Screening ONSMP active ingredient targets and disease proteins

Firstly, all ingredients were obtained from the HERB database (http://herb.ac.cn/) using the names of the nine ONSMP herbs as search terms. We utilized the PubChem database (https://pubchem.ncbi.nlm.nih.gov/) to query the composition of canonical smiles of the ONSMP components and subsequently imported it into the SwissADME database (http://www.swissadme.ch/). Based on the screening criteria shown in Table 1, we selected ONSMP active ingredients with good oral bioavailability and pharmacokinetic properties. After that, use the HERB database (http://herb.ac.cn/), SwissTargetPrediction database (http://old.swisstargetprediction.ch/), Similarity ensemble approach database (https://sea.bkslab.org/), and Super-PRED database (https://prediction.charite.de/index.php) to obtain the targets of ONSMP active ingredients. The disease proteins were obtained from HERB database (http://herb.ac.cn/), OMIM database (https://omim.org/), GeneCards database (http://www.genecards.org/), PharmGkb database (http://www.pharmgkb.org/), TTD database (http://db.idrblab.net/ttd), and DrugBank database (https://www.drugbank.ca/) with “chronic heart failure” and “myocardial fibrosis” as the search terms.

Table 1 Screening conditions of ONSMP active ingredientsScreening pathways and core targets of ONSMP on myocardial fibrosis in heart failure

We first installed the Bioconductor data package (including DOSE, clusterProfiler, enrichplot, and pathview) in R programming language 4.2.0 software, after which we imported the intersection proteins of heart failure proteins, myocardial fibrosis proteins, and ONSMP active ingredients target proteins were imported into this software to perform GO and KEGG enrichment analyses with a P-value of less than 0.05 as the screening condition. Obtain key signaling pathways and corresponding proteins of ONSMP for the treatment of myocardial fibrosis in heart failure from the results of KEGG enrichment analysis, then import these proteins into the string database (https://string-db.org/), with the settings of species as homo sapiens, confidence level ≥ 0.4, and hidden discontinuous nodes, to obtain the interactions of these proteins. Use Cytoscape-v3.9.0 software to calculate the betweenness centrality, closeness centrality, degree centrality, eigenvector centrality, local average connectivity, and network centrality of the prediction results of the string database and retain the proteins with these parameters greater than their respective mean values. After screening twice, the remaining nodes were the core proteins. Then, construct component-protein networks for these core proteins with the corresponding ONSMP active ingredients and calculate the degree of centrality by Cytoscape-v3.9.0 software.

Molecular docking of ONSMP active ingredients to pathway core targets

The molecular docking approach examines the ability of core proteins to interact with the corresponding ONSMP components and predicts the binding mode and affinity between them. Receptor proteins and ligand compounds were first obtained and processed: jointly using the Uniport database (https://www.uniprot.org/) and the AlphaFold Protein Structure Database (https://www.alphafold.ebi.ac.uk/) to acquire the receptor proteins β1-AR (Uniport ID: P08588), AC6 (Uniport ID: O43306), EPAC1 (Uniport ID: O95398), RAP1A (Uniport ID: P62834), STAT3 (Uniport ID: P40763), and CCND1 (Uniport ID: P24385) 3D structure files, and then use PyMOL 2.3.0 software to check the protein structures and remove water molecules, irrelevant protein chains and proto-ligands in the protein structure for docking; using the Pubchem database (https://pubchem.ncbi.nlm.nih.gov/) to acquire the ligand compounds baicalin (Pubchem CID: 64982), vitamin D (Pubchem CID: 12895043), resveratrol (Pubchem CID: 445154), Tanshinone IIA (Pubchem CID: 164676), apigenin (Pubchem CID: 5280443), calycosin (Pubchem CID: 5280448), capsaicin (Pubchem CID: 1548943), cryptotanshinone (Pubchem CID: 160254), emodin (Pubchem CID: 3220), rhein (Pubchem CID: 10168), ursolic acid (Pubchem CID: 64945), 15,16-dihydrotanshinone i (Pubchem CID: 11425923) 3D structure files, and then use Chem3D 2020 software to optimize the molecular force field of the ligand compound structure to finally obtain the optimal molecular structure in the lowest energy state. Molecular docking of receptor proteins and ligand compounds: Hydrogenation of proteins and hydrogenation and determination of torsion bonds of ligand compounds were performed using AutoDock Tools 1.5.6 and saved as pdbqt format files. Use the grid plate to set up molecular docking parameters, as shown in Table 2. Set the docking method as semi-flexible-docking and the docking algorithm as Lamarckian genetic algorithm. Run Auto Dock Vina1.2.0 for molecular docking to get the docking binding free energy as well as the docking result file, and then convert the PDBQT format of the complexes to PDB format by Open Babel GUI software, and then import them into PyMOL2.3.0 software and Discovery Studio2019 software for 3D and 2D transformation respectively.

