Animal model

Sprague–Dawley rats (weighted 200–220 g, male), were purchased from the Animal Center of Xiyuan Hospital (Beijing, certificate no. SYXK 2018-0018), and treated complying with Animal Research Committee’s guidelines for the Animal Center of Xiyuan Hospital. Rats were housed on a light/dark cycle (12:12 h) at 20 ± 2 °C with a humidity of 40 ± 5%, and fed standard rat chow & water. There were 12 rats in each group. Furthermore, the surgical procedures and experimental protocol were approved by Xi Yuan Hospital of China Academy of Chinese Medical Sciences (Protocol No. 2022XLC041). Induction of anesthesia in rats was achieved using intraperitoneal injection of sodium pentobarbital (80 mg/kg), maintained by inhalation of isoflurane (1%) and monitored with a pinch on the toe intraoperatively. The rat’s chest was opened between the second and fourth ribs, followed by LAD ligation using silk (4–0). The animals in the sham group received all surgical treatments except ligation.

High performance liquid chromatography (HPLC) for extracts of BYHWDDrugs and reagents

BYHWD (Drug approval number: B20050029; Batch number: 190203, 190322, 190403) was provided by Jining Huaneng Pharmaceutical Factory Co., Ltd. Salvianolic acid B (batch number: D1516060, content = 94.1%), paeoniflorin (batch number: A1712063, content ≥ 98%), tanshinone IIA (batch number: L1608056, content ≥ 98%), astragaloside IV (batch number: K1728029, content ≥ 98%) was purchased from Aladdin Biochemical Technology Co., Ltd.(Shanghai, China); the reference substance of amygdalin (batch number: Z28A6L2815, content ≥ 98%) was obtained from Shanghai Yuanye Biotechnology Co., Ltd.; M020170606, content ≥ 98%) and hydroxysafflor yellow A (batch number: A1610261, content ≥ 98%) were purchased from Chengdu Refensi Biotechnology Co., Ltd. Acetonitrile (lot number: LOT155898) and methanol (lot number: LOT095592) were chromatographically pure (Fisher Chemical), formic acid (lot number: LOT095224) and acetic acid (lot number: LOT104221) were chromatographically pure (MREDA, Beijing, China), and the experimental water was D11951 ultrapure water of Thermo Fisher Technology Co., Ltd. (Pittsburgh, PA, USA). Acetonitrile, methanol (HPLC grade), phosphoric acid as well as deionized water (analytical grade) were provided by Fisher Scientific (Shanghai, China).

Sample preparation

After being dissolved in methanol, the compounds were filtered with the membrane (0.20 μm) for the preparation of mixed standards, and then BYHWD was finely ground for sample solutions. Passing an 80-target quasisieve, the powder was precisely weighed (0.2 g) and then evenly dissolved in methanol (5.0 mL), and sealed with ultrasonic energy (frequency: 40 kHz, power: 200 w) for sixty minutes, followed by centrifugation at 400g for 10 min, take 4.5 mL of the supernatant to obtain a fat-soluble methanol extract. After adding 5.0 mL of triple distilled water and mixing with the residue, extract in a 70 °C water bath for 2 h, and centrifuge at 400g for 10 min. Subsequently, 4.5 mL of water-soluble extract and fat-soluble methanol extract were mixed in a 5:7 ratio, and diluted 5 times with the mobile phase; finally, filtered through a 0.22 μm organic filter.

Conditions for chromatography

To determine the active components in BYHWD, HPLC was used in combination with a binary low pressure gradient pump, a DAD UV detector, a column oven, as well as an automatic sampler (Agilent 1260, USA). Chromatographic separation was achieved on a CAPCELL PAK C18 column (2.0 × 150 mm, 5 μm, SHISEIDO, Japan) maintained at 40 °C. Mobile phase: phase A—5 mM ammonium acetate, 0.1% acetic acid in water, phase B—acetonitrile. The gradient program was set as follows: 0–25 min, 80–90% A; 25–30 min, 70–80% A; 30–35 min, 60–70% A; 35–40 min, 55–60% A; 40–45 min, 50–55% A; 45–60 min, 40–50% A; 60–65 min, remain 10% A. An aliquot of 20 μL sample solution was injected into the HPLC system for analysis, with a detection wavelength of 275 nm and a flow rate of 0.3 mL/min.

