbra [4,26]. Inflammation is a critical contributor to the pathogenesis of ischemic stroke. The deleterious effects of the inflammatory response following cerebral ischemia are significantly regulated by glial cells in brain, which release cytokines or

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    Yi Zhou and Yang Gu are co-first authors.

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