Table 2 Molecular docking parametersDrugs preparation

The Pharmacy Department of the First Affiliated Hospital of Tianjin University of Traditional Chinese Medicine provided all the herbs. Grind Salvia miltiorrhiza Bunge into powder, while Astragalus membranaceus (Fisch.) Bunge, Codonopsis pilosula (Franch.) Nannf, Eleutherococcus senticosus (Rupr. & Maxim.) Maxim., Salvia miltiorrhiza Bunge, Trionyx sinensis Wiegmann, Poria cocos (Schw.) Wolf, Lepidium apetalum Willd., Ophiopogon japonicus (Thunb.) Ker Gawl., and Citrus × aurantium L. were decocted with ten times the amount of water for 2 h and then filtered. Decocted dregs of the herbs with another ten times the amount of water for 2 h and then filtered. The two filtrates were combined, concentrated, and dried to make a powder and mixed with the powder of Salvia miltiorrhiza Bunge. 82 g ONSMP raw drug processed to obtain patent medicine is 31 g. We have examined the components contained in ONSMP by UHPLC-Q-Exactive-MS/MS in a previous study [9]. The positive drug of choice was the ACEI representative drug captopril Captopril (CAP, H31020564, Shanghai Xudong Haipu Pharmaceutical Co., Ltd., China), which inhibits ventricular remodeling by reducing Ang II production and has an activating effect on the β1-AR-cAMP signaling pathway [15,16,17].

Animals

All animal experimental procedures were performed by the National Institutes of Health Guidelines for Laboratory Animals (NIH Publications No. 8023, revised 1978) and approved by the Experimental Animal Ethics Committee of the Institute of Radiological Medicine, Chinese Academy of Medical Sciences. 55 healthy male SD rats were purchased from Beijing Viton Lihua Laboratory Animal Technology Co, Ltd, with animal production license No. SCXK (Beijing) 2021-0006. We housed rats at the Institute of Radiology, Chinese Academy of Medical Sciences, at a temperature of (22 ± 2) ℃ and a humidity of (40 ± 5) %, with a 12 h light–dark cycle and free access to water. Rats were divided into sham (n = 9) and operation (n = 46) groups, while later anesthetized using intraperitoneal injection of 1.5% tribromoethanol, tracheal intubated, and connected to a small animal ventilator (tidal volume of 6 ml, respiratory rate of 80 breaths/min, respiratory ratio 1:2). Two professionals opened the thoracic cavity at the 3rd and 4th intercostal spaces of rats in the operation group to expose the heart, then ligated the left anterior descending branch of the coronary artery with a 5–0 suture 2 mm below the left atrial appendage. Finally, sutured the thoracic cavity of the rats one layer at a time with a 2–0 suture and turned off the ventilator. Sham group rats underwent thoracotomy without ligation of the left anterior descending coronary artery, and the rest of the treatment was the same as above. Penicillin 800,000 units was injected intraperitoneally for three consecutive days to prevent infection. At the end of the 8th week after surgery, a total of 17 rats in the operation group died (the mortality rate was 37%), and no deaths occurred in the sham group. Then, using LVEF ≤ 50% as the inclusion criterion [18], 24 rats with heart failure were screened from the 29 rats surviving in the operation group and randomly divided into the model, ONSMP-L, ONSMP-H, and CAP groups (6 rats in each group). Based on the conversion formula for the body surface area of humans and rats, we derived that the equivalent drug dose in rats was 6.3 times that in humans. Thus, 6.3 times the ONSMP dose in humans is 2.79 g/kg, which we used for the gavage dose of rats in the ONSMP-L group, and later set the drug concentration of ONSMP-H to 2 times that of ONSMP-L (5.58 g/kg); the positive drug CAP gavage dose was 2.25 mg/kg. At the same time, six rats were randomly selected in the sham group and gavaged with saline along with the model group rats. The frequency of gavage in rats was once a day for 4 weeks.

