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Introduction

Myocardial fibrosis (MF) plays a critical role in the progression of cardiovascular diseases towards heart failure, often manifesting as cardiac systolic and diastolic dysfunction, arrhythmias and adverse cardiovascular events (1,2). Driven predominantly by coronary atherosclerosis and prolonged myocardial ischemia and hypoxia, MF involves substantial deposition of extracellular matrix components, including collagen (COL) I and COL III (3,4). Such excessive COL production disrupts cardiac function, potentially leading to myocardial infarction and heart failure, thereby posing severe risks to human health (5,6). Traditional therapies such as angiotensin-converting enzyme inhibitors and β-blockers are known to improve ventricular remodeling and enhance blood flow at ischemic sites, offering modest benefits in managing MF. However, the mechanisms of action of these agents are complex and their prolonged use is often associated with significant adverse effects, such as hypotension, dyspnea, bradycardia and shock (7).

Traditional Chinese medicine, with its multifaceted approach encompassing multiple components, pathways and targets, offers unique advantages in both the prevention and treatment of MF (8). Herbal treatments such as Astragalus (9), Salvia miltiorrhiza (10) and turmeric (11) are known to modulate collagen synthesis, prevent extracellular matrix collagen deposition and mitigate ventricular remodeling, showing potential therapeutic effects against MF. For example, research by Wang et al (12) demonstrated that Buyang Huanwu Decoction could attenuate MF by reducing the expression of inflammatory factors and influencing the IL-17 signaling pathway.

Rhodiola rosea, a member of the Crassulaceae family, is known for its diverse beneficial effects including anti-ischemia, anti-oxidation, anti-inflammation, as well as its ability to enhance lung function and relieve asthma (13,14). Notably, it is recognized for its cardiovascular benefits, particularly its capacity to protect cardiovascular endothelial cells and inhibit platelet aggregation (15). Salidroside (SAL), a key active component of Rhodiola rosea, has demonstrated properties that alleviate oxidative stress and improve MF. Studies, such as those by Ye et al (16), have shown that SAL can mitigate carbon tetrachloride-induced liver fibrosis in mice by inhibiting SK-HEP-1 exosome SphK1, which may reduce liver damage and enhance recovery from liver fibrosis. These findings suggest that Rhodiola rosea could similarly exert a protective effect against MF.

The present study examined SAL, the principal active ingredient in Rhodiola rosea, for its therapeutic potential against MF related to oxidative stress, identified using network pharmacology techniques. In addition, cell experiments were used to elucidate the effect and mechanism of SAL in the treatment of MF.

Materials and methods

Network pharmacology-related database and software

The present study used various databases and software to identify and analyze the bioactive components of Rhodiola rosea, including: HERB database (http://herb.ac.cn/), Encyclopedia of Traditional Chinese Medicine (ETCM) (http://www.tcmip.cn/ETCM/), PubChem database (http://pubchem.ncbi.nlm.nih.gov/), Swiss TargetPrediction (http://swisstargetprediction.ch/), STRING protein interactions information website (http://string-db.org/), GeneCards database (https://www.gege-cards.org/), OMIM database (http://omim.org/), DAVID database (http://david.ncifcrf.gov/summary.jsp), Cytoscape 3.9.1 software (http://cytoscape.org/), Wei Sheng Xin online mapping website (https://www.bioinformatics.com.cn/), Protein Data Bank database (http://www.rcsb.org/), AutoDock Tools 1.5.6 software (https://autodock.scripps.edu/), PyMOL 2.3.6 software (https://pymol.org/), OpenBabel 2.4.1 software (https://openbabel.org/), GraphPad Prism 9.4.1 software (Dotmatics), CytoNCA plugin (https://apps.cytoscape.org/apps/cytonca), and Image Lab 6.0.1 (Bio-Rad Laboratories, Inc.).

Network pharmacology research methods

Active ingredients from Rhodiola rosea were identified using the HERB database and the ETCM resource, employing screening criteria based on physicochemical properties: Molecular weight ≤500, logP ≤5, hydrogen bond donors ≤5 and hydrogen bond acceptors ≤10. Selected compounds were saved in simplified molecular input line entry specification (SMILES) format and imported into the SwissTargetPrediction database. Predictions were restricted to human research species with a prediction probability >0. Compounds that did not target any relevant proteins were excluded, resulting in the construction of a comprehensive component-target database for Rhodiola rosea.

