SYD was provided by Beijing TCM Hospital (Beijing, China, Z20053327), and thestock liquor of 20 mg/mL was prepared with 0.5% carboxymethyl cellulose sodium (CMC-Na) before the experiment. SYD consists of eight botanical drugs: Astragalus mongholicus Bunge [Fabaceae; Astragali radix] (60 g), Codonopsis pilosula (Franch.) Nannf. [Campanulaceae; Codonopsis radix] (60 g), Scrophularia ningpoensis Hemsl. [Scrophulariaceae; Scrophulariae radix] (60 g), Salvia miltiorrhiza Bunge [Lamiaceae; Salviae miltiorrhizae radix et rhizoma] (60 g), Corydalis yanhusuo (Y.H.Chou and Chun C. Hsu) W.T.Wang ex Z.Y.Su [Papaveraceae; Corydalis rhizoma] (40 g), Pheretima aspergillum (E. Perrier) [Megascolecidae; Pheretima] (40 g), Eupolyphaga sinensis Walker [Corydiidae; Eupolyphaga] (24 g), and Hirudo nipponica Whitman [Hirudinidae; Hirudo] (24 g). All botanical drugs were obtained from the Pharmacy of Beijing Hospital of Traditional Chinese Medicine (Beijing, China) and comply with the standards of the Pharmacopoeia of the People’s Republic of China (2020 Edition). The materials were authenticated by Professor Jiankun Wu (Pharmacy Department of Beijing TCM Hospital). Voucher specimens (No. Z20233327]) have been deposited in the Beijing Institute of Chinese Medicine.

To ensure chemical consistency, the SYD extract was characterized using ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC–MS/MS) on a Waters system with a HESI-II probe, Separation was performed on an Acquity UPLC HSST3 column (1.8 μm × 2.1 mm × 100 mm) at 45 °C, using a mobile phase of 0.1% formic acid in water (A) and 0.1% formic acid in methanol (B) with a flow rate of 0.3 mL/min. The HESI-II spray voltages were 3.7 kV (positive) and 3.5 kV (negative), with a vaporizer temperature of 320 °C. Data acquisition was processed via Waters Masslynx 4.1. The representative total ion chromatograms (TIC) and identified metabolites are provided in Supplementary Figure S1.

The botanical drugs were mixed and soaked in distilled water for 1 h, followed by boiling for 1 h and filtration. The residue was decocted again with distilled water for 30 min and filtered. The two extracts were combined, concentrated to 100 mL, and clarified. The supernatant was collected, pre-frozen at −80 °C, and freeze-dried using a vacuum freeze-dryer. The total weight of the starting botanical drugs was 368 g. After the extraction and lyophilization process, 68 g of freeze-dried powder was obtained. The final drug-extract ratio (DER) was 5.41:1, corresponding to a yield of 18.48% (w/w). The freeze-dried powder was stored in a desiccator at room temperature. Sacubitril/valsartan sodium tablet (SVST) (Lot #H20170363), was purchased from Novartis Singapore Pharmaceutical Manufacturing Private. Ltd. (Beijing, China).

