BEL (Lot: AFDE3105, Purity: 98%, Chengdu Alfa Biotechnology Co., Ltd., China) was used in this study. In the preliminary experiment, two gradient doses of BEL (25 mg/(kg$\cdot$d) and 50 mg/(kg$\cdot$d)) were selected based on literature data for similar drugs and species-equivalent dose conversion standards. Pharmacodynamic observations of cardiac function and myocardial injury indicators revealed that the cardioprotective effect of BEL was dose-dependent. Thus, 50 mg/(kg$\cdot$d) was chosen for further investigation [14, 15]. Based on previous research and references, DOX (Lot: NO.2541019001, Beijing Solarbio Science & Technology Co., Ltd., China) was administered at a dose of 5 mg/kg [14, 15, 18]. DEX (Lot: J14IS219829, Shanghai Yuanye Bio-Technology Co., Ltd., China), a Food and Drug Administration (FDA)-approved drug used to prevent DOX-induced cardiotoxicity in high-risk patients, was included as a positive control at a dose of 50 mg/kg, which is 10 times the DOX dose. This dose was chosen to ensure the accuracy and reliability of the results [5, 19].

Sixty specific-pathogen-free (SPF) male C57BL/6 mice (8 weeks old, body weight 18–22 g) were obtained from Beijing Sibeifu Biotechnology Co., Ltd. (License No.: SCXK (Beijing) 2019-0010). All mice were housed individually in cages with free access to food and water, in a well-ventilated environment with a 12-hour light/dark cycle, a temperature of 20–25 °C, and relative humidity maintained at 40%–70%. The experiment was approved by the Animal Welfare Committee of Hebei University of Chinese Medicine (Approval No.: DWLL202212032).

The mice were randomly divided into four groups (15 mice per group) using a random number table: Control, Model, BEL, and DEX groups. The Model, BEL, and DEX groups were intraperitoneally injected with 5 mg/kg DOX to establish a myocardial injury model, while the Control group received the same dose of normal saline via intraperitoneal injection once a week for 4 weeks. Echocardiography and myocardial tissue pathological staining were used to assess the success of modeling. In addition to DOX injection, the BEL group received 50 mg/(kg$\cdot$d) of BEL monomer suspension via intragastric administration for 28 consecutive days, while the DEX group was intraperitoneally injected with 50 mg/kg of DEX solution once a week for 4 weeks. The Control group was intragastrically administered normal saline at the same dose for 28 days.

As a ligand, the 2D structure of compound BEL was retrieved from the PubChem database and stored in “sdf” format. For selecting PDB files of core gene-related proteins, the RCSB PDB database (https://www.rcsb.org/) was utilized, with the following selection criteria: experimental method—X-ray crystallography (the primary method for determining protein tertiary structures), target species—Homo sapiens (human-derived proteins were chosen to accurately represent in vivo structural characteristics and avoid binding mode discrepancies due to species differences), resolution—0.5–2.5 Å (proteins with a resolution within this range provide detailed information on amino acid side chains and hydrogen bond networks, ensuring docking accuracy), presence of ligands in the structure (facilitating direct identification of the docking region and minimizing invalid structures without target pockets), pH range—7.35–7.50 (representing the physiological pH of the human body, simulating the native conformation of proteins in vivo). The proteins were screened according to these criteria and stored in “PDB” format. The online molecular docking software CB-Dock2 (https://cadd.labshare.cn/cb-dock2/php/blinddock.php) was then used to evaluate the binding activity between the compound and the target gene. In the molecular docking results, a Vina score lower than –5 kJ/mol indicates good docking between the protein and the ligand, with a lower Vina score reflecting a more stable ligand-receptor binding.

