Proteomics-based investigation of the protective effect and mechanism of Agari-5 in rats with myocardial infarction

Abstract

BACKGROUND

Myocardial infarction (MI) occupies a very high mortality and morbidity rate, and the search for effective pharmacological treatments has far-reaching implications for clinical research.

AIM

To explore the protective effects of Mongolian medicine Agari-5 on rats with MI.

METHODS

Sprague-Dawley rats were used, and both the Agari-5 and model groups had their coronary arteries clamped to induce MI. Proteomics was used to research the potential mechanism of action while ELISA, hematoxylin and eosin, and Masson’s staining were used to preliminarily investigate the protective impact of Agari-5 on rats with MI.

RESULTS

The current study has shown that Agari-5 might enhance cardiac function indicators, including echocardiography results of rats and creatine kinase, creatine kinase isoenzyme, and lactate dehydrogenase, in rats that had MI. According to the results of pathological staining, Agari-5 may lessen inflammatory cell infiltration and cardiomyocyte fibrosis, among other things. The proteome analysis revealed that there were 60 distinct proteins in total, four of which were associated with the heart. The expression of PSAT1, PDK1, SMAD4, and SDF2 proteins may be linked to the mechanism of their protective effects.

CONCLUSION

Potential therapeutic effects of Agari-5 for MI and its mechanism of action may be related to PSAT1, PDK1, SMAD4, and SDF2.

Key Words: Myocardial infarction; Agari-5; Proteomics; Protective effect; Mechanism

Core Tip: We demonstrated that myocardial infarction (MI) rats treated with Agari-5 expressed similar levels of SDF2, PDK1, SMAD4, and PSAT1 as the control group, suggesting that these proteins be important targets influenced by Agari-5. Through proteomic analysis the research delved deeper into the protective mechanisms of Mongolian traditional medicine formulas, finding that these traditional formulas may protect MI rats through the regulation of multiple protein targets and molecular mechanisms, such as multicomponent interactions and synergistic effects, providing a new direction for research on national medicines and offering a reference for clinical treatments of cardiovascular diseases.

INTRODUCTION

Myocardial infarction (MI) is a leading cause of death globally, particularly among middle-aged and elderly populations, and is one of the four major non-communicable diseases with high mortality and morbidity rates worldwide along with cardiovascular disease, cancer, diabetes mellitus, and chronic respiratory disease[1,2]. MI occurs when atherosclerotic lesions in the coronary arteries cause narrowing or occlusion, leading to hypoxia and ischemia, which result in the necrosis of myocardial tissue[3,4]. During the hypoxia-reoxygenation process of cardiomyocytes, a series of damaging changes occur in myocardial ultrastructure, energy metabolism, cardiac function, and electrophysiology, which continue to affect cardiac function and structural remodeling (key pathological processes in MI)[5]. Current treatments for MI include percutaneous coronary intervention and pharmacological therapies[6], and there is growing recognition of the potential of ethnomedicine in treating this condition.

Mongolian medicine Agari-5 is a widely used traditional remedy for cardiovascular diseases consisting of five herbs: (1) Descending incense; (2) Sumac; (3) Umeboshi; (4) Borax; and (5) Danshen. It primarily works by promoting blood circulation and resolving blood stasis and is used to treat conditions such as vascular fever, heart deficiency heat, heart stabbing pain, hypertension, and arteriosclerosis. It is particularly effective for treating heart diseases like Haye disease. The compound formula reflects the holistic approach and evidence-based principles of Mongolian medicine, offering a multicomponent and multitarget therapeutic effect.

Proteomics is the study of the entire set of proteins expressed in a cell or organism, utilizing techniques such as protein purification and mass spectrometry analysis to identify and quantify proteins[7,8]. Unlike genomics, proteomics is more complex as it seeks to understand not only the total protein expression but also various aspects like protein modifications, structures, and protein-protein interactions (PPIs). This comprehensive analysis makes proteomics a critical tool for understanding disease processes and cellular metabolism.