Isoflurane (R510) was purchased from RWD Life Science Co., Ltd. (Shenzhen, China). PE (S2569) was purchased from Slleck (Houston, United States). Dulbecco’s modified Eagle’s medium (DMEM) (SH30243.01) was purchased from Hyclone (Utah, United States). Fetal bovine serum (10091-148), penicillin-streptomycin (15140122), 0.25% Trypsin-EDTA (25200056), BCA Protein Assay Kit (23225) and TRIzol (15596026CN) were purchased from Thermo fisher (Massachusetts, United States). The ELISA-kit of BNP(LV20905), β-MHC (LV20784), TGF-β1 (LV20903), ANP (LV20904) and NTpro-BNP (LV30938) were purchased from Animal union Biotechnology Co., Ltd (Shanghai, China). Cell Counting Kit-8 (CK04) was purchased from Dojindo North. (Beijing, China). Paraformaldehyde, 4% (P1110), Hematoxylin-Eosin (H&E) Stain Kit (G1120), Modified Masson’s Trichrome Stain Kit (G1346), Triton X100 (IT9100), RIPA buffer (R0010), Protein Phosphatase Inhibitor (P1206), Methylene Blue Solution, 0.2% (G1301), Glutaraldehyde (P1127) were purchased from Solarbio (Beijing, China). Alpha Actin Polyclonal antibody (23660-1-AP), CoraLite488-conjugated Goat Anti-Rabbit IgG (H + L) (SA00013-2), m6A Monoclonal antibody (68055-1-Ig), Goat serum (B900780), Prestained Protein marker (PL00001), Extra Range Prestained Protein Marker (PL00003), Beta Tubulin Polyclonal antibody (10094-1-AP), HRP-conjugated Affinipure Goat Anti-Mouse IgG (H + L) (SA00001-1), HRP-conjugated Affinipure Goat Anti-Rabbit IgG (H + L) (SA00001-2) were purchased from Proteintech (Wuhan, China). LC3A/B Rabbit mAb (12741), SQSTM1/p62 Rabbit mAb (23214), Beclin-1 Rabbit mAb (3495), Phospho-mtor (Ser2448)Rabbit mAb (5536), Phospho-TFEB (Ser211) (E9S8N) Rabbit mAb (37681)TFEB Rabbit mAb (32361), METTL3 Rabbit mAb (96391), GAPDH Rabbit mAb (97166) were purchased from Cell Signaling Technology (Massachusetts, United States).

Six-week-old male C57BL/6 mice (weighing 21–23 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). During the feeding period, mice were kept under a 12-h alternating light cycle (8:00-20:00), with room temperature maintained at approximately 23 °C and humidity kept between 30% and 40%. Free access to food and water was provided throughout the experiment. All mice were reared in the animal room for 2 weeks and then used for subsequent animal experiments. All the animal experiments in this study were approved by the Animal Ethics Committee of the Beijing Institute of Traditional Chinese Medicine (No. BJTCM-M-2024-09-04).

The left anterior descending coronary artery (LAD) ligation was performed to establish the MI-induced HF model following the methods in our previous study (Li et al., 2023). Briefly, anesthetized mice were attached to an animal ventilator, a left thoracotomy was conducted, and the LAD was occluded by ligating the left ventricle 1–1.5 mm from the left atrial appendage with 6/0 silk yarn. Mice in the Sham group were subjected to the same thoracotomy without ligating the LAD. During the operation, the white of left ventricular anterior wall can be used as an important indicator to evaluate the success of the operation. Finally, the ST segment elevation greater than 0.2 mV indicates the success of myocardial infarction model.

The mice were randomly allocated into six groups: sham, model, low-dose SYD (SYD-L), medium-dose SYD (SYD-M), high-dose SYD (SYD-H), and positive control (SVST).

After surgery, the mice on the SYD-L group, SYD-M group, and SYD-H group were treated with 0.3, 0.6, and 1.2 g/kg/d of SYD by gavage, respectively. The dosage of SYD-M is converted according to the 9.1 conversion factor according to the clinical recommend dose (4.2 g/day) of adult daily medication (Li et al., 2022). The mice on the SVST group were treated 60 mg/kg/d of SVST (Liu et al., 2021). The Sham and Model groups were administered the equal amounts of 5% CMC-Na. All mice received corresponding drug intervention for 6 weeks.

Embryonic rat cardiomyocyte cell line (H9c2 cells, obtained from the Cell Resource Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, China) were maintained at 37 °C in a humidified atmosphere containing 5% CO2. The culture medium consisted of DMEM, 10% fetal bovine serum, 100 μg/mL penicillin, and 100 μg/mL streptomycin. Lentiviral vectors were constructed by Beijing XBHCbio Co (Beijing, China) and used to overexpress METTL3 in H9c2 cells. The lentivirus for overexpressing METTL3 (OE-METTL3) and the negative control lentiviral vector were prepared and titrated to 5 × 10⁸ TU/mL.