Based on the molecular docking results, YASARA 10.3.16 (YASARA Bioinformatics GmbH, Vienna, Austria) was used for molecular dynamics simulations. The protonation state of the residues was corrected by adding hydrogens and optimizing bond lengths and angles. Periodic boundary conditions (PBC) were applied, counterions were added to neutralize the system, and the physiological pH was set to 7.4. The appropriate force field was selected for the simulation. Through energy minimization and equilibrium simulations, steric clashes between atoms were resolved, and the energy of the initial structure was minimized. The predefined MD macro file was executed, and equilibrium simulations in the canonical ensemble (NVT) and isothermal-isobaric ensemble (NPT) were conducted for 100 ps each, at a temperature of 298 K and a pressure of 1 bar. Subsequently, a 100 ns molecular dynamics simulation of the complex system was performed, with conformations saved every 10 ps. Using YASARA’s built-in tools, parameters such as RMSD and RMSF were calculated to assess system stability. The binding free energy between the ligand and receptor was analyzed, and key interaction residues were identified through binding energy decomposition.

Statistical analysis was conducted using SPSS 27.0 (IBM Corp., Chicago, IL, USA), while GraphPad Prism 8.0.2 (GraphPad Software, San Diego, CA, USA) was used to generate statistical graphs. Data normality was tested, and comparisons of normally distributed data were performed using one-way analysis of variance (ANOVA). For homogeneous variances, post-hoc testing was done using the least significant difference (LSD) method. For heterogeneous variances, the Dunnett’s T3 test was applied. Data are presented as mean $\pm$ standard deviation ($\overline{x}$ $\pm$ s), and a p-value 0.05 was considered statistically significant.

During the experiment, compared to the blank control group, mice in the model group exhibited reduced water and food intake, yellowing fur, and lethargy, among other symptoms. These conditions improved to varying degrees in the treatment groups when compared to the model group. Echocardiography results revealed that the cardiac contour and internal structure in the blank control group were normal, with the ventricular wall showing a regular contraction/relaxation rhythm. In contrast, the model group displayed left heart enlargement, abnormal ventricular wall motion, and decreased EF and FS, along with increased values of LVIDs, LVPWs, LVVols, LVIDd, LVPWd, and LVVold (p 0.01). Each treatment group displayed significant improvement when compared with the model group (p 0.05 or p 0.01) (Fig. 1A,B). ECG results indicated that mice in the blank control group exhibited regular heart rhythms, while the model group showed arrhythmia, ST-segment depression, and other abnormalities. These issues improved to varying degrees in the treatment groups (Fig. 1C). The HW/BW% (heart weight-to-body weight ratio) in the model group was significantly elevated (p 0.01), but the HW/BW% values in the treatment groups were significantly reduced (p 0.01) (Fig. 1D). Indices of myocardial injury indicated that the levels of LDH and CK-MB were markedly increased in the model group in comparison with the control group (p 0.01). In contrast, the treatment groups exhibited significant reductions in these levels compared to the model group (p 0.05 or p 0.01) (Fig. 1E). Additionally, compared with the control group, the model group showed a significant decrease in the liver and kidney injury marker ALT (p 0.01), while UREA and CREA levels were significantly increased (p 0.01); after BEL treatment, UREA and CREA levels in mice decreased to varying degrees (p 0.05 or p 0.01), and CREA levels in the DEX group mice also showed a significant decrease (p 0.01) (Fig. 1F).These results indicate that DOX induces significant damage to cardiac and hepatorenal functions in mice, while BEL effectively mitigates this damage.

Fig. 1.

Effects of bellidifolin (BEL) on cardiac function, myocardial injury, and hepatorenal damage in mice. (A) Echocardiography of mice. (B) Cardiac function indices of mice. (C) Electrocardiogram of mice (The area pointed to by the arrow corresponds to the electrocardiographic features of arrhythmia). (D) Heart weight ratio of mice. (E) Serum indices of myocardial injury. (F) Serum indices of hepatorenal damage. (Data presented as mean $\pm$ SD. n = 6. Control (CON) vs. Doxorubicin (DOX): ^**p 0.01. DOX vs. BEL, DOX vs. DEX: ^#p 0.05,^#⁢#p 0.01).