In this study a rat MI model was constructed, and proteomics technology was applied to investigate the therapeutic effects of the Mongolian medicine Agari-5. By analyzing protein expression differences and utilizing bioinformatics methods, the study aimed to elucidate the molecular basis and advantages of Agari-5 in treating MI[9]. The research focused on uncovering the protein-level mechanisms behind the multitarget, multipathway, and multifunctional therapeutic effects of Agari-5, providing insights into its mechanism of action. These findings will offer a theoretical basis for the use of Agari-5 in treating cardiovascular diseases and guiding future research.

MATERIALS AND METHODS

Drugs and experimental reagents

Agari-5 (No. M20201334000) was obtained from DL-Dithiothreitol (Solarbio), Iodoacetamide (Aladdin), and Tetraethylammonium bromide (Sigma). Creatine kinase (CK), CK isoenzyme (CK-MB), and lactate dehydrogenase (LDH) were supplied by Mlbio Biotechnology Ltd. Other reagents, such as EDTA, xylene brilliant cyanine G, and sodium dodecyl sulfate, were sourced from Sinopharm Chemical Reagent Co.

Laboratory animals and ethics

Animal experiments were conducted in compliance with the guidelines set by the Experimental Animal Ethics Committee of the Affiliated Hospital of Inner Mongolia Minzu University (No. NM-LL-2024-05-16-08). The study followed the National Institute of Health Guidelines for the Ethical Use of Animals. Sprague-Dawley rats were obtained from the Changsheng Experimental Animal Centre of Liaoning Province, China [SCXK (Liao) 2020-0001] and housed in a specific pathogen-free experimental facility at Inner Mongolia Minzu University. The rats were kept under controlled conditions with a temperature of 23 ± 1 °C, humidity of 55% ± 5%, and a 12-h light-dark cycle. They were given ad libitum access to food, water, and physical activity throughout the study period.

Grouping of experimental animals and establishment of rat MI model

After 1 week of adaptive feeding, the rats were randomly assigned to a control group, model group, and Agari-5 group (n = 10). Rats in the model and Agari-5 groups underwent MI induction. Under isoflurane anesthesia the animals were disinfected with alcohol, and a thoracotomy was performed to expose the heart. The pericardium was carefully torn to fully expose the heart, and the left anterior descending coronary artery was ligated with a No. 4-0 black silk thread to induce MI[9-11]. The specific experimental flow is shown in Figure 1A.

Figure 1 Agari-5 restored cardiac function after myocardial infarction in rats. A: Overall experimental flowchart; B: Cardiac ultrasound of rats in each group (n = 3 hearts/group); C: Statistical histograms of ejection fraction, fractional shortening, left ventricular end-diastolic dimension, and left ventricular end-systolic dimension data of cardiac ultrasound in rats of each group. All data are expressed as mean ± SD. All statistics are shown in bar graphs. ^aP < 0.05. EF: Ejection fraction; FS: Fractional shortening; LVIDs: Left ventricular end-systolic dimension; LVIDd: Left ventricular end-diastolic dimension; MI: Myocardial infarction; NS: Not significant.

Experimental grouping and drug administration

In the control group and model group, an equivalent volume of saline was administered by gavage once daily. In the Agari-5 group a dose of 0.8127 g/kg was given daily for 4 weeks at a fixed time. The gavage doses for all rats were adjusted based on the equivalent doses for animals and humans.

Rat cardiac function assessment

Using a small animal ultrasound imaging system, echocardiograms were monitored for each group of rats, and several key cardiac function indicators were measured. These indicators included left ventricular ejection fraction, left ventricular short-axis shortening fraction, left ventricular end-systolic diameter, and left ventricular end-diastolic dimension.

Sample collection

Rats were anesthetized with isoflurane, and the abdominal cavity was opened to collect blood from the abdominal aorta. The blood was left at room temperature for 30 min and then centrifuged at 3500 rpm for 10 min. The supernatant was further centrifuged at 12000 rpm for 10 min at 4 °C and stored at -80 °C. Heart tissue from the rats was also rapidly collected and stored at -80 °C for further analysis.

