epz-6438 and Inflammation

Neuroinflammation has been proven to play an important role in the pathogenesis of early brain injury after subarachnoid hemorrhage (SAH). EZH2 (enhancer of zeste homolog 2)-mediated H3K27Me3 (trimethylation of histone 3 lysine 27) has been recognized to play a critical role in multiple inflammatory diseases. However, there is still a lack of evidence to address the effect of EZH2 on the immune response of SAH. Therefore, the aim of this study was to determine the role of EZH2 in SAH-induced neuroinflammation and explore the effect of EZH2 inhibition with its specific inhibitor EPZ6438.. The endovascular perforation method was performed on rats to induce subarachnoid hemorrhage. EPZ6438, a specific EZH2 inhibitor, was administered intraperitoneally at 1 hour after SAH. SOCS3 (Suppressor of cytokine signaling 3) siRNA and H3K27me3 CRISPR were administered intracerebroventricularly at 48 hours before SAH to explore potential mechanisms. The SAH grade, short-term and long-term neurobehavioral tests, immunofluorescence staining, and western blots were performed after SAH.. The expression of EZH2 and H3K27me3 peaked at 24 hours after SAH. In addition, inhibition of EZH2 with EPZ6438 significantly improved neurological deficits both in short-term and long-term outcome studies. Moreover, EPZ6438 treatment significantly decreased the levels of EZH2, H3K27Me3, pathway-related proteins TRAF6 (TNF [tumor necrosis factor] receptor family 6), NF-κB (nuclear factor-κB) p65, proinflammatory cytokines TNF-α, IL (interleukin)-6, IL-1β, but increased the expression levels of SOCS3 and anti-inflammatory cytokine IL-10. Furthermore, administration of SOCS3 siRNA and H3k27me3-activating CRISPR partly abolished the neuroprotective effect of EPZ6438, which indicated that the neuroprotective effect of EPZ6438 acted, at least partly, through activation of SOCS3.. In summary, the inhibition of EZH2 by EPZ6438 attenuated neuroinflammation via H3K27me3/SOCS3/TRAF6/NF-κB signaling pathway after SAH in rats. By targeting EZH2, this study may provide an innovative method to ameliorate early brain injury after SAH.

Topics: Animals; Benzamides; Biphenyl Compounds; Brain; Clustered Regularly Interspaced Short Palindromic Repeats; Disease Models, Animal; Enhancer of Zeste Homolog 2 Protein; Histone Code; Histones; Inflammation; Male; Microglia; Morpholines; Morris Water Maze Test; Neutrophil Infiltration; NF-kappa B; Pyridones; Rats; Rats, Sprague-Dawley; RNA, Small Interfering; Rotarod Performance Test; Signal Transduction; Subarachnoid Hemorrhage; Suppressor of Cytokine Signaling 3 Protein; TNF Receptor-Associated Factor 6

Multiple studies have documented that Enhancer of zeste homolog 2 (EZH2) could play a role in inflammation and a wide range of malignancies; however, the underlying mechanisms remain largely unaddressed. Microglial activation is a key process in the production and release of numerous pro-inflammatory mediators that play important roles in inflammation and neurodegeneration in the central nervous system (CNS). Therefore, our aim was to investigate whether inhibition of EZH2 with the selective small molecule inhibitor EPZ-6438 protects against neonatal microglial activation. First, in mouse primary microglial cells and a microglial cell line, we found that LPS can rapidly increase EZH2 mRNA level and we subsequently performed gene expression profiling and constructed networks in resting, EPZ-6438-treated, LPS-treated and LPS+EPZ-6438-treated primary microglial cells and a microglial cell line using transcriptome RNA sequencing and bioinformatics analyses. By examining the RNA sequencing, we identified EPZ-6438 target genes and co-regulated modules that were critical for inflammation. We also identified unexpected relationships between the inducible transcription factors (TFs), motif strength, and the transcription of key inflammatory mediators. Furthermore, we showed that EPZ-6438 controls important inflammatory gene targets by modulating interferon regulatory factor (IRF) 1, IRF8, and signal transducer and activator of transcription (STAT) 1 levels at their promoter sites. Our unprecedented findings demonstrate that pharmacological interventions built upon EZH2 inhibition by EPZ-6438 could be a useful therapeutic approach for the treatment of neuroinflammatory diseases associated with microglial activation.

Topics: Animals; Benzamides; Biphenyl Compounds; Cell Line; Cells, Cultured; Enhancer of Zeste Homolog 2 Protein; Gene Expression; Gene Regulatory Networks; Inflammation; Inflammation Mediators; Mice; Mice, Inbred ICR; Microglia; Morpholines; Pyridones