Myocardial HE staining results (Fig. 2A,B) revealed that, compared to the control group, mice in the model group exhibited varying degrees of eosinophilic changes, marked swelling of myocardial cells, significant rupture of myocardial striations, fibroblast proliferation, considerable thickening of myocardial cells compared to normal cells, deep-stained cytoplasm, pyknosis of myocardial cell nuclei, progressive nuclear disappearance, and visible apoptosis and necrosis, accompanied by inflammatory cell infiltration in necrotic areas. In contrast, each treatment group showed significant improvements compared to the model group.

Fig. 2.

BEL improves pathological changes in the myocardium, liver, and kidneys of cardiotoxic mice. (A,B) HE staining results of myocardial tissue (400$\times$, The area pointed to by the black arrow corresponds to the region characterized by fibroblast proliferation and increased spacing between myocardial cells). Scale bar: 50 μm. (C,D) HE staining results of liver tissue (400$\times$, The area pointed to by the black arrow corresponds to the region of hepatic steatosis). Scale bar: 50 μm. (E,F) HE staining results of renal tissue (400$\times$, The area pointed to by the black arrow corresponds to the region characterized by renal cell necrosis and interstitial edema). Scale bar: 50 μm. (G,H) Ultrastructure of myocardial tissue (5000$\times$, The area pointed to by the black arrow corresponds to the region characterized by mitochondrial disorganization and myofiber blurring). (CON vs. DOX: ^**p 0.01. DOX vs. BEL, DOX vs. DEX: ^#p 0.05, ^#⁢#p 0.01). Note: Pathological Scoring Criteria: (1) Myocardial Injury: Score 0: Myofibers are regularly arranged in bundles, with clear sarcomeres and neat alignment. Score 1: Scattered individual cells show vacuolation, myofibers are disorganized, and no obvious edema is present. Score 2: Myofiber fragmentation, partial inflammatory infiltration, and fibroblast proliferation are observed. Score 3: Extensive fibroblast proliferation, darkly stained cells, eosinophilic changes, and inflammatory infiltration are present. (2) Hepatic Injury: Score 0: No steatosis, no inflammatory cell infiltration, and no fibrosis. Score 1: Mild steatosis, inflammatory cell infiltration, and fibrosis. Score 2: Moderate steatosis, inflammatory cell infiltration, and fibrosis. Score 3: Severe steatosis, inflammatory cell infiltration, and fibrosis. (3) Renal Injury: Score 0: No cell necrosis and no interstitial edema. Score 1: Mild cell necrosis or interstitial edema. Score 2: Moderate cell necrosis or interstitial edema. Score 3: Severe cell necrosis or interstitial edema. (4) Mitochondrial Injury: Score 0: Mitochondria are neatly arranged, and myofibers are clear. Score 1: Mild disorganization of mitochondrial arrangement. Score 2: Disorganized mitochondrial arrangement and blurred myofibers. Score 3: Severe disorganization of mitochondrial size, structure, and arrangement, with blurred myofibers. Scale bar: 1 μm.

Liver HE staining results (Fig. 2C,D) indicated that liver tissue from mice in the model group exhibited extensive inflammatory cell infiltration around the portal vein, dilation of hepatic lobular venous lumens, and liver damage. The inflammatory infiltration in liver tissue was markedly reduced in all treatment groups compared to the model group.

Kidney HE staining results (Fig. 2E,F) showed that compared to the control group, renal tissue in the model group exhibited obvious tubular epithelial edema, swollen cells, vacuolated cytoplasm, visible connective tissue hyperplasia, and lymphocyte infiltration. Pathological damage in the renal tissue was significantly alleviated in all treatment groups compared to the model group.