Detection of cardiac enzyme markers by ELISA

After the final gastric lavage, the rats were fasted for 12 h and anesthetized. After routine disinfection the abdominal cavity was incised along the midline of the abdomen using surgical scissors. Postoperatively, the needle tip of the puncture needle was inserted at an angle of approximately 25° to 30° toward the heart end with a depth of approximately 5 mm, and blood was aspirated. Samples were centrifuged at room temperature (15000 × g, 20 min), and supernatant was removed. Then a second centrifugation step (15000 × g, 20 min) was performed at 4 °C. Supernatant was removed, and samples were then analyzed. Serum CK (No. ml107007, mlbio Co., Ltd., China), CK-MB isoenzyme (No. ml107008, mlbio Co., Ltd., China), LDH (No. ml106660, mlbio Co., Ltd., China) levels were assayed by commercially ELISA kits., following the manufacturer’s instructions. A plate reader was used to measure the absorbance of each well at a wavelength of 450 nm.

Hematoxylin and eosin staining and Masson’s staining

After euthanizing the rats under anesthesia, tissue samples were collected and fixed in 10% neutral formaldehyde for 24 h. The samples underwent gradient ethanol dehydration, xylene clearing, and paraffin embedding. Sections were then cut, stained with hematoxylin and eosin stain and Masson’s stain, and sealed with neutral gum. The lesions were subsequently observed under a light microscope.

Proteomics sample preparation

Cardiac samples were collected and ground to a powder in liquid nitrogen. An appropriate amount of the powder was transferred to a 1.5 mL centrifuge tube and incubated with lysis buffer (8 M urea) containing 1 mmol/L phenylmethylsulfonyl fluoride and 2 mmol/L EDTA (final concentrations) for 5 min. The mixture was then sonicated on ice for 5 min (2 seconds on, 3 seconds off). The lysate was centrifuged at 4 °C at 15000 g for 20 min, and the supernatant was collected. The total protein concentration was determined using the bicinchoninic acid protein quantification assay. An aliquot of the protein solution was taken based on the protein concentration and adjusted to a final volume of 200 µL with 8 M urea. The solution was reduced with 10 mmol/L dithiothreitol for 45 min at 37 °C and alkylated with 50 mmol/L iodoacetamide for 15 min in the dark at room temperature. Precooled acetone, four times the volume of the protein solution, was added to precipitate the proteins for 2 h at -20 °C. After centrifugation the protein precipitate was air-dried and resuspended in 200 µL of 25 mmol/L ammonium bicarbonate solution, followed by the addition of 3 µL of trypsin (Promega) for overnight digestion at 37 °C. After digestion the peptides from each sample were desalted using a C18 column, concentrated by vacuum centrifugation, and redissolved in 0.1% (v/v) formic acid[12].

Liquid chromatography-tandem mass spectrometry detection

Nanoliter liquid chromatography detection: Samples were separated using a Vanquish Neo ultra-high-performance liquid chromatography (UHPLC) system[13]. The mobile phase consisted of phase A, which was a 0.1% formic acid aqueous solution, and phase B, which was a 0.1% formic acid acetonitrile solution (100% acetonitrile). The injection mode utilized a trap-analytical dual-column method in which the trap column was a PepMap Neo Trap Cartridge (300 μm × 5 mm, 5 μm) and the analytical column was an Easy-Spray™ PepMap™ Neo UHPLC column (150 µm × 15 cm, 2 µm). The temperature of the analytical column was maintained at 55 °C using an integrated column oven. The sample volume was set at 200 ng with a flow rate of 2.5 µL/minute and an effective gradient duration of 6.9 min, resulting in a total run time of 8 min.

Orbitrap Astral mass spectrometer detection

Data-independent acquisition (DIA) analysis was carried out using a nanoscale Vanquish Neo system (Thermo Fisher Scientific) for chromatographic separation. Samples, following nanoscale UHPLC separation, were analyzed by DIA mass spectrometry using an Orbitrap Astral high-resolution mass spectrometer (Thermo Scientific). The detection mode was set to positive ion with a parent ion scanning range of 380-980 m/z and a primary mass spectral resolution of 240000 at 200 m/z. The normalized automatic gain control target was set at 500% with a maximum ion injection time of 5 ms. For MS² analysis the DIA data acquisition mode was employed, utilizing 299 scanning windows. The isolation window was set to 2 Th with a higher energy collision dissociation collision energy of 25%. The normalized automatic gain control target was also 500%, and the maximum injection time was set at 3 ms.

