TQ (purity ≥99.59%), 3-Methyladenine (3-MA; purity ≥99.91%) and GW9662 (PPAR-γ antagonist; purity ≥99.87%) were sourced from MedChemExpress, while pAD/14-3-3γ-short hairpin (sh)RNA was procured from Cyagen Biosciences, Inc. AngII was acquired from GLPBIO Technology LLC. PPAR-γ (cat. no. YT3836) antibody was supplied by ImmunoWay Biotechnology Company. Primary antibodies for LC3 (cat. no. 381544), p62 (cat. no. 380612), 14-3-3γ (cat. no. R381405) and secondary antibodies (goat anti-mouse, cat. no. 511103; goat anti-rabbit, cat. no. 511203) were purchased from Chengdu Zen-Bioscience Co., Ltd. Proteintech Group, Inc. provided antibodies against collagen I (cat. no. 14695-1-AP), atrial natriuretic peptide (ANP; cat. no. 27426-1-AP) and β-actin (cat. no. 66009-1-Ig). ABclonal, Inc. provided antibodies against brain natriuretic peptide (BNP; cat. no. A2179).

The rat H9C2 cell line, obtained from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences, was maintained in H-DMEM (HyClone; Cytiva), supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin-streptomycin (Gibco; Thermo Fisher Scientific, Inc.). The H9C2 cells were incubated at 37°C in a controlled environment (95% humidity, 21% O₂, and 5% CO₂) (16). The H9C2 cells were allocated into six distinct experimental groups: i) Control group, H9C2 cells were maintained in H-DMEM for 48 h under standard conditions; ii) AngII group: H9C2 cells were cultured in H-DMEM for 24 h, followed by exposure to 1 µM AngII for an additional 24 h (17); iii) AngII + TQ group: Cells were pretreated with 10 µM TQ for 24 h before subsequent exposure to 1 µM AngII for another 24 h; iv) AngII + TQ + 3-MA group: Cells were incubated with 10 µM TQ and 5 mM 3-MA (18) for 24 h, followed by 1 µM AngII treatment for 24 h; v) AngII + TQ + GW9662 group: Cells were pretreated with 10 µM TQ and 10 µM GW9662 (19) for 24 h, then exposed to 1 µM AngII for an additional 24 h; and vi) AngII + TQ + pAD/14-3-3γ-shRNA group: H9C2 cardiomyocytes were transfected with pAd/14-3-3γ-shRNA at 37°C for 48 h, incubated with 10 µM TQ for 24 h and subsequently treated with 1 µM AngII for another 24 h.

H9C2 cardiomyocytes were pre-cultured in fresh H-DMEM supplemented with 10% FBS and then transfected with either pAd/14-3-3γ-shRNA or pAd/negative control-shRNA (Cyagen Biosciences, Inc.; concentration not measured) using HighGene plus Transfection reagent (cat. no. RM09014; ABclonal Biotech Co., Ltd.). The transfection efficiency was ~85% after 48 h at 37°C, when the subsequent experiments were carried out. The shRNA sequences are provided in Table I.

Cell viability was determined via the Cell Counting Kit-8 (CCK-8; GLPBIO Technology LLC). H9C2 cells were cultured in 96-well plates at a density of 2×10⁴ cells/well and exposed to TQ at concentrations of 1, 5, 10 and 20 µM for 24 h. Subsequently, 10 µl CCK-8 reagent was introduced to each well for 1 h at 37°C, and the absorbance at 450 nm was recorded using a microplate reader (Thermo Fisher Scientific, Inc.).

ROS production levels were assessed via staining with dichlorofluorescein diacetate (DCFH-DA; Beyotime Institute of Biotechnology). H9C2 cells were exposed to 10 µM DCFH-DA for 20 min at 37°C in the dark, followed by three washes with H-DMEM lacking 10% FBS. Intracellular ROS was subsequently visualized using a fluorescence microscope (Olympus Corporation). Lyso-Tracker Red (Beyotime Institute of Biotechnology), a lysosomal red fluorescent probe, enables lysosome-specific staining in live cells by penetrating cell membranes. H9C2 cells were incubated with the Lyso-Tracker Red working solution for 30 min at 37°C in the dark, and fluorescence microscopy (Olympus Corporation) was employed for observation.

