Suppression of neuroinflammation using small molecule compounds targeting the key pathways in microglial inflammation has attracted great interest. Recently, increasing attention has been gained to the role of the second bromodomain (BD2) of the bromodomain and extra-terminal (BET) proteins, while its effect and molecular mechanism on microglial inflammation has not yet been explored. In this study, we evaluated the therapeutic effects of ABBV-744, a BD2 high selective BET inhibitor, on lipopolysaccharide (LPS)-induced microglial inflammation in vitro and in vivo, and explored the key pathways by which ABBV-744 regulated microglia-mediated neuroinflammation. We found that pretreatment of ABBV-744 concentration-dependently inhibited the expression of LPS-induced inflammatory mediators/enzymes including NO, TNF-α, IL-1β, IL-6, iNOS, and COX-2 in BV-2 microglial cells. These effects were validated in LPS-treated primary microglial cells. Furthermore, we observed that administration of ABBV-744 significantly alleviated LPS-induced activation of microglia and transcriptional levels of pro-inflammatory factors TNF-α and IL-1β in mouse hippocampus and cortex. RNA-Sequencing (RNA-seq) analysis revealed that ABBV-744 induced 508 differentially expressed genes (DEGs) in LPS-stimulated BV-2 cells, and gene enrichment and gene expression network analysis verified its regulation on activated microglial genes and inflammatory pathways. We demonstrated that pretreatment of ABBV-744 significantly reduced the expression levels of basic leucine zipper ATF-like transcription factor 2 (BATF2) and interferon regulatory factor 4 (IRF4), and suppressed JAK-STAT signaling pathway in LPS-stimulated BV-2 cells and mice, suggesting that the anti-neuroinflammatory effect of ABBV-744 might be associated with regulation of BATF2-IRF4-STAT1/3/5 pathway, which was confirmed by gene knockdown experiments. This study demonstrates the effect of a BD2 high selective BET inhibitor, ABBV-744, against microglial inflammation, and reveals a BATF2-IRF4-STAT1/3/5 pathway in regulation of microglial inflammation, which might provide new clues for discovery of effective therapeutic strategy against neuroinflammation.

使用针对小胶质细胞炎症关键途径的小分子化合物抑制神经炎症引起了人们的极大兴趣。近年来，溴结构域第二溴结构域（BD2）和额外末端（BET）蛋白的作用越来越受到关注，但其对小胶质细胞炎症的作用和分子机制尚未被探索。在本研究中，我们评估了BD2高选择性BET抑制剂ABBV-744对脂多糖（LPS）诱导的小胶质细胞炎症的体外和体内治疗作用，并探讨了ABBV-744调节小胶质细胞介导的关键通路。神经炎症。我们发现 ABBV-744 预处理浓度依赖性地抑制 BV-2 小胶质细胞中 LPS 诱导的炎症介质/酶的表达，包括 NO、TNF-α、IL-1β、IL-6、iNOS 和 COX-2。这些效应在 LPS 处理的原代小胶质细胞中得到了验证。此外，我们观察到，施用 ABBV-744 显着减轻了 LPS 诱导的小鼠海马和皮质中小胶质细胞的激活以及促炎因子 TNF-α 和 IL-1β 的转录水平。 RNA测序（RNA-seq）分析显示，ABBV-744在LPS刺激的BV-2细胞中诱导了508个差异表达基因（DEG），基因富集和基因表达网络分析验证了其对激活的小胶质细胞基因和炎症通路的调节。 我们证明，ABBV-744 预处理可显着降低 LPS 刺激的 BV-2 细胞中碱性亮氨酸拉链 ATF 样转录因子 2 (BATF2) 和干扰素调节因子 4 (IRF4) 的表达水平，并抑制 JAK-STAT 信号通路和小鼠实验结果表明，ABBV-744的抗神经炎症作用可能与BATF2-IRF4-STAT1/3/5通路的调节有关，这一点已通过基因敲除实验得到证实。该研究证明了BD2高选择性BET抑制剂ABBV-744对抗小胶质细胞炎症的作用，并揭示了BATF2-IRF4-STAT1/3/5通路在小胶质细胞炎症调节中的作用，这可能为发现有效的治疗方法提供新线索。对抗神经炎症的策略。