Kaixuan Qibi granules attenuate myocardial fibrosis through BRD4 blockade regulated by the NF-κB/NLRP3 signaling pathway

Figures

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  Fig. 1. Qualitative analysis of KXGs using UPLC-HRMS. (A) UPLC-HRMS chromatogram of KXGs in negative-ion mode. (B–C) UPLC-HRMS chromatogram of nine reference standards, namely rehmanosine D (1), amygdalin (2), paeoniflorin (3), ferulic acid (4), liquiritin (5), verbascoside (6), naringin (7), neohesperidin (8), and kaempferol (9). (D) Identification of 168 KXGs targets closely associated with myocardial fibrosis-related inflammation.

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  Fig. 2. KXGs exhibited remarkable efficacy in inhibiting ISO-induced MF while demonstrating no pathologic alterations in other organs. (A) Portrayal of the procedural framework involved in establishing the model of MF in C57BL/6 mice and evaluating the therapeutic efficacy of KXGs. (B) Sections of liver, heart, spleen, lung, and kidney tissue underwent histology with H&E staining (n = 4). The black arrow in the ISO group indicates the region of myocardial disarray with inflammatory infiltration and fibrotic changes. Scale bars = 100 μm.

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  Fig. 3. KXGs suppressed the progression of collagen deposition in vivo during MF. (A) Representative echocardiography graphs of NC, ISO, KX-L, KX-H, and FOS groups. Echocardiography was utilized for quantitative assessment of impaired cardiac function through (B) EF%, (C) FS%, and (D) LVESD (mm). (E) Myocardial tissue sections were subjected to staining (Masson's trichrome, Sirius red). Black arrows indicate collagen fibers (blue in Masson staining; red in Sirius Red staining). Scale bar: 100 μm. (F) The MF induced by ISO was assessed through immunofluorescent staining against collagen-І (green), collagen-ІІІ (green), and fibronectin (green), with counterstaining using DAPI (blue: nuclei) in the experimental groups indicated. Scale bar: 100 μm. Data are the mean ± SD (n = 4). Statistical significance was observed at p < 0.05∗ and p < 0.01∗∗. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

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  Fig. 4. Untargeted metabolomics analysis of serum following various treatments. (A) Venn diagrams depicting the metabolites of distinct treatment cohorts. (B) OPLS-DA of serum metabolites under different cohorts. (C) Volcano plots illustrating the differential abundance of metabolites in serum subjected to various treatments. (D) Heatmaps displaying significant variations in serum metabolite profiles among different treatment cohorts. (E) Bubble diagram depicting the enrichment of differential metabolites in cell metabolism-related pathways based on the KEGG database.

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  Fig. 5. KXGs inhibited the development of LPS-induced activation of inflammatory cytokines in vitro. (A) Cell viability was evaluated following a 48-h incubation of RAW264.7 cells with varying concentrations of KXGs. (B–H) Expression of proteins related to inflammatory cytokines, such as BRD4, ASC, caspase-1, NLRP3, NF-κB p65 activation index (p-NF-κB p65/NF-κB p65 ratio), and IL-1β, after KXGs treatment, were compared among the LPS group and KXGs group in RAW264.7 cells.

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  Fig. 6. KXGs inhibit the BRD4/NF-κB/NLRP3 signaling pathway during MF in vivo. (A) Immunofluorescence staining of ASC (green), BRD4 (green), NLRP3 (green), and p-NF-κB p65 (green) in myocardial tissue sections (n = 4). Scale bar: 100 μm. (B) Immunofluorescence staining of the proteinase caspase-1 (green) and inflammatory factor IL-1β (green) in myocardial tissue sections (n = 4). Scale bar: 100 μm. (C–I) Protein expression of ASC, BRD4, NLRP3, NF-κB p65 activation index (p-NF-κB p65/NF-κB p65 ratio), caspase-1, and IL-1β in myocardial tissue was assessed using western blotting, Data are the mean ± SD (n = 3). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

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  Fig. 7. Schematic of the potential mechanism underlying the impact of KXGs on attenuating MF. KXGs, comprising 16 traditional Chinese herbs, were found to consist of nine components by UPLC-HRMS, and these components have anti-inflammatory potential. Intraperitoneal injection of ISO in C57BL/6J mice elicited macrophage activation, which involved the BRD4/NF-κB/NLRP3 signaling pathway and triggered the secretion of a multitude of inflammatory mediators. KXGs inhibited the expression of BRD4 protein and subsequently modulated the NF-κB/NLRP3 signaling pathway, thereby attenuating inflammation and MF.