To further assess myocardial injury, the microstructure of the mouse myocardium was observed by transmission electron microscopy (TEM). As shown in Fig. 2G,H, myocardial mitochondria in the blank control group were neatly arranged, numerous, with intact mitochondrial membrane structures, clear cristae, and no rupture or obvious hypertrophy. In contrast, myocardial mitochondria in the model group were disorganized, condensed, scattered, with dissolution and disappearance of surrounding myofilaments, mitochondrial cristae rupture, hypertrophy, hyperplasia, and severe vacuolization. The administration groups showed significant improvements in mitochondrial structure compared to the model group.

Myocardial Masson staining results (Fig. 3A,B) revealed that myocardial cells appeared pink, while collagen fibers were stained blue-purple. Compared to the blank control group, myocardium in the other groups exhibited varying degrees of injury. In the model group, pathological sections showed disorganized myocardial cell arrangement, significant accumulation of blue-purple fibrous collagen, and destruction of myocardial structure. In contrast, the administration groups exhibited more organized myocardial cells with reduced collagen fiber accumulation compared to the model group. The Masson staining results indicated a strong correlation between cardiotoxicity and the development of myocardial fibrosis. To further assess collagen expression in myocardial tissues, Sirius red staining and Sirius red polarized light staining were performed. The results were consistent with the Masson staining, showing that collagen expression in myocardial tissues correlated with the severity of fibrosis, as shown in Fig. 3C–F.

Fig. 3.

BEL ameliorates myocardial fibrosis in cardiotoxic mice. (A,B) Masson staining results of myocardial tissue (400$\times$, Collagen fibers: blue. muscle fibers: red. cell nuclei: blackish blue, the region denoted by the arrow corresponds to the area of collagen fiber deposition). Scale bar: 50 μm. (C,D) Sirius red staining of myocardial tissue (200$\times$, Collagen fibers: red. muscle fibers: yellow, the region denoted by the arrow corresponds to the area of collagen fiber deposition). Scale bar: 100 μm. (E,F) Sirius red (polarized light) staining of myocardial tissue (200$\times$, Collagen I: yellowish-red/orange-red. Collagen III: green, the region denoted by the arrow corresponds to the area of collagen fiber deposition). Scale bar: 100 μm. (CON vs. DOX: ^**p 0.01. DOX vs. BEL, DOX vs. DEX: ^#p 0.05, ^#⁢#p 0.01).

Molecular docking of BEL with the 16 core targets was performed to verify its binding capacity to target proteins at varying degrees. Based on the aforementioned criteria, the PDB files of AKT1 (7MYX), EGFR (8A27), BCL2 (8H7B), CASP3 (7JI7), STAT3 (6TLC), IL6 (5GW9), TNF (6U66), PTGS2 (5F19), CCND1 (6P8E), ESR1 (7UJO), HSP90AA1 (3O0I), SRC (3EAC), TGF-$\beta$1 (5VQP), HDAC1 (7Z1K), CDK2 (6Q4G), and CDK4 (7OXW) were selected for docking with BEL (Fig. 5). The results demonstrated that BEL satisfied the criteria for docking with the key targets. Details such as binding energy and binding site information are presented in Table 3, confirming that BEL intervenes in DIC through core targets.

Fig. 5.

Molecular docking results of BEL and key target proteins. Note: Hydrogen bonds (blue dotted line), weak hydrogen bonds (white dotted line), hydrophobic interactions (gray dotted line), ionic interactions (yellow dotted line), cation-$\pi$ interactions (orange dotted line), and $\pi$-$\pi$ stacking forces (green dotted line).