Target collection and common target screening for oxidative stress and myocardial fibrosis

Using the GeneCards database, genes associated with MF were identified with the keywords ‘myocardial fibrosis’, selecting targets with a correlation score ≥ the second median. Genes related to oxidative stress were sourced using the keywords ‘oxidative stress’, selecting those with correlation scores ≥ the third median. The two sets were collated using the OMIM database. Duplicate targets were removed, leading to a refined prediction target library. The target proteins of Rhodiola rosea were intersected with disease targets using Wei Sheng Xin online mapping website, identifying shared targets visualized in a Venn diagram.

Construction and analysis of drug-component-target-disease network

A comprehensive drug component-target-disease network was created using Cytoscape 3.9.1 to visualize the interactions between active compounds of Rhodiola rosea and their potential targets in combating oxidative stress and MF. The network featured purple nodes for bioactive components and pink nodes for potential targets, with edges representing their interactions.

PPI network construction and analysis

A total of 91 potential targets of the antioxidant properties of SAL against MF were imported into the STRING database. The data were filtered by selecting ‘Homo sapiens’ as the organism with a minimum interaction score of ≥0.4. The resulting .tsv file was then imported into Cytoscape 3.9.1, where the CytoNCA plugin was used to compute the degree of each target, enabling the construction of a protein interaction network graph.

Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis

GO function and KEGG signal pathway enrichment analyses were performed using the DAVID database. Results were visualized using the Wei Sheng Xin online mapping website, drawing bar charts for GO function enrichment and bubble charts for KEGG pathway analysis.

Molecular docking analysis of core target and drug active ingredient

Molecular docking was used to evaluate the direct binding interaction between the key active ingredient, SAL, and core targets. Structural formulas and 3D structure files were sourced from the PubChem and the Protein Data Bank databases, respectively. Specific conditions applied to the docking simulations included: Biological Source: Homo sapiens; Experimental Method: X-ray diffraction; Resolution: ≤3Å. Using AutoDock Tools 1.5.6 and OpenBabel 2.4.1 software, docking simulations were performed and binding positions and energies were calculated. Structures were visualized with PyMOL 2.3.6 to generate 3D images.

Cell culture and treatment

H9c2 cells (American Type Culture Collection) were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc.), supplemented with 10% fetal bovine serum (TransGen Biotech Co., Ltd.). Cultures were incubated at 37°C in a 5% CO2 atmosphere. To minimize contamination, cultures were treated with a combination of penicillin-streptomycin-gentamicin (100X; cat. no. P1410-100ml; Beijing Solarbio Science & Technology Co., Ltd.) and a separate penicillin-streptomycin solution (100X; cat. no. P1400-100ml; Beijing Solarbio Science & Technology Co., Ltd.).

Methylthiazolyl tetrazolium (MTT) assay

Cells were seeded at a density of 5.0×04 cells per ml in a 96-well plate and exposed to various concentrations of AngII (0.01, 0.1 and 1 µM) in the presence of SAL (50, 100 and 150 µM) dissolved in DMEM for 24 h. Following incubation at 37°C for 4 h, 50 µl of MTT solution was added to each well. Following a further 4-h incubation at 37°C, the medium was aspirated and the wells were washed with PBS. Subsequently, 150 µl of dimethyl sulfoxide was added to solubilize the formazan crystals. The optical density (OD) at 570 nm was measured using a SpectraMax 190 microplate reader (Anthos Labtec Instruments GmbH).

Immunofluorescence assay

H9c2 cells were seeded in 24-well plates with slides at a density of 1×105 cells/ml and divided into three treatment groups: Control, 1 µM AngII and 150 µM SAL. After 24 h of treatment, cells were fixed with 4% paraformaldehyde for 15 min at room temperature and washed three times with PBS. Cells were permeabilized with 0.5% Triton-X for 20 min at room temperature, followed by three PBS washes. Wells were blocked with 5% goat serum (Beyotime Institute of Biotechnology) in PBS for 1 h at room temperature. Then, the cells were incubated overnight at 4°C with 100 µl primary antibody against COL III (1:100; cat. no. A3795; ABclonal Biotech Co., Ltd.). After washing three times with PBS, the cells were incubated with FITC-conjugated Goat anti-Rabbit lgG (H+L) secondary antibody (1:100 dilution; cat. no. AS011; ABclonal Biotech Co., Ltd.) for 1 h at room temperature, incubated with 10 µl DAPI (Beyotime Institute of Biotechnology) at room temperature for 10 min and visualized under a DMI8 inverted fluorescence microscope (Leica Microsystems GmbH) at magnification, ×20. The secondary antibody conjugate was FITC; the excitation wavelength was adjusted to 491 nm and FITC was detected under a DMI8 inverted fluorescence microscope at 516 nm (emission) according to the manufacturer's instructions.