Cells were harvested and fixed in 2% glutaraldehyde at 25°C for 2 h, followed by sequential washing with PBS, dehydration, embedding, sectioning and staining. The sections were 60-80 nm thick and were embedded at 37°C for 5-8 h using EPON 812. Staining was performed using 2% uranyl acetate and 2.6% lead citrate at 37°C for 8 min. Autophagosome structures in H9C2 cells were analyzed via TEM (Hitachi 7800; Hitachi, Ltd.).

Total proteins were isolated from H9C2 cells and mouse myocardial tissue using RIPA lysis buffer (Beijing Solarbio Science & Technology Co., Ltd.). Protein concentrations were determined via the BCA Assay Kit (GLPBIO Technology LLC). Equivalent amounts of protein (30-40 µg) were resolved via 10-12.5% gradient SDS-PAGE and subsequently transferred to polyvinylidene fluoride membranes (Pall Corporation). Membranes were blocked with 5% skimmed milk at room temperature for 2 h, followed by overnight incubation at 4°C with primary antibodies against LC3 (1:1,000), p62 (1:1,000), 14-3-3γ (1:1,000), β-actin (1:1,000), collagen I (1:500) and PPAR-γ (1:500) on a shaker. After washing three times using TBST (0.05% Tween 20), the membranes were incubated with HRP-conjugated secondary antibodies (1:5,000) for 2 h at room temperature. β-actin served as a loading control. Finally, protein bands were visualized using the Ultra High Sensitivity ECL kit (cat. no. GK10008; GLPBIO Technology LLC) and protein band densities were semi-quantified using ImageJ software (National Institutes of Health; v1.8.0.345).

RT-qPCR analysis was performed to quantify the mRNA expression levels of ANP, BNP, NADPH oxidase 4 (NOX4), superoxide dismutase 2 (SOD2) and collagen I. Total RNA was isolated with TRIzol reagent (Beijing Solarbio Science & Technology Co., Ltd.), followed by RT using the PrimeScript RT reagent kit (Monad Biotech Co., Ltd.) according to the manufacturer's instructions. qPCR was performed using the SYBR Green qPCR Master Mix (cat. no. B21203; Selleck Chemicals) on the BIO-RAD CFX Connect Real-Time PCR Detection System from Bio-Rad Laboratories, Inc. The qPCR protocol comprised an initial denaturation step at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 15 sec and annealing plus extension at 60°C for 1 min. β-actin served as the internal reference and the relative mRNA expression levels of the target genes were calculated employing the 2^−ΔΔCq method (20). The specific primers for each gene are detailed in Table II.

LC3 expression was evaluated using immunofluorescence staining. After washing the cell samples with PBS fixation was carried out with 4% paraformaldehyde (Biosharp Life Sciences) at room temperature for 10 min, then permeabilization for 10 min at room temperature using 0.2% Triton X-100 (Beijing Solarbio Science & Technology Co., Ltd.), followed by blocking with 2% BSA (cat. no. 9048-46-8; Shanghai Yuanye Biotechnology Co., Ltd.) at room temperature for 30 min. The cells were subsequently incubated overnight at 4°C with rabbit anti-LC3 primary antibody (1:500). Following three washes with TBST (0.05% Tween 20), fluorescent secondary goat anti-rabbit antibody (1:200; cat.no. A32732; Thermo Fisher Scientific, Inc.) was applied for 1 h at room temperature. DAPI counterstaining was performed at room temperature for 5 min, and fluorescence microscopy was used for observation.

Luciferase activity was assessed using a dual-luciferase reporter assay system (Promega Corporation; cat. no. E1910 Transfection of pcDNA3.1-PPAR-γ and psiCheck2-14-3-3γ (Hunan Fenghui Biotechnology Co., Ltd.) constructs into cells was carried out with LipoFiter (cat. no. HB-LF-1000; Hanbio Biotechnology Co., Ltd.) for 6 h at 37°C. Subsequently, 100 µl each of firefly and Renilla luciferase detection reagents were applied, followed by the measurement of relative light units. The luciferase activity ratio between firefly and Renilla was then calculated.