Mass spectrometry data analysis

The library search software used for DIA mass spectrometry data in this study was DIA-NN (v1.8.1). The library was searched using the library-free method with the following search parameters: (1) The database used was uniprot-proteome_UP000002494_Rat_20220719.fasta database, which contained a total of 46069 sequences; and (2) The iRT2.fasta database, which included 1 sequence. The deep learning-based parameter was enabled to predict a spectral library. Additionally, the multiple bypass retrieval option was selected to generate a spectral library using DIA data, which was then utilized for reanalyzing the DIA data for protein quantification. Both precursor ions and protein-level false discovery rates were filtered at 1%, ensuring that the filtered data could be used for subsequent raw data analyses[14,15].

Bioinformatics analysis

To gain a comprehensive understanding of the functional properties of the identified and differentially expressed proteins, a thorough enrichment analysis was conducted. This analysis included the following components: (1) Gene Ontology (GO) annotation; (2) EuKaryotic Orthologous Groups (KOG) functional classification; (3) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis; (4) Protein domain characterization; (5) Subcellular localization assessment; and (6) Signal peptide prediction (using SignalP). The differentially expressed proteins in each comparative group were enriched and analyzed at three levels: (1) GO classification; (2) KOG functional classification; and (3) KEGG pathway analysis.

Differently expressed protein interaction network

PPI analysis was performed using the StringDB protein interaction database (http://string-db.org/). If a corresponding species was available in the database, the protein sequences of that species were directly extracted. If no corresponding species was found, protein sequences from a closely related species were utilized. The differentially expressed protein sequences were compared with the extracted sequences using BLAST, and interactions among the differentially expressed proteins were identified based on a confidence score greater than 400 (medium confidence). Static and dynamic network diagrams were constructed using R packages, specifically qgraph for static visualizations and networkD3 for dynamic visualizations.

Western blot

Protein extraction was performed from the hearts of rats in each experimental group, and protein concentrations were quantified using a bicinchoninic acid kit. For protein analysis, 20 µg of each sample was loaded onto a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel in which the proteins were separated according to their molecular weight during electrophoresis. After separation the proteins were transferred from the gel to polyvinylidene fluoride membranes (Millipore, IPVH00010). To prevent nonspecific binding, the polyvinylidene fluoride membranes were subjected to a 2-h blocking process. Subsequently, the membranes were incubated overnight at 4 °C with primary antibodies diluted 1:1000 to ensure optimal specificity and sensitivity. After the primary antibody incubation, the membranes were washed three times with PBS to remove any unbound antibodies. The membranes were then incubated with secondary antibodies diluted 1:5000 for 1 h at room temperature, facilitating the detection of protein bands of interest. Finally, protein expression levels in the different experimental groups were compared.

Statistical analysis

Statistical analysis was conducted using GraphPad Prism 8.0 (GraphPad Software). The data are presented as mean ± SD. Student’s t-tests were used to evaluate differences between two groups while one-way analysis of variance was applied for multiple comparisons. Spearman’s correlation analysis and the χ² test were performed to assess correlations between different gene pairs. Survival differences were evaluated using the log-rank test. All statistical analyses were two-sided, with a significance level set at P < 0.05, indicating statistical significance.

RESULTS

Protective effect of Agari-5 on rats with a MI

General condition of rats: As shown in Figure 1A, this is the overall animal experiment flowchart. The rats in the control group exhibited bright, lustrous fur, were highly responsive, and demonstrated normal eating and drinking behaviors with no mortality occurring during the experimental period. In contrast, the rats in the model group displayed poor vitality, dull fur, cold skin, and a tendency to sleep excessively along with considerably reduced food and water intake; some of these rats also had scanty stools. The rats in the Agari-5 group appeared to have slightly better vitality though their fur lacked luster, and their complexion was somewhat warmer; however, they also exhibited slightly lower food and water intake compared with the control group. Body weight measurements revealed a steady increase in the control group each week while the model group experienced a significant decrease in body weight. In contrast, the Agari-5 group showed an upward trend in body weight compared with the model group, indicating weight gain as detailed in Supplementary Figure 1A.