Cellular RNA and reverse transcription-quantitative (RT-q) PCR

Cells were seeded in 6-well plates at 2×105 cells/ml and treated with drugs for 24 h. Post-treatment, cells were washed twice with PBS and RNA was extracted using EZol reagent (Shanghai GenePharma Co., Ltd.). RNA quality and quantity were assessed using a Nanodrop quantitative nucleic acid analyzer (Thermo Fisher Scientific, Inc.). cDNA was synthesized from RNA using Hifair III 1st Strand cDNA Synthesis SuperMix for qPCR (Shanghai Yeasen Biotechnology Co., Ltd.). qPCR was performed with Hieff UNICON Universal Blue qPCR SYBR Green Master Mix (Shanghai Yeasen Biotechnology Co., Ltd.). Cycling conditions were 95°C for 30 sec, followed by 40 cycles of 95°C for 5 sec and 60°C for 30 sec. RNA extraction, cDNA synthesis and qPCR were performed according to the manufacturers' protocols, and the experiment was repeated at least three times. β-actin served as the internal reference and relative gene expression was calculated using the 2−∆∆Cq method (17). Primer sequences for IL-6, MMP9, AKT serine/threonine kinase 1 (AKT1), albumin (ALB), caspase-3 (CASP3) and β-actin are given in Table SI.

Western blotting

Cells were lysed in RIPA buffer (Beyotime Institute of Biotechnology) containing phenylmethanesulfonyl fluoride to extract proteins. Protein concentration was determined using a BCA protein assay kit (Beyotime Institute of Biotechnology). Proteins (26 µg) were separated by 7.5% SDS-PAGE (Shanghai Yamei Biotechnology Co. Ltd.) and transferred to PVDF membranes (MilliporeSigma). Membranes were blocked with 5% skimmed milk for 1 h at room temperature, then incubated with primary antibodies against COL I (1:1,000; cat. no. 14695-1-AP; Wuhan Proteintech Biotechnology), alpha smooth muscle actin (α-SMA; 1:1,000; cat. no. A17910; ABclonal Biotech Co., Ltd.) and GAPDH (1:5,000; cat. no. ab181602; Abcam) overnight at 4°C. After washing, membranes were incubated with HRP-conjugated secondary antibody (1:10,000; cat. no. RGAR001; Wuhan Proteintech Biotechnology) for 1 h at room temperature and developed using an enhanced chemiluminescence substrate (Tanon Science and Technology Co., Ltd.) and ChemiDoc XRS+ Highly Sensitive Chemiluminescence Imaging System (Bio-Rad Laboratories, Inc.). COL I, α-SMA and GAPDH were 139, 42 and 36 kDa, respectively. Image Lab 6.0.1 was used to semi-quantify the blots.

Reactive oxygen species (ROS) level detection by immunofluorescence assay

Cells were directly inoculated into 24-well plates at 1×105 cells/ml and were divided into a control group, 1 µM AngII group and 150 µM SAL group. The cells were treated with the indicated drugs for 24 h. Post-treatment, cells were incubated with DCFH-DA (10 µmol/l) for 30 min at 37°C. Cells were then washed and visualized under a DMI8 inverted fluorescence microscope (Leica Microsystems GmbH) at ×10 magnification.

Determination of malondialdehyde (MDA) content

MDA levels were measured using an MDA assay kit (Beyotime Institute of Biotechnology) after treating cells in 6-well plates at 2×105 cells/ml for 24 h. The working solution from the MDA kit was heated and the absorbance was read at 532 nm on a SpectraMax microplate reader (Anthos Labtec Instruments GmbH).

Glutathione (GSH) content determination

GSH levels were assessed using a GSH assay kit (Beijing Solarbio Science & Technology Co., Ltd.). Cells were sonicated at 200 W at 4°C, they were sonicated for 3 sec, then stopped for 10 sec, and this was repeated 30 times. The supernatants were collected for analysis after centrifugation of 8,000 × g for 10 min at 4°C.

Flow cytometry

ROS levels were measured using a ROS assay kit (Beijing Solarbio Science & Technology Co., Ltd.). Post-treatment, cells were incubated with DCFH-DA, washed and analyzed on an Agilent flow cytometer (Agilent Technologies, Inc.).

Statistical analysis

Statistical analyses were performed using GraphPad Prism 9.4.1 software (Dotmatics). Differences were assessed using one-way ANOVA followed by Bonferroni post hoc test as appropriate. Results are expressed as mean ± standard deviation and P<0.05 was considered to indicate a statistically significant difference.