The three-dimensional structures of TQ and PPAR-γ were obtained from PubChem (Compound CID: 10281) and the Protein Data Bank (PDB) (PDB ID: 3PRG), respectively. The CB-Dock2 (https://cadd.labshare.cn/cb-dock2/php/index.php) server was utilized to carry out molecular docking.

The experimental procedures followed the guidelines established by the National Institutes of Health and were approved by the Animal Experimentation Ethics Committee of The First Affiliated Hospital, Jiangxi Medical College, Nanchang University (Nanchang, China; approval no. CDYFY-IACUC-202407QR115). Male C57BL/6 mice (22-24 g, 8 weeks old) were sourced from Changzhou Cavens Laboratory Animal Ltd. The mice were maintained in controlled environmental conditions, with a temperature of 23±1°C, a humidity of 40-50%, a 12-h light/dark cycle to simulate day and night, and ad libitum access to food and water to ensure their well-being and experimental accuracy.

The mice were randomly allocated into four groups: i) Sham operation, ii) TAC operation, iii) TAC operation with TQ treatment, and iv) TAC operation with TQ treatment alongside PPAR-γ antagonist (GW9662). Each group comprised 6 mice. The mouse model of pressure overload-induced cardiac hypertrophy was generated through TAC surgery, following the protocol outlined in a previous study (21). For the operation, animals were anesthetized in a chamber with isoflurane (induced with 2% and maintained with 1.5%), supplemented during surgery with 0.1 mg/kg buprenorphine HCl via subcutaneous injection. Post-surgery, the health and behavior of the mice were monitored twice daily. In the TQ treatment group, TQ (50 mg/kg, dissolved in corn oil) was administered via gavage for 6 weeks post-TAC (22). The PPAR-γ antagonist group received intraperitoneal injections of GW9662 (1 mg/kg) after TAC surgery, administered every 3 days for 6 weeks.

The animal was euthanized if predefined humane endpoints were met, including: i) Rapid weight loss of 15-20% of the original body weight (BW); ii) the inability to eat, drink or stand for up to 24 h; and iii) depression and hypothermia (<37°C) without anesthesia or sedation. No mice exhibited signs of reaching these endpoints during the experiment. Euthanasia was induced by an overdose of pentobarbital sodium (200 mg/kg). Animals were observed for 5-10 min post-injection to confirm death, assessed by respiratory and cardiac arrest, and loss of corneal reflex.

Mouse heart weight (HW) and BW were recorded. Heart tissue samples were preserved in formalin for 12 h at room temperature and embedded in paraffin following established protocols (23). Paraffin-embedded specimens were sectioned at 5 µm thickness and subjected to hematoxylin and eosin staining for 5 min at room temperature as well as Masson's trichrome staining for 5 min at room temperature, using standard procedures (24,25). The myocardial cell size was observed from ventricular slices stained with fluorescein isothiocyanate-labeled wheat germ agglutinin (WGA) at 37°C in the dark for 2 h. Finally, the sections were examined under an inverted fluorescence microscope (Nikon Eclipse Ti-SR), and images were subsequently captured.

After anesthetizing the mice with 1.5% isoflurane, echocardiography was performed. Cardiac function was assessed using a small animal ultrasound imaging system (VEVO2100; FUJIFILM VisualSonics, Inc.; FUJIFILM Sonosite, Inc.) featuring a 30-MHz probe.

Data analysis was conducted using GraphPad Prism 9.0 software (Dotmatics). For two-group comparisons, unpaired Student's t-test was applied, while one-way ANOVA followed by the Tukey's post hoc test was employed for multiple group comparisons. P<0.05 was considered to indicate a statistically significant difference.

H9C2 cell viability under varying concentrations of TQ (0, 1, 5, 10 and 20 µM) and AngII (1 µM) was evaluated using the CCK-8 assay. Treatment with TQ at different concentrations did not significantly alter cell viability compared with the control (0 µM) group (Fig. 1A). By contrast, AngII (1 µM) significantly reduced cell viability, an effect that was attenuated by TQ pretreatment (Fig. 1B). As the 10 µM concentration demonstrated the most notable protective effect, it was selected for subsequent experiments. The chemical structure of TQ is illustrated in Fig. 1C and D.