The effect of Agari-5 on cardiac function in rats with MI: As shown in Figure 1B and C, rat cardiac ultrasound images revealed a decreasing trend in left ventricular ejection fraction and left ventricular short-axis shortening fraction levels in the model group compared with the control group (P > 0.05), and an increasing trend in the Agari-5 group compared with the model group (P > 0.05). At the left ventricular diameter in diastole and left ventricular internal diameters levels, the model group showed an increasing trend compared with the control group (P < 0.05). Compared with the model group, the Agari-5 group showed significant differences (P < 0.05).

Cardiac function indicators: MI resulted in the destruction of cardiomyocytes, leading to increased activity levels of cardiac function enzymes, which are common indicators for detecting MI in rats. Compared with the control group, levels of CK, CK-MB, and LDH were significantly elevated in the model group (P < 0.05), confirming the successful establishment of the model. In contrast, treatment with Agari-5 significantly reduced the cardiac function markers in rats with MI compared with the model group (P < 0.05). For further details, refer to Figure 2A.

Figure 2 Common indicators for detecting myocardial infarction in rats. A: Agari-5 restored indicators of cardiac function after myocardial infarction in rats from left to right: (1) Creatine kinase; (2) Creatine kinase isoenzyme; and (3) Lactate dehydrogenase. All data are expressed as mean ± SD. ^aP < 0.05 vs model, ^bP < 0.01 vs model, ^cP < 0.05 vs control, ^dP < 0.01 vs control; B and C: Histopathological pictures of heart tissue sections were stained with hematoxylin and eosin and Masson’s staining. These parameters were recorded after 4 weeks of gavage administration of Agari-5 following myocardial infarction in rats (n = 3). CK: Creatine kinase; CK-MB: Creatine kinase isoenzyme; LDH: Lactate dehydrogenase.

Hematoxylin and eosin staining and Masson’s staining: As shown in Figure 2B, hematoxylin and eosin staining results indicated that the myocardial fibers in the control group were organized in bundles with intact myocardial tissue structure and no evidence of inflammatory cell infiltration. Meanwhile, the model group exhibited disordered myocardial tissue with blurred transverse striations and a significant enlargement of the myocardial interstitial space, accompanied by extensive inflammatory cell infiltration. Compared with the model group, the myocardial fibers in the Agari-5 group were more organized, showing improved cytoplasmic edema and more intact cytoplasmic nuclei. As illustrated in Figure 2C, Masson’s staining revealed no significant collagen fiber deposition in the myocardial tissue of the control group. In the model group normal myocardial fibers were damaged with extensive collagen fiber deposits observed, indicating pronounced myocardial fibrosis and atrophic degeneration along with inflammatory cell infiltration in the infarcted areas. In the Agari-5 group myocardial fibrosis was significantly reduced with fewer collagen fibers deposited and diminished inflammatory cell infiltration.

Quality assessment of quantitative proteomics results: In constructing the mathematical model, principal component analysis was initially employed to process the overall data, allowing for the observation of the natural distribution of experimental samples and group relationships. The distribution of each group, as shown in Supplementary Figure 1B and C, was classified satisfactorily, indicating clear differences in protein expression among the groups. Correlation analysis between samples within each group is presented in Supplementary Figure 1D. This analysis enabled the observation of biological replicates among samples within the same group. The results demonstrated that the correlation coefficients of samples within each group were higher than those of samples between groups, suggesting that the differential proteins identified in this experiment are more reliable.

Differential protein expression: In the analysis of differential protein expression, a total of 231 proteins were identified in the control group compared with the model group with 122 proteins being upregulated and 109 downregulated as shown in Figure 3A. In the comparison between the Agari-5 group and the model group, 190 proteins were identified of which 99 were upregulated and 91 were downregulated as displayed in Figure 3B. Finally, Figure 3C shows that in the Agari-5 group compared with the control group, 246 proteins were identified with 129 proteins upregulated and 117 downregulated.

Figure 3 Volcano plot of differential proteins between groups of rats. A: The control group compared with the model group with 122 proteins upregulated and 109 downregulated; B: In the comparison between the Agari-5 group and the model group, 190 proteins were identified of which 99 were upregulated and 91 were downregulated; C: In the Agari-5 group compared with the control group, 246 proteins were identified with 129 proteins upregulated and 117 downregulated. Horizontal coordinates represent differential folds taken logarithmically at the base of 2, vertical coordinates represent P values taken logarithmically at the base of 10, and red and green scatters represent upregulated and downregulated differential proteins, respectively.

