Regulatory Effects of Zhenxin Formula in Treating Doxorubicin-Induced Heart Failure: Network Pharmacology and Animal Experimental Verification

Abstract

Objective: This study aimed to elucidate the mechanisms underlying the protective effects of Zhenxin Formula (ZXF) against doxorubicin (Dox)-induced HF through the integration of network pharmacology, phospho-antibody array analysis, and experimental validation.

Methods: The active components and potential targets of ZXF were identified via the Traditional Chinese Medicine Systems Pharmacology (TCMSP) platform, while HF-associated target genes were retrieved from the OMIM, Genecards, and TTD databases. Protein-protein interaction (PPI) networks and compound-disease target networks were constructed using Cytoscape 3.7.2, with functional annotations performed through GO enrichment and KEGG pathway analyses using R software. Experimental validation involved the establishment of a Dox-induced HF model via intraperitoneal injection, with ZXF's therapeutic effects evaluated using cardiac ultrasound, morphological staining, and Western blot analysis. Additionally, a phospho-antibody array was utilized to screen over 300 molecules across 16 canonical signaling pathways in ZXF-treated HF models, with Western blot analysis confirming the specific pathways implicated in ZXF's therapeutic effects.

Results: Network pharmacology analysis identified 56 potential active ingredients in ZXF, 47 of which were associated with HF-related targets. AKT1 emerged as the target most strongly correlated with HF improvement. In vivo, ZXF significantly enhanced cardiac function and mitigated myocardial fibrosis and cardiomyocyte apoptosis. Phospho-antibody array analysis revealed that 16 phosphorylated proteins were upregulated and 3 downregulated in the Dox-treated group. ZXF intervention resulted in the upregulation of 10 phosphorylated proteins and downregulation of 5. Comparative analysis highlighted PDK1-Phospho and FOXO1/3/4-Phospho as pivotal phosphorylated proteins mediating ZXF's cardioprotective effects. Western blot analysis confirmed that ZXF enhanced phosphorylation levels of PI3K, PDK1, AKT, and FOXO1 in the Dox-induced HF model.

Conclusion: This study, employing network pharmacology, phospho-antibody array analysis, and experimental validation, demonstrates that ZXF ameliorates cardiac dysfunction and suppresses myocardial apoptosis in Dox-induced HF through modulation of the PI3K/PDK1/AKT/FOXO1 signaling pathway.

Keywords: PI3K/PDK1/AKT/FOXO1 signaling pathway; Zhenxin Formula; heart failure; network pharmacology; phospho-antibody array.

Figure 1

Screening of Drug-Disease Common Targets. In the figure, the purple circles represent drug targets of ZXF, the yellow circles denote heart failure (HF)-related disease targets, and their intersecting area indicates the shared drug-disease common targets.

Figure 2

Screening of Drug-Disease Common Targets. Hexagonal nodes represent herbal medicines; diamond nodes represent unique components of each herb; triangular nodes represent shared components (9 active components without target-disease intersections were removed, while 47 effective components are highlighted in red in Supplementary Table S1); circular nodes represent the 120 common targets. Node sizes vary according to their degree values.

Figure 3

Network Pharmacology Analysis Identifies Hub Targets of ZXF. (A) Node size and color intensity vary according to their Degree values, with the outermost circle representing targets having Degree ≥ 10. (B) Ranking of Core Targets Based on PPI Topological Analysis (Top 20 by Degree). The bar graph illustrates the Degree values of the targets.

Figure 4

Enrichment Analysis of Identified Targets and KEGG Pathway Analysis for ZXF. Enrichment Analysis of Identified Targets and KEGG Pathway Analysis for ZXF. (A) GO enrichment analysis of ZXF for heart failure treatment. The y-axis represents three categories: biological processes, cellular components, and molecular functions; the x-axis shows gene percentage; circle size corresponds to gene count, while color indicates significance. (B) KEGG enrichment analysis of ZXF compound for heart failure treatment. The y-axis displays pathways, the x-axis shows gene percentage, with circle size representing gene count and color indicating significance.

Figure 5

Chromatogram of catechin and paeoniflorin standards versus ZXF extract. Chromatogram of catechin and paeoniflorin standards versus ZXF extract. ((A) reference standard solution, (B) sample solution; 1 - catechin, 2 – paeoniflorin).

Figure 6

ZXF Improves Cardiac Function. (A) Animal experimental strategy. (B–G) Statistical results of EF, FS, LVESD, LVEDD, CO, SV. **P<0.01 vs Con group; ^ΔP<0.05, ^ΔΔP<0.01 vs Dox group. Sample sizes (n): Con=10, Dox=7, ZXF=8, Cap=7.

Figure 7

ZXF attenuated cardiac fibrosis and inflammation. (n=5). (A) Representative images of HE staining, Masson’s staining and Immunohistochemical staining of FAS; (B) Statistical results of CVF (%). (C) Statistical results of Fas protein expression levels (%). **P<0.01 vs Con group; ^ΔΔP<0.01 vs Dox group. Black arrows indicate characteristic histopathological changes, with their number reflecting lesion severity. Histochemistry analysis, magnification: 200×; scale=50 µm.

Figure 8

ZXF inhibited cardiomyocyte apoptosis. (n=5). (A) Western blot of protein levels of Caspase-3, Bcl-2, Bax and Fas. (B–E) Statistical results of Caspase-3/GAPDH, Bcl-2/GAPDH, Bax/GAPDH and Fas/GAPDH. **P<0.01 vs Con group; ^ΔΔP<0.01, ^nsP>0.05 vs Dox group.

Figure 9

Phospho-Antibody array results showing the regulatory effects of ZXF on HF. (n=6). (A) The overview of phospho-antibody array results. (B) The figure of PDK1 (Phospho-Ser241), PDK1 (Ab-241), FOXO1/3/4-pan (Phospho-Thr24/32) and FOXO1/3/4-pan (Ab-24/32). (C and D) Statistical results of p-PDK1/PDK1, and p-FOXO1/3/4/FOXO1/3/4. *P<0.05, **P<0.01 vs Con group; ^ΔP<0.05 vs Dox group.

Figure 10

ZXF regulated the PI3K/PDK1/AKT/FoxO1 signaling pathway. (n=5). (A) Western blot of phosphorylated and total protein levels of PI3K, PDK1, AKT, and FOXO1. (B–E) Statistical results of p-PI3K/PI3K, p-PDK1/PDK1, p-AKT/AKT, and p-FOXO1/FOXO1. **P<0.01 vs Con group; ^ΔP<0.05, ^ΔΔP<0.01 vs Dox group.

Figure 11

Illustration of the PI3K/PDK1/AKT/FoxO1 Signaling Pathway in Inhibiting Cardiomyocyte Apoptosis and the Role of ZXF.