Drug administration

Complying with the guidelines of Manufacturing Practice and Laboratory Practice, BYHWD (batch number: 190403) was produced from Jining Huaneng Pharmaceutical Factory Co., Ltd. (Shandong, China), with its major components and their contents determined by HPLC fingerprinting. BYHWD was dissolved in saline (2 mL) and administered 48 h after MI using a gavage tube with 0.8 g/kg for BYHWD-ld & 1.6 g/kg for BYHWD-hd, followed by administration every 24 h until day 30 [13, 14]. Aspirin dissolved in 2 mL of saline was administered as a positive control at 100 mg/kg [15]. The rats in the sham group were administered equivalent volumes of saline with the model group in the same way. Rats were anesthetized intraperitoneally 1.5 h after the final administration of BYHWD on day 30, followed by the surgical procedure, and the heart was removed for myocardial morphology and component analysis.

Echocardiography

Echocardiography for the assessment of cardiac function was carried out using a Vevo 2100 Imaging System (Visual Sonics, Toronto, Canada) equipped with a 30 MHz transducer. Rats were anesthetized with isoflurane in oxygen and put on a heated imaging platform, followed by calculation of these parameters: heart rate (HR), ejection function (EF), left ventricular stroke volume (SV), fractional shortening (FS), left ventricular end-systolic dimension (LVIDs), left ventricular end-diastolic dimension (LVIDd), anterior systolic wall thickness (LVAWs), and diastolic anterior wall thickness (LVPWd).

Hematoxylin–eosin (H&E) and Masson’s trichrome staining

After being collected from each group, the heart samples were fixed in 4% paraformaldehyde (Solarbio, Beijing, China) for twenty-four hours, embedded in paraffin wax, and sectioned into 4–5 µm thick slices, followed by H&E staining. Subsequently, slices were stained with Masson’s trichrome, showing red in normal tissues but blue in collagen tissues. A light microscope (Olympus BX51 microscope, Japan) was applied for high resolution image of heart sections. Quantitative analysis of collagen deposition was achieved using collagen volume fraction (CVF) with the application of Image Pro Plus (Media Cybernetics Inc, MD, USA).

Electron microscopy of myocardial tissue

Rat hearts were fixed with 2% glutaraldehyde (Solarbio, Beijing, China). Myocardial tissues were collected from the surrounding infarcted region of the left ventricle, cut into blocks (1 mm3), fixed overnight at 4 °C, washed for three times with phosphate-buffered solution (0.1 mol/L), and then post-fixed with osmium tetroxide (1%) for 2 h. Subsequently, we routinely prepared the ultrathin sections and photographed them by a transmission electron microscope (JEM 1400 plus, JEOL, Tokyo, Japan).

Detection of the Luminex liquid suspension chip

It was conducted by using the Bio-Plex Pro Chemokine Panel kit (40-plex) that complies with the instructions of the manufacturer. Specifically, sera with/without BYHWD treatment were incubated in 96-well plates which were embedded with microbeads for 1 h, detection antibody for thirty minutes, and streptavidin–phycoerythrin (PE) for 10 min, followed by detection using the BioPlex MAGPIX System (Bio-Rad).

Viscosity determination

Blood samples (200 µL) were collected in heparin and used to determine viscosity at different shear rates using a cone-plate viscometer (Model LG-R-80B, Steellex Co., China) at 37 °C. Whole blood viscosity was measured at a low shear rate of 5/S, a medium shear rate of 60/S and a high shear rate of 150/S.

Platelet number count and platelet isolation

After SD rats were anesthetized with intraperitoneal injection of 1% sodium pentobarbital, blood (10 mL) collection from the abdominal aorta was performed using a tube with acid-citrate-dextrose anti-coagulant (3.2% trisodium citrate, 0.109 mol/L). The ratio of anticoagulant to blood is 1:9. The whole blood was then centrifuged (120g, 15 min) at room temperature to obtain platelet-rich plasma (PRP). Subsequently, the PRP supernatant was pelleted and centrifuged (300g, 15 min). Finally, platelets were resuspended in modified Tyrode buffer (Leagene Ciotech. Co., Ltd., Beijing, China). Whole blood (20 µL) was diluted with blood diluent buffer (MEK640, NIHON KOHDEN, Japan) and tested on automated hematology analyzer (NIHON KOHDEN CORP., Japan) to calculate the number of platelets.

