Integrating Network Pharmacology and In Vitro 3D Cardiac Organoid Model Validation to Investigate the Action Mechanism of Shenfu Xiangshao Decoction on Heart Failure

Figures

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  FIGURE 1.. The flowchart of this study integrates computational prediction and in vitro 3D cardiac organoid model validation.

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  FIGURE 2.. Differentiation process of human COs and IF staining of cardiac-specific markers. A, The development of COs from day 0 to day 11 of culture. Scale bars, 50 μm. B, Schematic illustration of COs stepwise differentiation from hiPSCs. C, IF staining for cardiac-specific markers cTnT, CD31, and α-SMA. Scale bars, 100 μm.

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  FIGURE 3.. Network pharmacology flowchart of SFXSD in the treatment of HF. A, Venn diagram illustrating HF-related targets derived from the GeneCards, OMIM, TTD, and DrugBank databases. B, Venn diagram showing the intersection of SFXSD and HF-related targets. C, D, Visualization of drug–disease common target genes using the STRING 12.0 database and Cytoscape 3.9.1 software. E, F, Identification of 33 core genes through CytoNCA and CytoHubba plug-ins in Cytoscape 3.9.1. G, GO and KEGG enrichment analyses highlighting the key biologic processes of SFXSD in treating HF. H, The “active ingredient–target–signaling pathway” network of SFXSD against HF.

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  FIGURE 4.. Molecular docking and MD simulations of the 4 active ingredients bound to MAPK14. A–D, Binding modes of QU (A), KA (B), LA (C), and NA (D) with the MAPK14 from molecular docking. E–H, The RMSD, RMSF, Rg, and number of hydrogen bonds for complexes QU (E), KA (F), LA (G), and NA (H) during the MD simulations.

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  FIGURE 5.. Formation and validation of the HF cardiac organoid model stimulated with DOX. A, Morphologic changes in COs after 24 hours of DOX treatment. Scale bars, 50 μm. B, Cell viability of DOX-treated COs assessed by the CCK-8 assay. C, LDH levels in the supernatant of DOX-treated COs. D, E, The NT-proBNP and cTnT levels of DOX-treated COs measured by ELISA kits. Values are presented as mean ± SEM (n = 6). n.s.: P > 0.05, ***P < 0.001, ****P < 0.0001 versus Control group.

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  FIGURE 6.. Screening of effective concentrations of QU and KA against DOX-induced myocardial injury in COs using CCK-8, LDH, NT-proBNP, and cTnT. A–D, Cytotoxicity assessment of QU and KA by CCK-8 and LDH assays. E–L, The cardioprotective effects of QU and KA against DOX-induced myocardial injury by measuring CCK-8, LDH, NT-proBNP, and cTnT levels. Values are presented as mean ± SEM (n = 6). n.s.: P > 0.05, **P < 0.01, ****P < 0.0001 versus Control group, #P < 0.05, ##P < 0.01, ####P < 0.0001 versus Model group.

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  FIGURE 7.. The protein expression and quantification of p38/ERK and BAX/BCL-2 in HF COs. A–C, The protein expression and quantification of p-P38, p-ERK, and BAX/BCL-2 treated by QU. D–F, The protein expression and quantification of p-P38, p-ERK, and BAX/BCL-2 treated by KA. Values are presented as mean ± SEM (n = 3). *P < 0.05, ***P < 0.001, ****P < 0.0001 versus Control group, #P < 0.05, ##P < 0.01 versus Model group.

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  FIGURE 8.. The effects of QU and KA on inflammation, apoptosis, and fibrosis in HF COs. The levels of IL-6 and TNF-α in the supernatant are examined by ELISA kits under the treatment of QU (A, B) or KA (C, D). E, TUNEL staining is applied to detect cardiomyocyte apoptosis after QU or KA treatment. Red fluorescence indicates apoptotic cells, while blue fluorescence marks cell nuclei. Scale bars, 100 μm. The relative fluorescence intensities of QU (G) or KA (H) treatments are quantified. F, The Masson staining of COs sections treated by QU or KA. Collagen fibers are blue, and the cytoplasm of cardiomyocytes is red. Scale bars, 200 μm (upper panel) and 50 μm (lower panel). The relative collagen content of QU (I) or KA (J) treatments is quantified. Values are presented as mean ± SEM (n = 3). *P < 0.05, **P < 0.01, ****P < 0.0001 versus Control group, #P < 0.05, ##P < 0.01, ###P < 0.001, ####P < 0.0001 versus Model group.