Bioinformatics analysis

Functional annotation of differentially expressed proteins: GO is an international standard classification system for gene functions, comprising three main components: (1) Biological process; (2) Molecular function; and (3) Cellular component. The differentially expressed proteins annotated to secondary GO entries were counted, and each classification displayed the top 20 GO terms with the highest-ranked differentially expressed proteins (in descending order). The results are presented in Supplementary Figure 2A-C. In the biological process category, the groups include cellular process, metabolic process, biological regulation, and cellular component. Molecular function is primarily characterized by cellular anatomical entities and protein-containing complexes. Cellular component is mainly represented by binding activities.

This study was annotated using the KOG database, and the number of differentially expressed proteins within each KOG category was counted and displayed in a bar chart (Supplementary Figure 2D-F). The categories included posttranslational modification, protein turnover, chaperones, general function prediction only, signal transduction mechanisms, and others, highlighting the functional groups of proteins identified in each experimental comparison.

KEGG pathways mainly encompass areas such as metabolism, genetic information processing, environmental information processing, cellular processes, human diseases, and drug development. Pathway analysis allows for the identification of critical biochemical, metabolic, and signal transduction pathways in which the differentially expressed proteins are involved. Supplementary Figure 2G-I presents the top KEGG pathways identified, including biosynthesis of amino acids, carbon metabolism, purine metabolism, thermogenesis, and T helper type 17 cell differentiation.

Functional enrichment of differentially expressed proteins: In the enrichment analysis the 50 GO terms with the highest P values (in descending order) were selected and visualized in bar charts as shown in Figure 4A-C. For biological processes the dominant terms included signal transduction, negative regulation of transcription by RNA polymerase II, and innate immune response. In molecular function the prominent categories were metal ion binding, ATP binding, and protein homodimerization activity. In cellular component key categories included extracellular space, integral component of the membrane, and Golgi apparatus. From the KOG enrichment analysis, 20 functional categories with the top P value rankings (sorted from smallest to largest) were selected and visualized using bubble diagrams (Figure 4D-F). The dominant pathways included general function prediction only, signal transduction mechanisms, amino acid transport and metabolism, and carbohydrate transport and metabolism. For the KEGG enrichment analysis results (Figure 4G-I), bubble diagrams were used to represent the degree of enrichment based on fold enrichment, P value, and the number of differentially expressed proteins involved. Key enriched pathways included biosynthesis of amino acids, cell adhesion molecules, drug metabolism, T helper type 17 cell differentiation, and biosynthesis of cofactors.

Figure 4 Bubble diagram of enrichment analysis of differentially expressed proteins for Gene Ontology, EuKaryotic Orthologous Groups, and Kyoto Encyclopedia of Genes and Genomes. A: Gene Ontology (GO) enrichment analysis (control vs model); B: GO enrichment analysis (Agari-5 vs model); C: GO enrichment analysis (Agari-5 vs control); D: EuKaryotic Orthologous Groups (KOG) enrichment analysis (control vs model); E: KOG enrichment analysis (Agari-5 vs model); F: KOG enrichment analysis (Agari-5 vs control); G: KEGG enrichment analysis (control vs model); H: Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis (Agari-5 vs Model); I: KEGG enrichment analysis (Agari-5 vs Control). The horizontal coordinate represents the enrichment multiplicity, and the vertical coordinate represents the name of the entries of GO, KOG, and KEGG.

Interaction network analysis of differentially expressed proteins: The results of the PPI interaction network analysis are displayed in Figure 5. These networks can be imported into Cytoscape software for further visual editing and exploration. In the comparison between the control group and model group, 144 nodes were identified. In the Agari-5 group vs the model group, 112 nodes were found. Between the Agari-5 group and the control group, 168 nodes were identified. In total, the analysis revealed 60 differential proteins, detailed in Table 1.