To assess the potential protective effects of TQ against AngII-induced hypertrophy in H9C2 cells, cells were pretreated with AngII (1 µM) for 24 h to induce hypertrophy, followed by evaluation of the cell surface area through WGA immunostaining. Pretreatment with TQ significantly decreased the cell surface area compared with the AngII group (Fig. 2A). In addition, analysis of the protein and mRNA expression levels of key hypertrophic markers, ANP and BNP, indicated that TQ significantly downregulated the expression of these markers (Fig. 2B-D). These results collectively indicated that TQ mitigated the hypertrophic response triggered by AngII in H9C2 cells.

To assess the cardioprotective effects of TQ on cardiac hypertrophy, a pressure overload hypertrophy mouse model was induced via TAC surgery. Cardiac function was analyzed through echocardiography (Fig. 3A). Mice in the TAC group exhibited a decline in left ventricular ejection fraction and fractional shortening, alongside an increase in left ventricular internal dimension diastole and posterior wall dimension, compared with the sham group (Fig. 3B-E). However, post-TAC administration of TQ improved cardiac function and attenuated hypertrophy in mice (Fig. 3A-E). Furthermore, pretreatment with TQ alleviated TAC-induced hypertrophy, as indicated by a reduction in cardiac size (Fig. 3G). Additionally, a marked increase in heart size and myofibril degeneration was observed in the TAC group, while the TQ-treated group exhibited reduced heart size and improved myofibril integrity, as shown by hematoxylin and eosin staining (Fig. 3H). The HW/BW ratio was significantly lower in the TAC + TQ group compared with the TAC group (Fig. 3F). Additionally, the protein and mRNA expression levels of ANP and BNP were notably down-regulated in the TAC + TQ group relative to the TAC group (Fig. 3I-L). These findings indicated that TQ attenuated pressure overload-induced cardiac hypertrophy.

The inter-relation between cardiac fibrosis and hypertrophy has been well-established, with fibrosis progression impairing myocardial contractility, ultimately contributing to heart failure and potential mortality (26,27). Masson staining, immunoblotting and RT-qPCR were employed to assess the effects of TQ on cardiac fibrosis. Masson staining showed a marked increase in fibrosis in mice subjected to TAC surgery, which was markedly attenuated by TQ treatment (Fig. 4A). The protein and mRNA levels of type I collagen were significantly upregulated in the TAC group compared with the control but were reduced following TQ administration (Fig. 4B-D). Similarly, in vitro experiments using H9C2 cells revealed elevated expression of type I collagen in the AngII group, which was mitigated by TQ, aligning with the in vivo findings (Fig. 4E-G).

Excessive generation of ROS is recognized as a key mechanism contributing to the progression of cardiac hypertrophy (5). Elevated ROS accumulation in cardiomyocytes has been shown to aggravate cardiac hypertrophy and myocardial fibrosis, ultimately leading to heart failure (28). To explore the potential antioxidative effect of TQ in cardiac hypertrophy, ROS levels were measured in treated cells. A notable increase in ROS levels were observed in the AngII group, while TQ treatment markedly reduced ROS levels (Fig. 5A). Additionally, RT-qPCR and western blot analysis indicated that TQ significantly downregulated the mRNA and protein expression of the oxidation gene, NOX4, and significantly upregulated the mRNA and protein expression of antioxidation gene, SOD2, in both the TAC-induced and AngII-induced groups (Fig. 5B-G).

Autophagy plays a central role in the pathogenesis of cardiac hypertrophy, with its dysregulation contributing to the worsening of the condition (29,30). To evaluate the effect of TQ pretreatment on adaptive autophagy in H9C2 cells, the expression levels of autophagy markers, LC3II and p62, as well as lysosome counts, were analyzed. As shown in Fig. 6A-C, TQ + AngII treatment significantly increased LC3II expression while reducing p62 levels compared with AngII alone. This modulation was attenuated by the autophagy inhibitor, 3-MA. These results suggested that autophagy was downregulated during the onset of hypertrophy in cardiac cells and that TQ pretreatment restored autophagic activity under these conditions. Furthermore, LysoTracker Red staining revealed a reduction in lysosome numbers following AngII-induced treatment of H9C2 cells, which was reversed upon TQ administration. Notably, the addition of 3-MA markedly reduced fluorescence intensity in H9C2 cells (Fig. 6D). Immunofluorescence analysis indicated an elevation in LC3 expression following TQ pretreatment, which was markedly reversed by 3-MA (Fig. 6E). Thus, we found that TQ activated adaptive autophagy in H9C2 hypertrophy cardiomyocyte.