Clot retraction

The washed platelets (100 μL) were mixed with platelet poor plasma (PPP, 300 μL) and induced to coagulate with thrombin (0.2 U/mL) [16]. After the incubation at 37 °C for different time intervals, the mixture was photographed and the degree of clot retraction was further analyzed by Image J software (version 1.4.3.67). Retraction (%) = 100 − [(sample clot area/intact sample area) × 100].

Flow cytometry analysis

Whole blood was incubated with anti-rat CD61 antibody labeled with phycoerythrin (104315) and anti-rat CD62P antibody labeled with PE (148305, BioLegend, California, USA), with the concentration finally reaching up to 20 mg/mL. After incubation for thirty minutes in darkness, the mixture was analyzed using a flow cytometer (LSRFortessa with FASCDiva software; Becton Dickinson, USA). Platelets were identified based on their characteristic light scatter as well as CD61 antibody binding. The activated platelets were calculated using the percentage of 10,000 CD61 platelets to exhibit Allophycocyanin-CD62P fluorescence.

Platelet aggregation

Fresh platelets were isolated from each group as described above and treated with adenosine diphosphate (ADP) for 15 min. Subsequently, platelet aggregation was detected using a single-channel aggregometer (Chrono-Log Corp, USA) and reported using the percent of aggregation at 3 min and normalized to a standard deflection, corresponding to light transmission through PPP.

Platelet spreading

As previously described [17], 96-well cell plates were precoated with fibrinogen at 4 °C overnight and then blocked with bovine serum albumin (BSA) (1%, room temperature) for an hour. Subsequently, fresh platelets were diluted using Ca2+-free modified Tyrode solution consisting of Mg2+ (2 mmol/L) and were spread on immobilized fibrinogen at 37 °C. 200 μL saline control, BYHWD-ld, BYHWD-hd and aspirin were correspondingly added to the wells of each group and incubated for 30 min. Following fixation with PFA (4%), adhesion was stopped and platelets were permeabilized with Labeling solution (0.2% Triton X-100, 0.5% BSA). The adherent platelets were stained with Calcein. Finally, the fluorescence intensity was captured at Ex/Em = 496/515 nm.

Oxygen consumption rate (OCR)

Fresh platelets from the sham, model, BYHWD-hd and positive groups were extracted by the method described above and used in this assay. PRP was diluted into 10^8 per well using PPP. Medium (80 µL) was added to wells B-G, and the same amount of culture medium was added to wells A and H as controls, followed by centrifugation (5 min, at 200g and 20 ℃) for cell adhesion. After 30 min, the culture medium was taken place by the same amount of assay medium. Substances, including Oligomycin (10 µM), carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) (0.5 µM), antimycin A along with rotenone (0.5 µM), were added into the kit pack to block the mitochondrial respiration chain complex. Quantification of OCR was performed using an extracellular flux analyzer (Seahorse, Agilent, USA).

Platelet RNA isolation and RNA sequencing

The platelets from the sham, model, and BYHWD-hd groups were extracted by the method described above and used in this assay. After the samples were treated with Trizol (Invitrogen, California, USA), total RNA was purified, followed by deoxyribonuclease. Quality assessment of the resulting RNA was conducted using a bioanalyzer assay. Based on the Ovation RNA Seq v.2 kits (NuGen, Inc., San Carlos, CA), total RNA (DNA-free) was adopted as input for library construction. Following the final step for amplification, libraries (250- 450 base pairs) were selected. After mixed, barcoded RNA-seq libraries were subjected to 75 base pair paired-end sequencing using an Illumina HiSeq-2000 machine (Illumina, San Diego, California, USA). Additionally, each sample has the equivalent of 1 dedicated flow cell. After demultiplexed, the sequencing data was converted to the format of FASTQ. Paired-end reads were aligned with RefSeq using TopHat2. RSeQC v2.3.9 (Boston, USA) was adopted for the calculation of RNA reads per kilobase per million mapped reads (RPKM). To ensure that the actual transcript levels obtained from the measurements, all the downstream analysis of the RNA-seq data was on the basis of a minimum RPKM threshold (0.3).