Based on the molecular docking results, the TGF-$\beta$1-BEL complex was selected for molecular dynamics simulation to evaluate the binding stability of the protein-ligand complex. Fig. 6A illustrates the binding mode of the TGF-$\beta$1-BEL complex. The root-mean-square deviation (RMSD), a key indicator of conformational atomic deviations, was used to monitor the stability of the complex system. As shown in Fig. 6B, the RMSD of the TGF-$\beta$1-BEL complex stabilized between 50,000 and 100,000 ps, indicating minimal conformational changes in the TGF-$\beta$1 protein and relatively stable binding between TGF-$\beta$1 and BEL. The root-mean-square fluctuation (RMSF) reflects the flexibility of amino acid residues. Fig. 6C shows significantly higher flexibility in the amino acid residues between positions 180–220 and 300–340 of TGF-$\beta$1 in the TGF-$\beta$1-BEL complex compared to other regions. Fig. 6D presents the binding free energy calculated by the YASARA program, confirming that BEL binds stably to TGF-$\beta$1 with strong binding energy and affinity.

Fig. 6.

Molecular dynamics simulation results of BEL and key target proteins. (A) Binding mode of the TGF-$\beta$1-BEL complex. (B) RMSD. (C) RMSF. (D) Binding energy.

The expression of TGF-$\beta$1 was assessed in different groups of mice in vivo using immunohistochemistry, RT-qPCR, and Western blotting (WB). Results showed a significant increase in TGF-$\beta$1 expression in the model group (p 0.01), while all treatment groups exhibited a significant decrease in TGF-$\beta$1 levels (p 0.01) (Fig. 7A–C). These results confirm that TGF-$\beta$1 is a key target for BEL in the treatment of DIC.

Fig. 7.

Expression of TGF-$\beta$1, $\alpha$-SMA, Col I, and Col III in myocardial tissue of mice. (A) Immunohistochemical detection of TGF-$\beta$1 (200$\times$). Scale bar: 100 μm. (B) RT-qPCR detection of TGF-$\beta$1. (C) Western blotting detection and grayscale analysis of TGF-$\beta$1. (D) Immunohistochemical detection of $\alpha$-SMA (200$\times$). Scale bar: 100 μm. (E) RT-qPCR detection of $\alpha$-SMA. (F) WB detection and grayscale analysis of $\alpha$-SMA. (G) Immunohistochemical detection of Col I and Col III (200$\times$). Scale bar: 100 μm. (H) RT-qPCR detection of Col I and Col III. (I) WB detection and grayscale analysis of Col I and Col III. (Antibody dilution: TGF-$\beta$1 = 1:20,000. $\alpha$-SMA = 1:5000. Col I = 1:1000. Col III = 1:1000) (CON vs. DOX: ^*p 0.05, ^**p 0.01. DOX vs. BEL, DOX vs. DEX: ^#p 0.05,^#⁢#p 0.01).

Following the same verification methods as for TGF-$\beta$1, the expression of fibrosis-related markers $\alpha$-SMA, Col I, and Col III was further assessed at the tissue, protein, and gene levels. The results revealed a significant increase in $\alpha$-SMA expression in the model group (p 0.01), which was significantly reduced after intervention with BEL and DEX (p 0.01) (Fig. 7D–F). The expression trends of Col I and Col III mirrored those of $\alpha$-SMA (Fig. 7G–I).

Given that TGF-$\beta$1 exerts its effects via regulation of the PI3K-AKT signaling pathway, which was also identified in the KEGG enrichment analysis (Fig. 3E), the expression of PI3K, AKT, and their phosphorylated forms in myocardial tissues was further investigated. The results showed that p-PI3K/PI3K and p-AKT/AKT were significantly decreased in the model group (p 0.01), but were significantly restored after treatment with BEL and DEX (p 0.01), as shown in Fig. 8.

Fig. 8.

Expression of the PI3K-AKT signaling pathway in myocardial tissue of mice. (A) WB detection strip chart. (B) Grayscale analysis of WB detection. (Antibody dilution: PI3K = 1:1000. p-PI3K = 1:1000. AKT = 1:1000. p-AKT = 1:1000) (CON vs. DOX: ^*p 0.05, ^**p 0.01. DOX vs. BEL, DOX vs. DEX: ^#p 0.05, ^#⁢#p 0.01).