Results

Establishment of a Rhodiola rosea database and visualization of the drug-active ingredient relationship network diagram

Using the HERB and ETCM databases, the present study identified 25 unique ingredients, listed in Table I. The 2D structures and CAS numbers of these components were retrieved from the PubChem database and imported into the SwissTargetPrediction database, resulting in the identification of 312 individual targets. The present study then compiled a comprehensive dataset of targets relating to oxidative stress and MF, gathering 12,835 oxidative stress-related targets from the GeneCards database. Of these, 1,242 targets had correlation scores above the median value and 5,090 targets were specifically linked to MF. From this subset, the present study isolated 1,667 targets with correlation scores above the third median. Using a Venn diagram, the overlap between Rhodiola rosea targets and those related to oxidative stress and MF was visualized, identifying 157 intersecting targets as potential mediators of the effects of Rhodiola rosea (Fig. 1A). Employing Cytoscape 3.9.1 software, a drug-component-target-disease network diagram was created (Fig. 1B). Using CytoNCA, the present study furthered identified the top five active ingredients based on their degree ranking: SAL, kaempferol, β-sitosterol, tanshinone IIA and ginsenoside Rh2 (Table II). Notably, SAL was the primary component that mitigated MF through its antioxidant properties. The potential targets of Rhodiola rosea's SAL component for counteracting MF were subsequently imported into the STRING protein interaction database, which enabled it to identify 91 protein nodes and 586 interaction edges, with a local clustering coefficient of 0.602 and a PPI enrichment P-value of <1.0×10−16. After importing the data into Cytoscape and using the CytoNCA plugin, the protein interaction network was visualized and the top 32 core interaction targets identified for further analysis (Fig. 1C and D). The primary five core targets were identified as serum ALB (Degree=81.0), IL6 (Degree=71.0), AKT1 (Degree=68.0), MMP9 (Degree=59.0) and CASP3 (Degree=57.0). Using the DAVID database, these targets were analyzed and 317 biological processes (BP), 52 cellular components (CC) and 76 molecular functions (MF) predominantly involved in the negative regulation of apoptosis, protein hydrolysis and cell proliferation were identified (Fig. 1E). Additionally, 142 KEGG pathways enriched at P<0.05 were identified, highlighting the top 20 signaling pathways involved in the cardioprotective effects mediated by SAL (Fig. 1F). This analysis highlighted the role of SAL in regulating crucial cellular processes such as cell proliferation, apoptotic processes and inflammation and its involvement in cardiovascular and metabolic pathways associated with conditions such as cancer, atherosclerosis and diabetes complications.

Figure 1. Role of Rhodiola rosea in mitigating myocardial fibrosis through antioxidative stress mechanisms employing network pharmacology. (A) Venn diagram of intersection targets of Rhodiola antioxidative stress in improving myocardial fibrosis. (B) Drug-component-target-disease network. (C) STRING protein interaction network (D) Visualization histogram of top 32 intersection targets of Rhodiola. (E) GO enrichment analysis of core targets. (F) KEGG pathway enrichment analysis of core targets. STRING, Search Tool for the Retrieval of Interacting Genes; GO, gene ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes.

Table I. Basic information on the chemical composition of Rhodiola rosea.

Table I.

Basic information on the chemical composition of Rhodiola rosea.

Number	Compound name	Molecular formula	CAS number
1	1,8-menthadien-4-ol	C10H6O	3419-02-1
2	1-octanol	C8H18O	111-87-5
3	1-octen-3-ol	C8H16O	3391-86-4
4	3-methyl-2-buten-1-ol	C5H10O	556-82-1
5	3-methyl-2-butenal	C5H8O	107-86-8
6	3-octanol	C8H18O	589-98-0
7	6-methyl-5-hepten-2-ol	C8H16O	1569-60-4
8	6-methyl-5-hepten-2-one	C8H14O	110-93-0
9	Benzeneethanol	C8H10O	60-12-8
10	Formic acid hexyl ester	C7H14O2	629-33-4
11	Hexanal	C25H44N14O8	2610-10-8
12	Limonene oxide	C10H16O	1195-92-2
13	Linalool	C10H18O	78-70-6
14	Linalool oxide	C10H18O2	34995-77-2
15	Myrtanol	C10H18O	514-99-8
16	Myrtenol	C10H16O	515-00-4
17	Octanal	C8H16O	124-13-0
18	Octanoic acid	C8H16O	124-07-2
19	Salidroside	C14H20O7	78-70-6
20	7-hydroxycoumarin	C9H6O3	93-35-6
21	Caffeic acid	C9H8O4	501–16–6
22	Kaempferol	C15H10O6	520-18-3
23	Gallic acid	C7H6O5	149-91-7
24	Ethyl gallate	C9H10O5	831-61-8
25	Tyrosol	C8H10O2	501-94-0

Table II. Top five Chinese medicine components in the degree network diagram of drug component target disease.