The H9c2 cell hypertrophy model was constructed by inducing PE (50 μM) for 24 h (Chen et al., 2024). The cell hypertrophy model evaluates the success of the model by increasing the cell surface area.

Weigh 0.1 g of SYD freeze-dried powder and dissolve it in 10 mL of DMEM. Vortex thoroughly and filter the solution using a 0.22 μm filter to obtain a sterile stock solution of SYD freeze-dried powder. Prior to the experiment, dilute the stock solution to the desired concentration.

The experimental cell treatments were as follows: Control group (no intervention), Model group (induced with 50 μM PE for 24 h), SYD-L group (treated with 0.5 mg/mL SYD with 50 μM PE for 24 h), SYD-M group (treated with 1 mg/mL SYD with 50 μM PE for 24 h) and SYD-H group (treated with 2 mg/mL SYD with 50 μM PE for 24 h).

Blank group, Negative vector control group (VE-C) and Overexpression-METTL3 control group (OE-C) treated with no intervention; Negative vector model group (VE-M) and Overexpression-METTL3 model group (OE-M) treated with 50 μM PE for 24 h; Negative vector SYD group (VE-S) and Overexpression-METTL3 SYD group (OE-S) treated with 1 mg/mL SYD with 50 μM PE for 24 h.

On 6 weeks after surgery, the mice were anesthetized with 1.5% isoflurane and echocardiography was conducted with a Vevo 3100 Imaging System (Visual Sonics Inc, Canada). Briefly, an MI-mode echocardiogram was used to analyze cardiac function indicators, including left ventricular ejection fraction (EF), left ventricular fractional shortening (FS), left ventricular internal dimension at end-diastole (LVIDd), and left ventricular internal dimension at end-systole (LVIDs).

On 6 weeks after surgery, mouse hearts were removed and fixed in 4% paraformaldehyde for 24 h. The hearts were then dehydrated, embedded in paraffin, and sectioned for 5 μm. Masson’s trichrome staining was used to examine cardiac fibrosis. H&E staining was used to explore basic pathological changes. After staining, sections were observed and photographed under a digital pathology slide scanner (Aperio CS2, Leica, Germany).

The mouse serum or H9c2 cardiomyocyte culture supernatants were collected and used to measure protein levels of specific biomarkers using ELISA kits according to the manufacturer’s instructions. Briefly, samples and standards were added to 96-well plates pre-coated with capture antibodies. The plates were incubated at room temperature for 1–2 h, followed by washing to remove unbound components. Next, detection antibodies were added and incubated, followed by the addition of enzyme-linked secondary antibodies. After thorough washing, a substrate solution was applied to develop the color reaction. The reaction was stopped using a stop solution, and absorbance was measured at 450 nm using a microplate reader. The concentration of the target protein was calculated based on the standard curve.

Heart tissues or H9c2 cells were removed and immediately washed with PBS. After fixation with electron microscope solution for 3 h, fixation with PBS containing 1% osmic acid for 2 h, dehydration was carried out in gradient ethanol. Finally, it was embedded and sliced by an embedding agent, uranium-lead double dyeing, and observed by TEM.

Heart tissues or H9c2 cells were homogenized in radio-immunoprecipitation assay lysis buffer, and total protein concentrations were determined by using a bicinchoninic acid protein assay kit. Equal amounts of protein extracts were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were then transferred to methanol-preactivated polyvinylidene difluoride membranes. Membranes were then blocked with 5% skim milk for 1 h at room temperature and incubated with primary antibodies overnight at 4 °C. After washing with TBS-T, membranes were incubated with a horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Finally, proteins were visualized using an enhanced chemical luminescent system (Pierce Biotechnology, Inc., Rockford, United States), and bands were imaged using a chemiluminescence imaging system.