The 14-3-3 proteins, a group of highly conserved acidic proteins found in all eukaryotic cells, comprise seven isoforms (31). Research has established their role in regulating autophagy and their protective function in cardiomyocytes (16,32). To investigate whether TQ conferred protection via autophagy regulation mediated by 14-3-3γ, the expression of 14-3-3γ was knocked down in H9C2 cells, the success of which was verified by western blot analysis (Fig. S1). As shown in Fig. 7A and B, TQ pretreatment led to a significant upregulation of 14-3-3γ expression, an effect that was reversed by pAD/14-3-3γ shRNA. In addition, knockdown of 14-3-3γ significantly influenced autophagic activity (Fig. 7A, C and D). Specifically, pAD/14-3-3γ shRNA reduced LC3II expression while simultaneously increasing p62 expression compared with the TQ group. TEM further confirmed a reduction in autophagic vesicles and therefore a suppression of autophagy in H9C2 cells treated with pAD/14-3-3γ shRNA (Fig. 7E). Additionally, pAD/14-3-3γ shRNA upregulated the mRNA and protein expression levels of ANP and BNP compared with the AngII + TQ group (Fig. 7F-H). These experiments confirmed that TQ alleviated cardiac hypertrophy by upregulating 14-3-3γ.

PPAR-γ, a protective regulator, is essential for maintaining cardiac homeostasis and preventing heart failure (33-35). We hypothesized that TQ could specifically target PPAR-γ and activate adaptive autophagy, thereby alleviating cardiac hypertrophy. To test this conjecture, the protein expression level of PPAR-γ was detected using western blot analysis. The results demonstrated that PPAR-γ expression in hypertrophic cardiac tissue revealed was significantly downregulated compared with the control group. However, TQ treatment effectively restored PPAR-γ levels. Notably, the co-administration of a PPAR-γ inhibitor (GW9662) reversed the TQ-mediated upregulation of PPAR-γ (Fig. 8A-D). Molecular docking techniques were employed to explore the regulatory interaction between TQ and PPAR-γ, simulating their binding. This method is widely used to evaluate compound interactions and activity (36). The analysis demonstrated that TQ formed hydrogen bonds with several key sites on PPAR-γ (Fig. 8F), suggesting that PPAR-γ modulation may be a potential mechanism of TQ's effects. Furthermore, similar results in AngII-pretreated H9C2 cells as those obtained from in vivo experiments were observed. The results indicated that the autophagy levels were suppressed as LC3II expression decreased and p62 expression increased in the AngII + TQ + GW9662 group treated compared with the AngII + TQ group (Fig. 8A-D). TEM further confirmed the reduction of autophagic vesicles in the PPAR-γ inhibitor-treated group, consistent with the western blot results (Fig. 8E). Additionally, PPAR-γ inhibition reversed the protective effects of TQ on cardiac hypertrophy, resulting in an enlarged cardiomyocyte surface area (Fig. 8G) and elevated the protein and mRNA expression of ANP and BNP (Fig. 8H-J).

Inhibition of 14-3-3γ expression was observed upon treatment with the PPAR-γ inhibitor, GW9662. The suppression of PPAR-γ resulted in a corresponding reduction in 14-3-3γ levels (Fig. 9A and C). Similarly, knockdown of 14-3-3γ via pAD/14-3-3γ shRNA also diminished PPAR-γ expression (Fig. 9B and D). Moreover, the dual-luciferase assay demonstrated that PPAR-γ upregulated 14-3-3γ promoter activity (Fig. 9E). These results suggest that TQ mitigates cardiac hypertrophy by promoting adaptive autophagy through the PPAR-γ/14-3-3γ signaling axis (Fig. S2).