Figure 5 Interaction network diagram of differentially expressed proteins among groups. A: Control group; B: Model group; C: Agari-5 group. Each node in the interaction network represents a differentially expressed protein; node red (up) indicates that the expression level of differentially expressed proteins is upregulated, and node blue (down) indicates that the expression level of differentially expressed proteins is downregulated.

Table 1 Statistical table of differential protein expression.

Gene	Agari5 vs model regulation	Model vs control regulation	Agari5 vs control regulation
Neb	Up	Down	Insig
Pnkp	Up	Down	Insig
Papss1	Up	Down	Insig
Lonrf1	Up	Down	Insig
Psat1	Down	Up	Insig
Ccser2	Up	Down	Insig
Man1c1	Down	Up	Insig
Smoc1	Down	Up	Insig
AC095947.1	Up	Down	Insig
Nxn	Up	Down	Insig
Tanc1	Up	Down	Insig
Sdf2	Up	Down	Insig
Syap1	Up	Down	Insig
Dmc1	Up	Down	Insig
Nom1	Up	Down	Insig
Decr1	Down	Up	Insig
Rnpep	Down	Up	Insig
Pdk1	Up	Down	Insig
Isg15	Up	Down	Insig
Trio	Up	Down	Insig
Trim32	Up	Down	Insig
Csnk2a2	Up	Down	Insig
Chgb	Up	Down	Insig
Cdk18	Down	Up	Insig
Pold1	Down	Up	Insig
Smad4	Up	Down	Insig
Otc	Up	Down	Insig
Mbp	Down	Up	Insig
Psbpc1	Down	Up	Insig
Ptprc	Up	Down	Insig
Grb10	Up	Down	Insig
Pth1r	Up	Down	Insig
Slc6a7	Down	Up	Insig
Vcam1	Down	Up	Insig
Map1a	Down	Up	Insig
Afm	Up	Down	Insig
Il6st	Up	Down	Insig
Padi3	Up	Down	Insig
Ermard	Down	Up	Insig
Gramd1a	Up	Down	Insig
Znf830	Down	Up	Insig
Lztfl1	Down	Up	Insig
Cep70	Down	Up	Insig
Armcx3	Up	Down	Insig
Synj1	Up	Down	Insig
Nrxn1	Down	Up	Insig
Prx	Down	Up	Insig
Ces1f	Down	Up	Insig
Isg20 L2	Up	Down	Insig
Dync2 Li1	Down	Up	Insig
Blvra	Up	Down	Insig
Krt42	Up	Down	Insig
Tepp	Down	Up	Insig
Fmo2	Up	Down	Insig
Wls	Up	Down	Insig
Rsl1d1 L1	Up	Down	Insig
Asrgl1	Down	Up	Insig
Ttl	Up	Down	Insig
Nme7	Down	Up	Insig
Pde4dip	Up	Down	Insig

Insig: Insignificant.

Immunoblot analysis of differentially expressed proteins in rat heart tissue: The levels of differentially expressed proteins in rat heart tissues were evaluated through western blot analysis. Based on the proteomics data, four key proteins were identified: (1) SDF2; (2) PDK1; (3) SMAD4; and (4) PSAT1. For SDF2, PDK1, and SMAD4, protein expression levels in the model group were decreased compared with the control group though the differences were not statistically significant (P > 0.05). In contrast, the Agari-5 group showed an upregulation trend in the expression levels of these proteins compared with the model group again without statistical significance (P > 0.05). Regarding PSAT1, its expression in the model group was upregulated relative to the control group (P > 0.05). However, the Agari-5 group exhibited a trend of downregulated PSAT1 expression compared with the model group although this change was not statistically significant (P > 0.05). The protein bands are shown in Figure 6A, and the corresponding statistical bar graphs are displayed in Figure 6B-E.

Figure 6 Protein bands. A: Protein bar graph; B-E: The statistical histograms of each protein (n = 3, mean ± SD). ^aP < 0.05; NS: Not significant.

DISCUSSION

In recent decades natural medicines have gained significant use in clinical treatment for various diseases[16-18]. Cardiovascular disease remains a leading cause of mortality and morbidity worldwide. Studies have demonstrated that traditional Mongolian medicine preparations, such as Sanwei Tanxiang Decoction, can alleviate acute ischemia-reperfusion injury by modulating the electrophysiological properties of rat cardiomyocytes[19]. Similarly, the extract from Sugmul-3, another Mongolian medicine, has shown protective effects against isoproterenol-induced heart failure in Wistar rats by regulating mitochondrial dynamics[20].