After the total RNA was extracted from the mice heart tissue or H9c2 cells, 2ul of total RNA was added to the NC membrane in a unified concentration. Crosslinking 1 h under 254 nm UV lamp. Overnight incubation using 0.1% m6A antibody following closure with TBST containing 5% skim milk powder. The next day, imaging was performed using chemiluminescence.

Cell viability was detected by using the CCK-8 assay kit. Briefly, H9c2 cells were seeded at a density of 1 × 10⁶ cells per well in a 96-well plate and incubated overnight to allow for adherence. Subsequently, H9c2 cells were treated with varying concentrations of PE (0, 25, 50, 100, and 200 μmol/L) and SYD (0, 1, 2, 3, 4, 5, and 6 mg/mL) for 24 h. After treatment, CCK-8 solution was added, and the H9c2 cells were incubated at 37 °C for 1 h. The absorbance at 450 nm was measured using a microplate reader, and cell viability was calculated accordingly.

Cells were fixed, permeabilized, and blocked, followed by incubation with an α-actin antibody overnight at 4 °C. The next day, cells were incubated with a fluorescently labeled secondary antibody for 1 h. A mounting medium containing DAPI was added to prevent fluorescence quenching. The cells were then examined using an inverted immunofluorescence microscope.

Total RNA was extracted from experimental cells using TRIzol reagent. The extracted RNA was then reverse transcribed into cDNA using the HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper). The mRNA levels of METTL3, mTOR, and TFEB were quantified using the SYBR Green I incorporation method. The reaction program was set as follows: Initial Denaturation: 95 °C for 10 min. Amplification Cycles (45 cycles): Denaturation: 95 °C for 15 s. Annealing: 60 °C for 60 s. Melting Curve Analysis: 95 °C for 15 s. Cooling to 60 °C for 1 min. Heating to 95 °C for 30 s. Fluorescence readings were recorded. GAPDH was used as the internal control. The relative mRNA expression levels were determined using the 2^−ΔΔCT method. Primer sequences are listed in Table 1.

The m6A MeRIP-Seq service was provided by CloudSeq Inc. (Shanghai, China). Total RNA was subjected to immunoprecipitation with the GenSeq® m6A MeRIP Kit (GenSeq Inc.) by following the manufacturer’s instructions. Briefly, RNA was randomly fragmented to ∼200 nt by RNA Fragmentation Reagents. Protein A/G beads were coupled to the m6A antibody by rotating at room temperature for 1 h. The RNA fragments were incubated with the bead-linked antibodies and rotated at 4 °C for 4 h. After incubation, the RNA/antibody complexes were washed for several times, and then, captured RNA was eluted from the complexes and purified. RNA libraries for IP and input samples were then constructed with GenSeq® Low Input Whole RNA Library Prep Kit (GenSeq, Inc.) by following the manufacturer’s instructions. Libraries were qualified using Agilent 2100 bioanalyzer (Agilent) and then sequenced.

Raw reads (Raw Data) are generated after sequencing on a sequencer, image analysis, base identification and QC. Q30 was first used for quality control. Then, cutadapt software (v1.9.3) was used to remove splice information and remove low quality reads to obtain high quality clean reads. The clean reads were matched to the reference genome using Hisat2 (v2.0.4). Methylated genes in each sample were then identified using MACS (1.4.2). Differential methylation gene identification was performed using diffReps (1.55.6). The peaks located mRNA were screened using our own programs and annotated accordingly.

Drug ingredients were obtained from TCMSP database and previous literature. Search for drug ingredient targets through HERB and SwissTargetPrediction databases. Relevant HF targets were collected through GeneCard, DISGENET and TTD databases. Finally, the “Botanical drug-Ingredient-Target network” was constructed by cytoscape 3.7.0. The core targets were imported into David database for KEGG and GO enrichment analysis.

Quantitative data are expressed as mean ± SEM. Data were statistically analyzed using Graphad Prism 9.5 software, and Ordinary one-way ANOVA was used for three or more groups, and then Tukey’s multiple comparison test was conducted. P<0.05 was considered statistically significant.