Cardiac function, organ coefficients, and Elisa assay

At the end of the gavage, we measured the LVEF, LVFE, IVSd, IVSs, LVIDd, LVIDs, LVPWd, and LVPWs of the rats by ultrasound. At the end of the experiment, 1.5% tribromoethanol was injected intraperitoneally into the anesthetized rats. Then, open the abdominal cavity of the rats and collect blood from the abdominal aorta with a sterile blood collection needle and procoagulant tube. Removed the heart and right-leg tibia, washed with normal saline, dried with filter paper, weighed, and calculated HWI and HW/TL. After separating the right heart, weighing the left ventricular weight, and measuring the length of the short and long axis of the left ventricle, we calculate LVWI, LVW/TL, and SI. The blood of the rats was left at room temperature for 30 min and then centrifuged at 3500 rpm (10-cm radius) for 15 min, and the serum was collected; 50 mg of myocardium was homogenized by mixing it with 450 ml of PBS and then centrifuged at 5000 rpm (10 cm radius) for 15 min to collect the supernatant. The levels of ANP, BNP, NT-ProBNP, PICP, MMP-2, MMP-9, TIMP-1, and cAMP were detected by an enzyme-linked immunosorbent kit (ANP: MM-21097R1, BNP: MM-0067R1, NT-ProBNP: MM-0329R1, PICP: MM-0621R1, MMP-2: MM-0111R1, MMP-9: MM-20918R1, TIMP-1: MM-0056R1; cAMP: MM-0549R1, Jiangsu Meiman Industrial Co., Ltd.) using a multifunctional enzyme labeling instrument (MULTISKANFC. Thermo Scientific, Massachusetts, USA) to analyze the absorbance of the test wells and calculate the corresponding index levels.

Histopathological staining and immunohistochemistry

Myocardial tissues from the infarcted area of rats were immersed in 4% paraformaldehyde for 3 days, paraffin-embedded, and cut into 4 or 6 μm sections. In contrast, the myocardium was later stained for histopathological examination using Hematoxylin–eosin (HE), Sirius red, and Masson staining kits (HE: BP-DL001-100 mL, Sirius red: BP-DL030-500 mL, Masson: BP-DL022-50 mL, Nanjing Sempega Biotechnology Co., Ltd., Nanjing, China) for histopathological examination of the myocardium. For HE staining, 4 μm sections in an electrically heated blast drying oven at 60 ℃ for 3 h, anhydrous ethanol immersion twice (5 min/time), 75% ethanol immersion for 5 min, distilled water rinsing for 5 min, hematoxylin solution staining for 5 min, distilled water rinsing for 5 min, 1% hydrochloric acid alcohol wash for 30 s, distilled water wash for 15 min, 0.5% Eosin staining solution for 2 min, and distilled water wash for 30 s, 80% ethanol wash for 30 s, 95% ethanol immersion twice (1 min/time), anhydrous ethanol immersion twice (3 min/time), xylene immersion twice (3 min/time), and neutral gum sealing. For Sirius red staining, 6 μm sections were dewaxed to water, then immersed in Sirius red staining solution for 60 min, rinsed in distilled water for 30 s, rinsed in weak acidic working solution for 1 min, immersed in Mayer’s hematoxylin staining solution for 10 min, rinsed in distilled water for 10 min, dehydrated, transparent, and sealed. For Masson staining, 4 μm sections were dewaxed to water, stained with an equal mixture of Weigert hematoxylin A and Weigert hematoxylin B for 10 min, washed in water for 1 min, and then stained with Masson's complex staining solution for 15 min, douche briefly in distilled water, and treated with 1% phosphomolybdic acid solution for 5 min, aniline blue staining solution for 5 min, and 1% glacial acetic acid in water for 1 min, dehydrated, transparent and sealed.

Subsequently, a universal immunohistochemistry kit (WE0316, Beijing Boosun Biotechnology Co, Ltd, Beijing, China) detected positive expression of α-SMA and FSP-1. The 4 μm sections were dewaxed and rinsed 3 times (5 min/time) with PBS, added 3% H2O2 (80% methanol) dropwise, and left at room temperature for 10 min before being rinsed again with PBS and cooled to room temperature. Placed section in hot Sodium Citrate Antigenic Repair Solution for 10 min for hot antigen repair, cooled to room temperature, and rinsed 3 times (5 min/time) with PBS, later immersed in goat serum sealing solution for 20 min, followed by a drop of 50 μL of antibody and kept at 4 ℃ for 12 h before being rinsed again with PBS. The antibodies α-SMA (ab124964) and FSP-1 (ab220213) were purchased from Abcam (Cambridge, UK).

The sections for HE, Sirius red, Masson, and immunohistochemistry were magnified 400 times on an orthogonal light microscope (Nikon Eclipse E100, Nikon Corp., Tokyo, Japan) with 3 random fields of view, and then the CVF of Sirius red and Masson stained sections and the α-SMA and FSP-1 positive expression of immunohistochemistry sections were calculated by using Image J image analysis software.