Quantitative real-time PCR (RT-qPCR) for total RNA

Platelets of sham, model, BYHWD-ld, and BYHWD-hd groups were extracted by the method described above and used in this experiment. On the basis of a PrimeScript RT Reagent Kit (Takara, Japan), reverse transcription of total RNA was conducted. RT-qPCR was conducted with the application of QuantStudio™ Test Development Software (Thermo Scientific, USA) as well as SYBR Green RT-qPCR Master Mix (EZBioscience, USA). Besides, data analysis was carried out using the 2 − ΔΔCT method. All primers were purchased from Sangon Biotech (Shanghai, China). Sequences of the primers are listed in Supplementary Material Table 1.

Telomere quantitative FISH (Q-FISH), immunofluorescence microscopy, and image acquisition

Telomerase activation alleviated interstitial fibrosis in the non-infarcted region of the LV, which significantly reduced the mortality from heart failure after MI [18]. Therefore, we conducted the corresponding tests to explore whether BYHWD alleviates the degree of telomere damage during myocardial infarction. After blocked with calf serum (4%)/Triton X-100 (0.1%)/phosphate buffer saline (PBS), tissues were stained with prediluted troponin-T (cTnT) antibody (ab47003, Abcam) for 2 h at room temperature overnight at 4 °C, then washed with PBS, followed by incubation with 1:400 goat anti-rabbit Alexa 488 (Abcam, Cambridge, UK) for 1 h, and counterstaining with 4',6-diamidino-2-phenylindole (DAPI, 1 μg/mL) in PBS for 5 min, washed with ddH2O, air-dried, and then mounted with ProLong Gold Antifade (Life Technologies, Pittsburgh, USA). In addition, the images were captured by an Olympus spinning-disk confocal microscope based on a PLAN APO 40× objective. The measurement of telomere Q-FISH was performed using TelC-Cy3 peptide nucleic acids (PNA) probe (F1002, PNA Bio, Daejeon, South Korea) [19]. A PNA signal that normalized to nuclear DAPI among telomere fluorescence units (TFUs) among cTnT + cardiomyocytes (CMs) were recognized as telomere signal intensity, and then captured using the ImageJ plugin Telometer. Subsequently, telomere levels were blindly scored within 5–6 interested regions for 2 non-consecutive sections. This study uses the method of Chang et al. [19] and the description of the methods partly reproduces their wording.

Protein extraction and western blot analysis

The whole protein of 50 mg platelet precipitation descried above was extracted using a protein extraction kit (Applygen Technologies, Beijing, China) complying with the instructions of the manufacturer, separated on SDS polyacrylamide gel electrophoresis (10%) and transferred to the membrane of polyvinylidene difluoride. Membranes were firstly blocked with 5% BSA dissolved in Tris buffered saline (TBS) with Tween for a hour, and then probed with appropriate antibodies at 1:1000 against phosphoinositide-3-kinase (PI3K) (ab154598, Abcam, Cambridge, UK), phospho-phosphoinositide-3-kinase (p-PI3K) (341468, ZEN BIO, Chengdu, China), AKT serine/threonine kinase (Akt) (ab179463, Abcam, Cambridge, UK), phospho-Akt serine/threonine kinase (p-Akt) (ab38449, Abcam, Cambridge, UK), Rap1(#2399, CST, Boston, USA), cell division cycle 42 (CDC42) (ab187643, Abcam, Cambridge, UK), nonreceptor tyrosine kinase (Src) (2019S, CST, Boston, USA), phospho-nonreceptor tyrosine kinase (p-Src) (6943S, CST, Boston, USA), beta-actin (ab6276, Abcam, Cambridge, UK) at 4 ℃ with gentle agitation overnight. After incubation with horseradish peroxidase-conjugated IgG (1:10,000) as secondary antibody for 1 h at room temperature, blots were detected using an enhanced chemiluminescence detection system.

Statistical analysis

Statistical analysis was conducted using GraphPad Prism 7 software (GraphPad software Inc., USA). All data were described with means ± standard deviation (SD). Comparisons for groups was performed using one-way ANOVA, followed by Bonferroni test. A P value less than 0.05 indicates statistical significance.