Table II.

Top five Chinese medicine components in the degree network diagram of drug component target disease.

Ranking	Components	Degree:score
1	Salidroside	14.0
2	Kaempferol	9.0
3	β-sitosterol	7.0
4	Tanshinone IIa	6.0
5	Ginsenoside Rh2	6.0

These findings provided valuable insights into the underlying mechanisms of the cardioprotective actions of Rhodiola rosea, emphasizing SAL as the key active ingredient in combating MF through its antioxidant capabilities.

Molecular docking analysis

Molecular docking was performed following the preparation of the target protein using AutoDock Tools 1.5.6. SAL and its corresponding core target molecules were docked to evaluate the binding efficacy. Adjustments were made to the mesh size to optimize the analysis of binding positions and docking energies. Binding energies were found to be <-1.2 kcal/mol, indicating a strong potential for binding between the drug and its targets. Specifically, a binding energy <0 kcal/mol suggests a favorable binding interaction, with values ≤-1.2 kcal/mol indicating effective docking and potential therapeutic efficacy. Lower binding energies denoted increased stability and a higher likelihood of a successful interaction between the ligand and the receptor. From a molecular perspective, these results supported the potential therapeutic effects of SAL on MF, given its robust interactions with key molecular targets such as ALB, IL6, AKT1, MMP9 and CASP3 in Table III. Fig. 2 illustrates the 3D interaction model between SAL and the core target molecules, underscoring the potential mechanism by which SAL may inhibit MF in H9c2 cells through these key interactions.

Figure 2. Salidroside treats myocardial fibrosis through multiple targets. Salidroside is represented in pink, core targets are represented in green and grey, linked residues are represented in purple (residue names are represented in text) and hydrogen bonds are represented in yellow dashed lines. (A) ALB and salidroside molecular docking (B) IL6 and salidroside molecular docking. (C) AKT1 and salidroside molecular docking (D) MMP9 and salidroside molecular docking. (E) CASP3 and salidroside molecular docking. ALB, albumin; AKT1, AKT serine/threonine kinase 1; CASP3, caspase-3.

Table III. Molecular docking binding energy of SAL and core targets.

Table III.

Molecular docking binding energy of SAL and core targets.

Targets	PDB ID	Ingredients	Pubchem ID	Affinity, kcal/mol
ALB	7VR0	Salidroside	159278	−3.23
IL6	7NXZ	Salidroside	159278	−4.31
AKT1	1UNQ	Salidroside	159278	−3.27
MMP9	6ESM	Salidroside	159278	−3.8
CASP3	1RHJ	Salidroside	159278	−3.11

[i] SAL, salidroside; ALB albumin; AKT1, AKT serine/threonine kinase 1; CASP3, caspase-3.

Efficacy of SAL in ameliorating myocardial fibrosis

To evaluate the effect of various concentrations of AngII and SAL on H9c2 cardiomyocyte viability after 24 h of treatment, an MTT assay was employed. The results demonstrated that incubating H9c2 cells with 0.01, 0.1 and 1 µM AngII did not significantly alter cell viability (Fig. 3A). Similarly, treatment with SAL at concentrations of 50, 100 and 150 µM did not affect cell viability (Fig. 3B). Then, the cells were treated with 0.01, 0.1 and 1 µM AngII for 24 h to verify whether AngII induced MF successfully. Subsequent western blotting revealed a dose-dependent increase in the levels of COL I and α-SMA, which are biomarkers indicative of MF (3,18), following 24 h of exposure to AngII. This dose-dependent increase confirmed the successful establishment of the MF model with AngII, particularly with significant elevations in both COL I and α-SMA levels in the 1 µM AngII group compared with lower concentrations (P<0.01; Fig 3C). AngII at 1 µM was the most significant in establishing MF model. Further experiments employed H9c2 cells treated with 1 µM AngII for 24 h as the MF model. The next step was to verify the therapeutic effect of different concentrations of SAL on MF. Cells were pre-treated with 1 µM AngII for 2 h before adding SAL at 50, 100 and 150 µM concentrations and co-incubating for an additional 24 h. Western blotting was conducted to assess changes in COL I and α-SMA levels. Results indicated a dose-dependent reduction in these fibrosis markers in the SAL treatment groups, demonstrating the capacity of SAL to effectively reverse AngII-induced MF. Notably, the levels of both COL I and α-SMA were significantly lower in the 150 µM SAL group compared with lower concentrations of SAL, highlighting the robust therapeutic potential of SAL at higher doses (P<0.001; Fig. 3D). This evidence substantiated the therapeutic efficacy of SAL in reducing MF markers, suggesting its potential utility as a treatment option in MF management.