Western blotting

Protein samples were electrophoresed on a 10% electrophoresis gel and then transferred to PVDF membranes, immersed in rapid containment solution for 30 min, then washed three times with 1 × TBST (10 min/wash), and engaged in polyclonal primary antibody for 12 h at 4 ℃. The antibodies β1-AR (ab85037), AC6 (ab14781), Epac1 (ab124162), STAT3 (ab68153), p-STAT3 (ab76315), CCND1 (ab16663), α-SMA (ab124964), FSP-1 (ab220213), and GAPDH (ab9485) purchased from Abcam (Cambridge, UK); Rap1A (DF6157) purchased from Affinity Biosciences (Jiangsu, China). Washed PVDF membrane with 1 × TBST three times (10 min/time), immersed with a secondary antibody for 2 h and washed with 1 × TBST three times (10 min/time). The strips were immersed in ECL developer for 1 min, then detected by a multifunctional imager (97-0827-02, Jena Analytics, Germany), and analyzed for grayscale values with Image J image analysis software.

Assayed Rap1A activity levels were according to the kit instructions (Abcam, ab212011, Cambridge, UK). Briefly, 50 mg of myocardial tissue was clipped from the rat apical infarct site and washed with ice-cold PBS, then 1 ml of 1 × analytical buffer was added and homogenized. The supernatant was collected by centrifugation in a high-speed cryo-centrifuge at 14,000 × g for 10 min at 4 ℃. After that, incubate the supernatant for 30 min with agarose beads coupled to the Rap‐binding domain of RalGDS, which binds specifically to the active form of Rap1. Subsequently, the precipitated GTP‐Rap1A was detected by Western blot analysis using anti‐Rap1A antibody, and analyzed for grayscale values with Image J image analysis software.

qPCR

Incorporate 1 mL of RNA extract (G3013, Servicebio, Wuhan, China), 20 mg of myocardial tissue, and grinding beads into a grinding tube, and subject to thorough grinding using a three-dimensional freeze grinder. At 4 ℃, centrifuge at 12,000 rpm for 10 min. Take the supernatant and add 400 μL of chloroform, mix well, and let stand for 3 min. Then, centrifuge again at 12,000 rpm for 10 min at 4 ℃, and transfer 400 μL of supernatant to a new centrifuge tube. Add 550 μL of isopropanol to the supernatant mix well and let stand for it at – 20 ℃ for 15 min. Centrifuge it at 12,000 rpm for 10 min at 4 ℃. The white sediment at the bottom of the tube is the RNA. After removing the liquid, add 1 mL of 75% ethanol and mix by inverting the tube to wash the sediment. The centrifuge tube will be centrifuged at 4 ℃ at 12,000 rpm for 5 min and placed on a super-clean table for 3 min after removing the supernatant. Then, 15 μL of RNA solubilizing solution was added and incubated at 55 ℃ for 5 min. The RNA concentration and purity were measured using an ultra microspectrophotometer (NanoDrop2000, Thermo Scientific, Massachusetts, USA). Using the reverse transcription reagent kit (G3337, Servicebio, Wuhan, China) to prepare the reverse transcription reaction system, gently mix, then centrifuge. Processed reaction mixture was on the PCR instrument (DS-11, Thermo Scientific, Massachusetts, USA) with the program set to 25 ℃ for 5 min, 42 ℃ for 30 min, and 85 ℃ for 5 s. Take 0.1 mL of the PCR reaction plate to establish the reaction system, and set the PCR amplification program on the fluorescence quantitative PCR instrument (CFX Connect, Bio-rad, California, USA): 95 ℃ for 30 s, 95 ℃ for 15 s and 60 ℃ for 30 s (40 cycles), with fluorescence signals collected at 0.5°C per temperature rise from 65 ℃ to 95 ℃. The sequences of the primer sets used for this analysis were as follows (Table 3). Use the 2−ΔΔCt method to quantify each gene's relative expression level.

Table 3 Primers’ sequences used in this studyStatistical analysis

Data were analyzed using SPSS 210 statistical software. Measurement information was expressed as mean ± standard deviation when it conformed to normal distribution and median (interquartile spacing) when it did not work to normal distribution. When analyzing the influencing factors, one-way ANOVA was used for between-group comparisons when the measures conformed to a normal distribution with a chi-square variance. If the difference was statistically significant, compare the data two by two using the Bonferroni test. The Kruskal–Wallis H-test was used to compare multiple groups when the information did not fit a normal distribution, or the variance was not homogeneous, and the Mann–Whitney U test was further used for two-by-two comparisons when the difference was statistically significant. A statistical difference was indicated by P < 0.05. Finally, use statistical graphs to create GraphPad Prism 9.5 software.