Figures & data
Figure 1. The selective BET inhibitor compound 38 inhibits the LPS-induced inflammatory response.
(a, b) Four-hour administration with the selective BET inhibitor compound 38 downregulated the mRNA levels of Il-1β, Il-6, and Tnf-α in mouse Raw264.7 (a) and primary BMDM (b) cells induced by LPS. (c, d) Exposure for 24 h decreased the mRNA levels of Il-1β, Il-6, and Tnf-α in Raw264.7 (c) and BMDM (d) cells induced by LPS. (e, f) ELISA demonstrated the inhibitory effect of compound 38 on IL-1β, IL-6, and TNF-α expression of Raw264.7 cells after 4 h (e) and 24 h (f) exposure. Data represent the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to the LPS-treated group. #P < 0.05, ##P < 0.01, ###P < 0.001, ####P < 0.0001 compared to the control group.
Figure 2. Compound 38 treatment attenuates inflammatory responses in the LPS/ GalN-induced acute liver injury (ALI) mouse model in vivo.
(a) The chemical structure of compound 38. (b) Survival curves for mice in the normal control (NC), ALI model, and compound 38-treated groups (n = 12 per group). (c) Serum ALT and AST levels in mice from the NC, ALI model, and compound 38-treated groups (n = 10 per group). (d) Morphological changes in the livers of mice from the indicated groups. (e and f) Representative images of the histological examination of liver tissues. H&E (e) and TUNEL (f) staining of the indicated groups of mice. (g and h) Representative images of immunohistochemical staining of F4/80+ macrophages (g) and LY-6 G+ neutrophils (h) within the liver tissue. (i) Il-1β, Il-6, and Tnf-α mRNA levels in the liver tissues of different groups quantified by RT-qPCR analysis. Data represent the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to LPS/GalN-stimulated mice. #P < 0.05, ##P < 0.01, ###P < 0.001, ####P < 0.0001 compared to the control group.
Figure 3. Compound38 inhibits pro-inflammatory gene expression and blocks the inflammatory pathway.
(a–d) Total RNA for RNA-seq analysis was extracted from the liver tissues of mice in the control group (n = 5), LPS/GalN-exposure group (n = 6), and compound 38-treated group (n = 5). (a) Heatmap showing the different clustering gene expression patterns among the control, LPS/GalN exposure, and compound 38-treated groups; differentially expressed genes were filtered for an adjusted P value < 0.05 and absolute value of log2 fold-change > 1. (b) The overlap between the gene set upregulated by LPS/GalN and the gene set subsequently downregulated by compound 38 in vivo presented as a Venn diagram. (c) Gene-annotation enrichment analysis performed using differentially expressed genes between the LPS/GalN model group and the compound 38-treated group, revealing that the differentially expressed genes were enriched in pathways associated with the inflammatory response. (d) Visualization of the top 10 hub genes and their PPI network. (e) GSEA results revealing the biological processes affected by compound 38. (f) Western blot analysis validating that compound 38 regulates the JAK-STAT and MAPK signaling pathways in Raw264.7 and primary BMDM cells. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to LPS/GalN-stimulated mice. #P < 0.05, ##P < 0.01, ###P < 0.001, ####P < 0.0001 compared to the control group.
Figure 4. Compound 38 treatment inhibited hepatic stellate cell (HSC) activation.
(a) The human HSC cell line LX-2 was starved in serum-free medium for 24 h and then treated with compound 38 and TGF-β1 (10 ng/mL) for 24 h, after which α-SMA, COL1A1, and TGF-β mRNAs were quantified by RT-qPCR analysis. (b) The rat HSC cell line HSC-T6 was cultured in serum-free medium for 24 h and then treated with compound 38 and TGF-β1 (10 ng/mL) for 24 h, after which α-Sma mRNAs were quantified by RT-qPCR analysis. Data represent the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001,****P < 0.0001 compared to the TGF-β1-stimulated group. #P < 0.05, ##P < 0.01, ###P < 0.001, ####P < 0.0001 compared to the control group.
Figure 5. Compound 38 treatment reduces liver inflammation and fibrosis.
Mice received intraperitoneal injections of CCl4 (10% CCl4 dissolved in olive oil at a dose of 1 mL/kg body weight) twice a week. (a) Serum ALT and AST levels in mice from the normal control (NC; n = 11), CCl4-induced liver fibrosis model (n = 9), and model with compound 38 treatment (n = 9) groups. (b) Representative images of Masson staining from the mouse liver tissues of the indicated groups. (c and d) Representative images of immunohistochemical staining for COL1α1 (c) and α-SMA (d) in liver sections. (e) Quantification of different stainings (n = 10 images per group from the NC, CCl4, and CCl4 + compound 38 groups; six samples per group). Data represent the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to CCl4-induced mice. #P < 0.05, ##P < 0.01, ###P < 0.001, ####P < 0.0001 compared to the control group.
Figure 6. Compound 38 blocks fibrosis-related pathway activation in LX2 cells.
(a–c) Total RNA for RNA-seq analysis was extracted from LX2 cells in the control group (n = 3), TGF-β exposure group (n = 2), and compound 38-treated group (50 nM, n = 3). (a) Heatmap showing the different clustering gene expression patterns among the control, TGF-β1 exposure, and compound 38-treated groups; differentially expressed genes were filtered for an adjusted P value < 0.05 and absolute value of fold-change > 1.5. (b) Gene-annotation enrichment analysis performed on differentially expressed genes between the TGF-β exposure group and the compound 38-treated group. (c) Heatmap revealing the key genes of biological processes and pathways affected by compound 38. (d) Western blot analysis to validate activation of the TGF-β/SMAD and Wnt/β-catenin signaling pathways in the human HSC cell line LX-2 and the rat HSC cell line HSC-T6. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to the TGF-β-induced group. #P < 0.05, ##P < 0.01, ###P < 0.001, ####P < 0.0001 compared to the control group.
Figure 7. (a) The viability of four kinds of cells was assessed using the Cell Titer-Glo assay after 24-h administration by compound 38. (b) Four-hour administration with the selective BET inhibitor compound 38 downregulated the mRNA levels of Il-1β, Il-6, and Tnf-α in mouse Kupffer cells.*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to the TGF-β-induced group. #P < 0.05, ##P < 0.01, ###P < 0.001, ####P < 0.0001 compared to the control group.
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