Figure 3. SAL effectively mitigates myocardial fibrosis. (A) The activity of different concentrations of AngII on H9c2 cells by MMT Assay. (B) The activity of different concentrations of SAL on H9c2 cells by MMT Assay. (C) The protein levels of myocardial fibrosis markers COL I and α-SMA induced by AngII were detected by western blotting. (D) The protein levels of myocardial fibrosis markers COL I and α-SMA after SAL treatment by western blotting. *P<0.05, **P<0.01, ***P<0.001. SAL, salidroside; AngII, angiotensin II; MTT, methylthiazolyl tetrazolium; COL, collagen; α-SMA, alpha smooth muscle actin.

Salidroside mitigates myocardial fibrosis through multi-target mechanisms

For subsequent experiments, the present study established a treatment regimen where H9c2 cells were exposed to 150 µM SAL for 24 h as the primary drug treatment group. Simultaneously, another set of cells were treated with 1 µM AngII alone, followed by an additional 24-h treatment with 150 µM SAL. Using immunofluorescence assays, the level of COL III, a well-established marker of MF (4), was assessed. The results showed that 1 µM AngII significantly elevated COL III levels compared with the control group (P<0.05). Notably, treatment with 150 µM SAL markedly reduced COL III levels relative to the AngII-only treated group (P<0.05), illustrating the potent antifibrotic effects of SAL (Fig. 4A). To elucidate the underlying mechanisms of the antifibrotic action of SAL, RT-qPCR was used to verify its effect on multiple targets associated with MF (Fig. 4B). The RT-qPCR results revealed elevated mRNA levels of IL6, MMP9 and CASP3 in the 1 µM AngII group compared with controls, while ALB and AKT1 mRNA levels were significantly decreased. Markedly, treatment with 150 µM SAL not only reduced the mRNA levels of IL6, MMP9 and CASP3 but also increased those of ALB and AKT1 relative to the AngII group. These findings confirmed that SAL counters AngII-induced MF by modulating the expression of critical fibrosis-related genes. The multi-target action of SAL highlighted its potential as a therapeutic agent for the treatment of MF, effectively reversing the pathological processes through interactions with key molecular pathways.

Figure 4. SAL improves myocardial fibrosis through multiple targets. (A) The level of myocardial fibrosis marker COL III by immunofluorescence (magnification, ×20; scale bar, 100 µm) (B) The mRNA levels of IL6, MMP9, AKT1, ALB and CASP3 by reverse transcription-quantitative PCR. *P<0.05, **P<0.01 , ***P<0.001. SAL, salidroside; COL, collagen; AKT1, AKT serine/threonine kinase 1; ALB, albumin; CASP3, caspase-3.

Antioxidative effects of SAL in mitigating myocardial fibrosis

To validate the antioxidative properties of SAL, assessments were conducted on several oxidative stress biomarkers. The MDA assay results showed that MDA levels were significantly elevated in the 1 µM AngII group compared with controls, indicating increased oxidative stress. However, treatment with 150 µM SAL resulted in a notable reduction in MDA levels compared with the 1 µM AngII group (P<0.05), highlighting the ability of SAL to suppress MDA production and counteract oxidative stress induced by AngII (Fig. 5A). Further analysis using a GSH assay revealed a significant decrease in GSH levels in the 1 µM AngII group compared with controls (P<0.05). Conversely, GSH levels were significantly elevated in the 150 µM SAL group compared with the 1 µM AngII group (P<0.05), suggesting that SAL enhanced GSH production, thereby protecting against oxidative stress (Fig. 5B). The basis for assessing the increase in ROS is to study the extent of cellular oxidative stress by detecting the levels of ROS within cells. Immunofluorescence and flow cytometry are the commonly used methods. These methods have high sensitivity, high selectivity, high throughput and are non-destructive and can detect trace changes in intracellular ROS without interference from other substances, which can improve experimental efficiency and is suitable for long-term and continuous monitoring. Therefore, immunofluorescence assay and flow cytometry were employed to detect ROS levels. These confirmed that ROS levels were substantially higher in the 1 µM AngII group compared with controls (P<0.01). Markedly, the 150 µM SAL group showed a significant decrease in ROS levels compared with the 1 µM AngII group (P<0.01 in immunofluorescence and P<0.05 in flow cytometry), illustrating the potent antioxidative activity of SAL (Fig. 5C and D). Collectively, these findings demonstrated that SAL effectively inhibited ROS production and enhanced antioxidant defenses in H9c2 cells, thus mitigating AngII-induced oxidative stress. This multifaceted antioxidative response underlined the therapeutic potential of SAL in the treatment of MF.

