Uncovering the molecular mechanisms of tonifying kidney and activating blood Decoction against myocardial fibrosis using network Pharmacology and experimental validation

Figures

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  Fig. 1. Tonifying Kidney and Activating Blood decoction demonstrates potential therapeutic effects by inhibiting ventricular fibrosis and the activation of the p38MAPK/NF-κB signaling pathway, as well as repairing the blood-brain barrier in chronic heart failure rats.This suggests a significant role of the p38MAPK/NF-κB signaling pathway in the pathogenesis of ventricular fibrosis and blood-brain barrier damage.Abbreviations: MAPK, mitogen-activated protein kinase; NF-κB, nuclear factor kappa-B.(By BioRender (app.biorender.com)).

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  Fig. 2. The application of network pharmacology is utilized for the analysis of Tonifying Kidney and Activating Blood decoction and chronic heart failure.This includes a Venn diagram illustrating the targets of active constituents of Tonifying Kidney and Activating Blood decoction and chronic heart failure-related targets (A), a PPI network diagram of core targets (B), and an enrichment analysis diagram of GO (left) and KEGG pathway (right) (C). The “Active ingredients-key targets-pathway” network is also presented (D).Abbreviations: GO, gene ontology; KEGG, kyoto encyclopedia of genes and genomes; PPI, protein-protein interaction.

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  Fig. 3. Molecular docking technology is employed to examine the interaction between tanshininone IIA in the monarch drug Salvia miltiorrhiza of Tonifying Kidney and Activating Blood decoction and its targets.An analysis of the binding patterns of p38MAPK, I-κBα, p65 NF-κB, AQP4, Occludin, ZO-1, AKT, and STAT3 to tanshinone IIA is conducted, with the docking scores provided(A-H).Abbreviations: MAPK, mitogen-activated protein kinase; I-κBα,nuclear factor-kappa B inhibitor alpha; NF-κB, nuclear factor kappa-B; AQP4, aquaporin-4;ZO-1,zonula occludens 1;AKT, protein kinase B; STAT3,signal transducer and activator of transcription 3.

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  Fig. 4. Tonifying Kidney and Activating Blood decoction delays the onset of ventricular fibrosis in chronic heart failure. (A) Pathological and fibrosis images of heart, as demonstrated by Masson’s trichrome staining (scale bar = 200 μm) and hematoxylin and eosin staining (scale bar = 200 μm).Pathological markers of myocardial hematoxylin and eosin staining include the following: a black arrow (→) indicating the area of myocardial infarction; an asterisk (*) denoting significant infiltration of lymphocytes and neutrophils; a blue arrow (→) pointing to swollen cardiomyocyte in the marginal area of myocardial infarction; and a green arrow (←) highlighting dense fibrous connective tissue, which contains collagen fiber. (B) Representative images of fibrosis and mitochondrial structures in myocardial tissues, as visualized by Sirius Red staining (scale bar = 200 μm) and transmission electron microscopy (scale bar = 1.0 μm).The ultrastructural markers of myocardial tissue observed through transmission electron microscopy are as follows: the black arrow (→) indicating an inordinance myofibrils; the blue arrow (→) denoting mitochondria vacuolization; and the green arrow (←) signifing…

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  Fig. 5. Tonifying Kidney and Activating Blood decoction mitigates the disruption of the blood-brain barrier induced by chronic heart failure and to inhibit the expression of hippocampal p38MAPK/NF-κB pathway.(A) Pathological and intercellular junctions images, as captured using transmission electron microscopy(scale bar = 1.0 μm), hematoxylin and eosin staining(scale bar = 100 μm), and Nissl staining(scale bar = 100 μm).The blue arrow(“→”) indicates capillary pericytes; the black arrow(“→”) points to astrocyte foot board; the purple arrow(“→”) identifies capillary endothelial cells; the asterisk(“*”) highlights erythrocytes; the red arrow(“→←”) denotes density change of pyramidal neurons in hippocampal CA1 region; the green arrow(“→”) indicates intercellular tight junction change in hippocampal blood-brain barrier and its disruption in model rats. (B-C)The expression levels of proteins, including (p-)p38MAPK, (p-)p65 NF-κB, AQP4, (p-)I-κBα,STAT3,TNF-α, PI3 K, and AKT in hippocampal tissues of five groups of rats(including KTBA + S group) using western blot,n = 3–4. (D-E) The levels of (p-)p65 NF-κB, STAT3,AQP4, Occludin, and ZO-1 were examined in hippocampal tissues from the five groups…

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  Fig. 6. Tanshinone IIA has been observed to regulate proteins associated with inflammation, water metabolism, and gut barrier in CCD-841 CoN cells subjected to ischemia/hypoxia. (A) Western blot analysis was conducted to examine The expression levels of inflammation proteins (PI3 K, AKT, p38MAPK, p65 NF-κB, I-κBα, and STAT3), water metabolism protein (AQP4), and gut barrier proteins (ZO-1, ZO-2, and Claudin-1) at 48 h across different experimental groups. (B) The relative expression intensities of the inflammation proteins p38MAPK, p65 NF-κB, I-κBα, STAT3, PI3 K and AKT, water metabolism protein AQP4, gut barrier proteins ZO-1, ZO-2, and Claudin-1 at 48 h using western blot,n = 3. (C) The relative intensity of p38MAPK, p65 NF-κB, AQP4, I-κBα, ZO-1,and Occludin mRNA at 48 h, as measured by RT-qPCR,n = 3. (D) Immunofluorescence images of p38MAPK, p65 NF-κB, and AQP4 gene at 48 h. (E) The level of p38MAPK, p65 NF-κB, and AQP4 proteins at 48 h, as quantified by immunofluorescence staining, n = 3. Note: *p < 0.05, **p < 0.01.AKT, protein kinase B; AQP4,aquaporin-4; DAPI,4’,6-diamidino-2-phenylindole, dihydrochloride; I-κBα,nuclear factor-kappa B inhibitor alpha; mRNA, messenger ribonucleic…

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  Fig. 7. (A) Technology Roadmap for Animal Experimentation In Vivo. (B)The ECG of rats were analyzed both before and after the ligation of the LAD artery. Figure Lead I A and Lead II A illustrates the ECG recorded prior to the surgical procedure, whereas Figure Lead I B and Lead II B displays the ECG obtained post-surgery. The red arrow (↓) indicates the elevation of the ST-segment, a hallmark of acute myocardial infarction, specifically anterior lateral infarction, implicating the LAD as the responsible vessel. The blue arrow (↑) denotes ST-segment depression and T wave inversion, while the green arrow (→)signifies the presence of a pathologic QS complex. (C) Tanshinone IIA was found to inhibit the dysregulation of water metabolism, disruption of the gut fluid barrier, and p38MAPK/NF-κB pathway. These processes are typically observed in hypoxia-induced CCD-841 CoN cells, with the p38MAPK/NF-κB signaling pathway playing a significant role in the dysbiosis of water metabolism and disruption of the gut fluid barrier. Therefore, the potential of Tanshinone IIA as an inhibitor of these processes warrants further investigation. (By app.biorender.com). (D)Technology Roadmap for Cell…