Mongolian medicine emphasizes a holistic view of the human body, treating diseases through the interaction of multiple drugs. This approach offers advantages such as fewer adverse reactions and side effects, particularly in preventing and treating cardiovascular diseases. Agari-5 is a widely used traditional compound in Mongolian medicine for managing cardiovascular diseases. Its multicomponent and multitarget characteristics enhance its effectiveness in treating cardiovascular diseases[21].

Among the key components of Agari-5 is Salvia miltiorrhiza, which plays a critical role in reducing myocardial fibrosis, delaying cardiac remodeling[22], reducing myocardial inflammation and injury[23,24], and decreasing oxidative stress and injury[25,26]. Therefore, understanding the protective mechanisms of Agari-5 is essential for guiding clinical use, improving patient outcomes, and enhancing the prognosis, treatment, and overall quality of life for individuals with cardiovascular diseases.

In this study an MI rat model was established to assess cardiac function indices and the pathological changes in cardiac tissues. The results demonstrated that compared with the control group the cardiac function indices in the model group were significantly elevated while pathological findings showed disorganized myocardial tissue structure with blurred transverse striations, markedly enlarged myocardial gaps, and extensive infiltration by inflammatory cells. However, following treatment with Agari-5, both cardiac function and myocardial tissue pathology improved. Notably, the degree of myocardial fibrosis was markedly reduced with less collagen fiber deposition and decreased inflammatory cell infiltration. These findings suggest that the therapeutic effect of the Agari-5 compound may be attributed to the interactions of its components.

In the proteomics study the results showed a clear distribution and high dispersion between groups, indicating reliable findings for differential protein expression. Comparisons between the groups revealed a range of 190 to 246 differentially expressed proteins while enrichment analyses suggested that the therapeutic mechanism of Agari-5 in rats following MI may involve alterations in processes such as fatty acid degradation, metabolism, elongation, carbon metabolism, and protein digestion and absorption, all of which are vital for energy metabolism. Furthermore, differential protein function annotation analysis identified cardiovascular-related proteins in biological processes. These findings also indicated that Agari-5 may act on pathways related to myocardial fibrosis and mitochondrial energy metabolism in cardiomyocytes after MI. Together, the differential protein annotations and enrichment analyses suggest that Agari-5 may exert a therapeutic effect on MI by modulating these pathways.

In the PPI analysis a total of 60 proteins were identified with four specific proteins (SDF2, PDK1, SMAD4, and PSAT1) receiving attention due to their potential role as biomarkers for MI diagnosis. The SMAD4 signaling pathway has been reported to have a close association with cardiovascular disease in which its activation can lead to myocardial injury[27] and is significantly involved in the progression of myocardial fibrosis. PDK1 plays a key role in cellular phosphorylation processes related to mitochondrial energy metabolism, such as maintaining mitochondrial membrane potential, ATP generation, and calcium uptake[28].

In this study we used western blotting to investigate the expression levels of these differential proteins. These results showed that in MI rats treated with Agari-5 the expression levels of these proteins were comparable with those in the control group, suggesting that SDF2, PDK1, SMAD4, and PSAT1 may be important targets influenced by Agari-5. This study applied a logical approach of “disease-drug-target” to preliminarily verify the efficacy of Agari-5 in treating MI in rats.

Moreover, through proteomic analysis the research delved deeper into the protective mechanisms of Mongolian traditional medicine formulas, finding that these traditional formulas may protect MI rats through the regulation of multiple protein targets and molecular mechanisms, such as multicomponent interactions and synergistic effects, providing a new direction for research on national medicines and offering a reference for clinical treatments of cardiovascular diseases.

CONCLUSION

This study successfully identified the potential therapeutic benefits and mechanisms by which Agari-5 exerts its effects against MI. The findings provide initial evidence supporting the use of Agari-5 as a treatment for cardiovascular diseases and cardiac injuries, highlighting its possible role as an effective strategy in clinical settings.