Figure 5. SAL exerts an antioxidative stress effect and thereby mitigates myocardial fibrosis. (A) MDA levels in H9c2 cells (B) GSH levels in H9c2 cells (C, D) ROS levels in H9c2 cells were determined by immunofluorescence and flow cytometry (magnification, ×10; scale bar, 200 µm). *P<0.05, **P<0.01. SAL, salidroside; MDA, malondialdehyde; GSH, glutathione; ROS, reactive oxygen species; AngII, angiotensin II.

Discussion

The present study investigated the protective effects of Rhodiola rosea against MF-induced cardiac injury and relevant mechanisms. The top five active ingredients of Rhodiola rosea against MF via anti-oxidative stress were screened by network pharmacology, and SAL ranked first, indicating that SAL was the main component of Rhodiola rosea in its anti-oxidative stress mechanism to improve MF. The PPI network identified the top 32 core interaction targets, among which five key targets ALB, IL-6, AKT1, MMP9 and CASP3 were considered to have high research value. Hence, the principle components of Rhodiola rosea are able to play a crucial role in MF through the aforementioned targets. Among 317 BPs, 52 CCs and 76 MFs and 142 KEGG enriched signaling pathways, SAL regulated crucial cellular processes primarily involved in cell proliferation, apoptotic processes and inflammation and demonstrated involvement in cardiovascular and metabolic pathways associated with conditions such as cancer, atherosclerosis and complications in diabetes. The network pharmacology analysis was confirmed by SAL reducing MF markers COL I, COL III and α-SMA levels in H9c2 cells induced by AngII. In parallel, SAL significantly decreased the inflammatory factors IL6, MMP9 and CASP3 levels and suppressed oxidative stress MDA, ROS and increasing GSH expressions, the underlying mechanisms of which might be related to increasing levels of ALB and AKT1.

Network Pharmacology analysis was performed to investigate the bioactive ingredients of the Rhodiola rosea. The results demonstrated the top five active constituents were SAL, kaempferol, β-sitosterol, tanshinone IIA and ginsenoside Rh2. These compounds have promising ability in their antioxidant (19), anti-tumor (20) and anti-inflammatory effects (21,22). Additionally, they may suppress AngII-induced inflammatory in atrial fibrosis and atrial fibrillation (23) and cardiotoxicity induced with 5-FU (24). In terms of mitigating MF, SAL was the principle component due to its antioxidant properties. Furthermore, as signaling molecules, these compounds can influence angiogenesis (25) and inhibit apoptosis (26) and pyroptosis (27). SAL has the effects of protecting endothelial barrier disruption induced by intermittent hypoxia and acts against atherosclerosis (28). In addition, SAL enhances anti-apoptosis cardiomyocyte and ventricular remodeling in a cardiomyopathy mouse model (29). Based on the aforementioned findings, the present study was mainly concerned with SAL from Rhodiola rosea to observe the critical influence in MF by its anti-oxidant activity.

MF is the hallmark of cardiovascular diseases with regard to heart failure, leading to cardiac systolic and diastolic dysfunction, arrhythmias and adverse cardiovascular events (1,2). MF generates substantial deposition of extracellular matrix components in the cardiac microenvironment (3,4). The present study showed that COL I and COL IIII were abundant in H9c2 cells induced by AngII, illustrating the weakened elasticity of the vascular wall. SAL treatment increased the proportion of COL I and COL IIII, suggesting that SAL partly contributed to the recovery of ventricular compensation. The aforementioned findings showed that SAL alleviated MF, supporting the evidence for the potential of SAL to defeat MF. It has been reported that Rhodiola granules significantly strengthen cardiac function and reduce the fibrotic area in an ischemia/reperfusion injury model of rats (30). α-SMA is the marker of mature myofibroblast in cardiac tissues (31). Cardiac fibrosis is normally quiescent, but is activated when triggered by various stimuli and transformed into α-SMA-positive myofibroblasts (32). In the present study, α-SMA levels were markedly upregulated by AngII, while SAL lowered α-SMA levels. A previous study indicated that appropriate activation of myofibroblasts is promising for tissue repair; however, excessively activated myofibroblasts may even result in heart failure (33). Yang et al (34) found that SAL clearly decreased α-SMA expression in the renal interstitium, which is in line with the data of the present study.

