Multi-Target Cardioprotection from Berberis kaschgarica Extract in Zebrafish via AMPK Pathway Activation

Figures

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  Figure 1. Identification of components in BKR extracts. (A) Depicts the total ion current (TIC) chromatogram of the samples in positive ion mode; (B) illustrates the TIC chromatogram of the samples in negative ion mode. The blue triangle in the lower-left corner indicates the starting point of LC-MS data acquisition.

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  Figure 2. Two complementary in vitro assays were employed to assess the antioxidant potential of the BKRE extract. (A) DPPH radical scavenging capacity of the BKRE extract was evaluated, which serves as a direct indicator of its free radical neutralization ability. (B) ABTS radical eliminating activity of the extract was further determined to clarify its antioxidant efficacy under in vitro conditions.

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  Figure 3. Network pharmacology analysis. (A) Ingredient target network diagram: The innermost dark green circle denotes BKR, with 14 surrounding green diamonds corresponding to its 14 bioactive compounds, whereas the two outer light green square circles and adjacent right-side square cluster denote 421 associated targets. (B) Venn diagram. (C) PPI network diagram. (D) Volcano plot integrating fold change values and adjusted p-values; this plot highlights major gene expression alterations. Red dots indicate upregulated genes, blue dots downregulated genes. The horizontal dashed line marks the significance threshold, distinguishing genes with significant vs. non-significant expression changes. (E) Differential gene expression heatmap.

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  Figure 4. Bioinformatics analysis: (A) Venn diagram; (B) KEGG pathway enrichment diagram; (C) GO analysis: BP; (D) GO analysis: CC; (E) GO analysis: MF; Red boxes highlight core pathways.

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  Figure 5. Molecular docking. (A) Heatmap: depicts the binding results between 14 bioactive components and 5 core targets. Color intensity reflects the receptor–ligand binding affinity: darker shades indicate stronger binding activity. (B) This diagram shows structural models of 8 component–target molecular pairs with the lowest binding energies.

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  Figure 6. Effects of BKRE exposure on morphology, survival rate, malformation rate, and heart rate of zebrafish larvae. (A) Representative morphological phenotypes of zebrafish larvae following exposure to different BKRE concentrations (Ctrl, 1, 10, 50, 200 μg/mL; top to bottom). (B) Survival rate of zebrafish larvae under different BKRE exposure concentrations. Data are presented as mean ± SEM (n = 3 biological replicates per group); ** p < 0.01 vs. Ctrl group. (C) Malformation rate of zebrafish larvae under different BKRE exposure concentrations. Malformations included body axis curvature and edema. Data are presented as mean ± SEM (n = 3); ** p < 0.01 vs. Ctrl group. (D) Heart rate (beats/20 s) of zebrafish larvae under different BKRE exposure concentrations. Data are presented as mean ± SEM (n = 3); * p < 0.05 vs. Ctrl group.

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  Figure 7. Protective effects of BKRE on isoproterenol ISO-induced cardiac dysfunction in zebrafish larvae. (A) Representative fluorescence images of cardiac morphology (cardiomyocytes labeled with MF20 antibody, green) in Ctrl, model, and BKRE-treated (5, 10, 20 μg/mL) groups. (B) Heart rate of zebrafish larvae in each group. Data are mean ± SEM (n = 3 biological replicates per group). (C) Cardiac output of zebrafish larvae in each group. Data are mean ± SEM (n = 3). (D) Atrial–ventricular (AV) distance of zebrafish larvae in each group. Data are mean ± SEM (n = 3). (E) Cardiac edema volume of zebrafish larvae in each group. Data are mean ± SEM (n = 3). Statistical significance: * p < 0.05, ** p < 0.01 vs. Ctrl; ## p < 0.01 vs. model; ns: not significant.

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  Figure 8. The impact of BKRE on ISO-induced cardiac apoptosis in zebrafish larvae. (A) Fluorescence microscopy visualized apoptotic cells in zebrafish larvae from Ctrl, ISO-induced model, and BKRE-treated (5, 10, 20 μg/mL) groups. Apoptotic cells were labeled using the Annexin V-FITC Apoptosis Detection Kit (Cat. No. A13201, Thermo Fisher Scientific, USA) and exhibited green fluorescence; increased fluorescence intensity indicated enhanced apoptotic activity. (B) Quantitated the relative apoptotic fluorescence intensity for each group (n = 3 biological replicates per group). Apoptosis levels were significantly elevated in the ISO-induced model group compared to the Ctrl group (## p < 0.01); BKRE treatment (5–20 μg/mL) led to a dose-dependent reduction in apoptotic fluorescence intensity relative to the model group (* p < 0.05, ** p < 0.01).

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  Figure 9. ROS accumulation. (A) Fluorescence imaging assessed ROS levels in zebrafish larvae from Ctrl, ISO-induced model, and BKRE-treated (5, 10, 20 μg/mL) groups. (B) Quantification of relative ROS fluorescence intensity (n = 3 biological replicates per group) showed significantly elevated ROS levels in the ISO group vs. Ctrl (## p < 0.01). BKRE (10, 20 μg/mL) significantly reduced ROS intensity vs. the ISO group (* p < 0.05), while the 5 μg/mL group showed no statistical significance (ns).

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  Figure 10. Oxidative stress and inflammatory responses: (A) GSH content; (B) NO content; (C) T-SOD activity; (D) IL-6; (E) TNF-α and (F) CAT. Data are presented as mean ± SEM (n = 3 per group). Statistical significance: ns, not significant; * p < 0.05, ** p < 0.01 vs. model group; ## p < 0.01 vs. Ctrl group.

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  Figure 11. Transcriptome analysis. (A) Volcano plot: green = downregulated, red = upregulated. (B) Heatmap: color intensity reflects relative expression. (C) Bubble plot: red boxes = key pathways, bubble size = gene count, color = p-value. Relative mRNA expression of target genes: (D) AMPK-α; (E) HK2; (F) PFKL; (G) PPARα; (H) PPARGC-1α; (I) mTOR. Data: mean ± SEM (n = 3/group). Statistics: one-way ANOVA + Tukey’s test. * p < 0.05, ** p < 0.01 vs. model; # p < 0.05, ## p < 0.01 vs. Ctrl; ns = not significant.

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  Figure 12. Mechanism diagram: In the ISO-induced zebrafish HF model, BKRE exerts multifaceted cardioprotection by activating the core regulatory axis AMPKα-PPARα-PGC-1α: BKRE binds to core targets (ALOX5, NQO1) to activate AMPKα, which triggers two parallel regulatory branches—activated AMPKα directly upregulates PGC-1α, a key coactivator regulating energy metabolism, while also phosphorylating and activating PPARα; subsequently, PPARα and PGC-1α form a coactivator complex that synergistically upregulates the transcription of glycolysis-related key genes (HK2, PFKL), thereby alleviating myocardial energy metabolism disorders. Concurrently, this regulatory axis enhances SOD activity and promotes GSH biosynthesis to scavenge excess ROS, suppresses the NF-κB pathway to reduce secretion of pro-inflammatory cytokines (IL-6, TNF-α) and maintain NO homeostasis; collectively, these effects inhibit cardiomyocyte apoptosis, alleviate atrioventricular dilation and pericardial edema, ultimately improving cardiac function and attenuating ISO-induced HF in zebrafish. Arrows indicate activation/promotion of molecular/pathway regulatory relationships; upward arrows (↑) represent increased…