In vivo, we established a murine MI model through permanent left anterior descending (LAD) coronary artery ligation. Six-week pharmacological intervention revealed cardioprotective effects of SYD. Initial evaluation of cardiac remodeling indices demonstrated that MI mice exhibited significantly elevated heart weight-to-body weight (HW/BW), heart weight-to-lung weight (HW/LW), and heart weight-to-tibia length (HW/TL) ratios compared to sham controls (Figures 1A–D), indicative of pathological cardiac enlargement. These ratios were substantially normalized by medium/high-dose SYD and SVST treatment. Echocardiographic assessment revealed deteriorated systolic function in MI mice (Figure 1E), characterized by reduced ejection fraction (EF) and fractional shortening (FS), along with increased left ventricular internal diameter during systole (LVIDs). Remarkably, SYD medium/high doses and SVST significantly ameliorated these functional impairments (Figures 1H–K). Histopathological analysis via hematoxylin-eosin (HE) and Masson’s trichrome staining demonstrated extensive myocardial necrosis, interstitial edema, inflammatory infiltration, and collagen deposition in MI mice, which were mitigated by SYD and SVST treatments (Figures 1F,G). ELISA quantification of serum biomarkers associated with HF and remodeling including B-type natriuretic peptide (BNP), β-myosin heavy chain (β-MHC), and transforming growth factor-β1 (TGF-β1) revealed significant reductions in SYD-M, SYD-H, and SVST groups compared to MI controls (Figures 1L–O). Collectively, these findings substantiate SYD’s therapeutic potential in ameliorating post-MI cardiac remodeling and functional deterioration.

After confirming the anti-HF effects of SYD in MI mice, we screened the drug concentrations of PE (50 μM) and SYD (0.5/1/2 mg/mL) through CCK-8 assays to determine the intervention doses, which were used to induce hypertrophy in H9c2 cells and anti-hypertrophy (Figures 2A–C). Immunofluorescence staining of α-actin in H9c2 cells was performed to measure the cell surface area, which showed a significant reduction in cell size after SYD intervention (Figures 2D,E). ELISA quantification of hypertrophic biomarkers (β-MHC, BNP, NT-proBNP, ANP) in the culture supernatant indicated that SYD treatment decreased the levels of these markers (Figures 2F–I). Collectively, these results demonstrate that SYD can suppress PE-induced cardiomyocyte hypertrophy.

Having clarified the therapeutic effects of SYD on post-MI HF mice and hypertrophic cardiomyocytes, we proceeded to investigate its underlying mechanisms. Dot blot and immunoblot analyses demonstrated elevated myocardial m6A levels and METTL3 expression in post-MI HF mice, which were reversed by SYD treatment (Figures 3A–D). Consistently, SYD normalized PE-induced m6A hypermethylation and METTL3 upregulation in H9c2 cells (Figures 3E–H). Methylated RNA immunoprecipitation sequencing (MeRIP-seq) identified differential m6A peak distribution among control, PE-induced, and SYD-treated groups. Motif analysis confirmed conserved RRACH sequences (R = purine, A = m6A, H = non-guanine) across groups with intergroup variations (Figure 3I). Comparative analysis revealed 7,254 vs. 6,290 m6A peaks between control and model groups (4,947 shared), and 6,288 vs. 4,400 peaks between model and SYD groups (3,565 shared) (Figure 3J). Metaplot analysis showed distinct 5′UTR methylation patterns across groups (Figure 3K), with peak distribution differences in coding sequences (CDS) and start/stop codon regions (Figure 3L). These findings establish SYD-mediated regulation of differential m6A methylation landscapes.