In addition, the anti-inflammatory properties of Rhodiola were highlighted. MF is closely associated with inflammation and fibrosis is the last stage of a chronic inflammatory response. IL6 is a proinflammatory cytokine in MF. Fibroblasts maintain this potential pathogenic change by regulating the production of IL6. The results of the present study demonstrated that SAL downregulated IL6 levels. IL6 overexpression induces myofibroblast proliferation, differentiation and fibrosis (1). In addition, SAL downregulated the expressions of MMP9 and CASP3. Hence, SAL prominently reduced pro-inflammatory cytokines, which prolong exacerbation of collagen deposition and MF. Chinese medicinal compounds, including ginsenoside and berberine, have also been shown to offer protective effects against MF by targeting similar pathways, thereby reinforcing the therapeutic potential of natural compounds in cardiac fibrosis management (35,36).

In the data of network pharmacology, the present study found that ALB and AKT1 were also mostly involved in MF. Jäntti et al (37) reported that ALB is critical for cardiac function. The results of the present study showed that SAL increased the ALB and AKT1 levels in H9c2 cells treated with AngII. It has been reported that the cardiac function of patients improves with an increase in ALB level and that subsequently their quality of life is improved. The Akt signaling pathway is associated with the cardiac hypertrophy (38). The present study showed that SAL increased AKT1.

The pathogenesis of MF involves complex mechanisms, including the activation of the renin-angiotensin-aldosterone system (RAAS) (39,40), with AngII being a pivotal mediator not only in the cardiovascular system but also in other organs, such as the kidneys and liver. RAAS inhibitors have shown efficacy in mitigating liver fibrosis and reducing portal hypertension (41), illustrating the broad pathophysiological effect of RAAS. In addition, AngII stimulates fibroblast cell migration and proliferation, enhancing collagen production and contributing to myocardial structural disarray (42).

The present study explored the oxidative stress pathways involved in MF, revealing that AngII intensifies oxidative stress by increasing ROS and MDA levels while reducing GSH levels, indicating an imbalance between pro-oxidant and antioxidant mechanisms. Oxidative stress further triggers pathways leading to cell apoptosis and necrosis, accelerating MF progression. The findings using network pharmacology identified 25 active components in Rhodiola rosea, with SAL emerging as a key antioxidant agent. SAL demonstrated significant efficacy in modulating the expression of oxidative and fibrotic markers such as COL I, COL III, α-SMA, IL6, MMP9 and CASP3, underscoring its potential therapeutic benefits in MF treatment.

The present study possessed some limitations. First, it only used H9c2 cells to study the myocardial markers, basic structure and function induced by SAL. It would be more precise to extract rat neonatal cardiomyocytes and determine the effect of SAL on cardiomyocyte activity and proliferation. Second, the present study used H9c2 cells to explore the effect of SAL on the MF process and the underlying mechanisms associated with fibrosis. In future studies cardiac fibroblast cells should be applied to illustrate the SAL treatments in progress of MF. Third, the present study used immunofluorescence and western blotting to detect the expressions of COL I and COL III. Using RT-qPCR for the detection of COL I and COL III should be in a future study.

In conclusion, the present study elucidated the complex interplay of oxidative stress, inflammation and fibrosis in MF and highlighted the efficacy of Rhodiola rosea, particularly SAL, in ameliorating these pathological processes. These insights provided a strong foundation for further development of targeted therapies for MF, using the multifaceted actions of natural compounds to address this debilitating condition.

Supplementary Material

Supporting Data

Acknowledgements

Not applicable.

Funding

The present study was supported by the National Natural Science Foundation of China (grant no. 82304868 for LY) and the Dalian Chinese Medicine Scientific Research Project (grant no. 22Z12006 for HL).

Availability of data and materials

The data generated in the present study may be requested from the corresponding author.

Authors' contributions

HL contributed to the conception and design of the study. LZ and FD conducted data curation and formal analysis. YZ and KW was responsible for sample collection. YX was responsible for data analysis. HY and LY collected data, discussed and revised the manuscript. LZ, HY and YX confirm the authenticity of all the raw data. All authors read and approved the final version of the manuscript.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Glossary

Abbreviations

Abbreviations:

References