To systematically explore the pharmacological basis of SYD in treating post-MI HF and identify potential targets regulated by m6A modification, we performed an integrative analysis of MeRIP-seq data and network pharmacology. Differential m6A methylation analysis identified 638 DMGs between the Model and Control groups, and 767 DMGs between the Model and SYD groups (Figure 4A), with 139 overlapping genes identified as potential key targets mediating the cardioprotective effects of SYD (Figure 4B). Functional enrichment analyses (GO and KEGG) for these 139 genes significantly highlighted biological processes related to autophagosome assembly, autophagy regulation, and the mTOR signaling pathway (Figures 4C,D). Concurrently, network pharmacology was employed to provide a global profile of SYD’s multi-target landscape. A total of 186 potential bioactive metabolites from SYD were identified (Table 2), yielding 634 component targets, which intersected with 1,309 disease-related targets to produce 206 potential therapeutic targets (Figure 4E). The “drug-metabolite-target” network and topological analysis further positioned central autophagy regulators, such as mTOR and SQSTM1 (p62), as core nodes (Figures 4F,G). Importantly, enrichment analyses of these network targets independently confirmed the prominence of cardiac contractility regulation and the mTOR/autophagy axis (Figure 4H), showing high consistency with the MeRIP-seq findings. Collectively, these dual bioinformatic perspectives converge on the regulation of autophagic homeostasis through the mTOR pathway, providing a robust rationale for the subsequent mechanistic validation of the METTL3/m6A -mTOR/TFEB axis.

Ultrastructural analysis via transmission electron microscopy (TEM) revealed SYD-mediated mitigation of myocardial hypertrophy and organelle swelling, accompanied by increased autolysosomes (Figure 5A). Immunoblotting confirmed SYD dose-dependently upregulated autophagy markers (LC3-II/I ratio, Beclin-1) while reducing p62 accumulation (Figures 5B–E). Concurrently, SYD suppressed mTOR phosphorylation and enhanced TFEB expression (Figures 5F–I), indicating activation of autophagy-lysosomal pathways.

Contrasting with in vivo findings, PE-induced autophagic hyperactivity in H9c2 cells was counteracted by SYD treatment, as evidenced by TEM (Figure 6A) and immunoblot analysis of autophagy markers (Figures 6B–E). Subsequently, we examined the expression levels of the mTOR/TFEB pathway and found that SYD intervention enhanced mTOR phosphorylation and reduced TFEB protein expression in hypertrophic H9c2 cells (Figures 6F–I). Interestingly, the autophagy levels exhibited opposing trends in vivo and in vitro, which we believe reflects disrupted autophagic homeostasis and highlights the bidirectional nature of autophagy.

To verify whether METTL3-mediated m6A modification affects the mTOR/TFEB autophagy pathway, we examined the MeRIP-seq results and found that the m6A methylation profiles of mTOR and TFEB were altered across different groups (Table 3). To further validate this mechanism, we constructed a lentiviral overexpression vector, FV035-rMETTL3-3FLAG-OE, to stably upregulate METTL3 expression in H9c2 cells. The integrity of the recombinant plasmid was confirmed by DNA sequencing and agarose gel electrophoresis, which showed a distinct band at approximately 9.9 kb. Following lentiviral packaging and transduction (titer: 5 × 10⁸ TU/mL), the successful upregulation of METTL3 was confirmed by RT-qPCR analysis (Figures 7A–C). Functional assays revealed that METTL3 overexpression exacerbated PE-induced cardiomyocyte hypertrophy, as evidenced by the significant elevation of hypertrophic markers, including β-MHC, BNP, NT-proBNP, and ANP (Figures 7D–G). Conversely, SYD treatment markedly attenuated these hypertrophic responses in both the vector-control and METTL3-overexpressing cells (Figures 7D–G). Furthermore, RT-qPCR analysis demonstrated that SYD reversed the METTL3-driven transcriptional suppression of mTOR and TFEB (Figures 7H,I). These findings establish METTL3 as a critical mediator of SYD’s effects on the mTOR/TFEB autophagy axis, delineating a novel epigenetic mechanism through which SYD modulates autophagic homeostasis in an m6A-dependent manner.