Fifteen-week-old male SHRs (n = 16, weighing 280–320 g) and age/sex-matched Wistar-Kyoto (WKY) rats (n = 8, weighing 290–330 g) were obtained from Vital River Laboratory Animal Technology (Certificate: SCXK (Jing) 2016-0006, Beijing, China). The zoological study was performed in the Animal Experimental Center of the Affiliated Hospital of Shandong University of Traditional Chinese Medicine (Shandong, China) at controlled temperature (21 ± 1°C). The study was approved by the Animal Ethics Committee of the Affiliated Hospital of Shandong University of Traditional Chinese Medicine (Jinan, China), and the ethics approval number is AWE-2019-021.

After acclimatization for 1 week, 16 SHRs were randomly divided into the SHR (SHR, n = 8) and quercetin (Que, n = 8) groups; eight WKY rats were used as the normal control group (WKY, n = 8). According to a research and our previous study, intragastrical administration of quercetin at a dose of 20 mg/kg/d for 8–12 weeks prevented cardiac hypertrophy in established cardiac hypertrophic rat model and alleviated atherosclerotic lesion in ApoE^-/- mice, respectively (Chen et al., 2018; Jiang et al., 2020). Therefore, in this study, Rats in the quercetin group were administered 20 mg/kg/d quercetin (Meilun Biotechnology, Dalian, China) intragastrically, while the other two groups received the same volume of saline intragastrically for 8 weeks.

Systolic blood pressure (SBP) and diastolic blood pressure (DBP) were measured by the tail-cuff method with Mouse and Rat Tail Cuff Blood Pressure Systems (MRBP-10, IITC Life Science, United States) before and after treatment every 2 weeks. Rats were placed on a heated platform (35°C) for 10 min to keep them warm before measurement. The measurements were repeated three times for each rat at each time point, with the mean value recorded.

Eight weeks after treatment, rats were anesthetized and their chests were shaved with a depilatory agent. Transthoracic echocardiography was performed using a Veterinary Ultrasonic system Ultrasound Scanner (M5 Vet, Mindray, Guangdong, China). Left ventricular structure and function were assessed by measuring left ventricular end-diastolic internal diameter (LVIDd), left ventricular end-diastolic posterior wall thickness (LVPWd), end-diastolic interventricular septal thickness (IVSd), left ventricular end-systolic internal diameter (LVIDs), left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) via two-dimensional and M-mode echocardiography.

After echocardiography, the heart was quickly extracted. The heart and body weights of each rat were recorded. Heart tissues were collected, fixed with 4% formaldehyde for 48 h at room temperature, embedded in paraffin, and sectioned at 5 μm. Sections were stained with hematoxylin and eosin (H&E) and Masson’s trichrome using standard protocols to assess general histology and interstitial fibrosis. In order to determine the cardiomyocyte cross-sectional area, heart sections were deparaffinized, rehydrated, and processed for antigen retrieval with EDTA antigen retrieval solution (Servicebio, G1206, Wuhan, China). Afer washes, sections were incubated with diluted wheat germ agglutinin (WGA) solution (Sigma-Aldrich, L4895, United States) at 37°C in the dark for 30 min, and then incubated with DAPI (Servicebio, G1012, Wuhan, China) at room temperature for 10 min. Following spontaneous fluorescence quenching, slides were mounted with anti-fade mounting medium. Images were captured using fluorescent microscope (Nikon, ECLIPSE C1, Japan). Four images were taken from four different rats in each group. The quantification of collagen volume fraction and cardiomyocyte cross-sectional area were analysed using Image-Pro Plus 6.0 software (Media Cybernetics, United States).

The mitochondrial ultrastructure was observed by TEM. Fresh cardiac tissues were cut into 1–3 mm blocks, fixed with 2% glutaraldehyde at 4°C overnight. After dehydration, resin penetration and embedding, the ultrathin sections were stained with 2% uranium acetate-saturated alcohol solution. Ultrastructural images were captured with a TEM (JEOL-1200, Japan).

Mitochondrial superoxide production was measured by MitoSOX™ Red mitochondrial superoxide indicator (Thermo Fisher (China), M36008, Shanghai, China) according to the manufacturer’s instructions. Freshly prepared frozen heart sections were incubated with 5 μM MitoSOX™ reagent working solution for 10 min at 37°C protected from light. Images were captured using fluorescence microscope (Nikon, Japan) with excitation and emission at 514 and 585 nm, respectively. Four images were taken from four different rats in each group. ImageJ (National Institutes of Health, Bethesda, Maryland, United States) was employed to analyze the relative levels of mitochondrial superoxide in the heart.

Blood samples were obtained from the inferior vena cava, and serum was obtained by centrifugation. Serum T-SOD, MDA and GSH-PX amounts were detected with the T-SOD assay (Jiancheng Biological Engineering Institute, A001-1, Nanjing, China), MDA assay (Jiancheng Biological Engineering Institute, A003-1) and GSH-PX assay (Jiancheng Biological Engineering Institute, A005-1) kits, respectively, according to the manufacturer’s instructions.

Immortalized rat myoblast H9c2 cells were purchased from Procell Life Science and Technology (Wuhan, China). H9c2 cells were cultured in DMEM (Procell) supplemented with 10% fetal bovine serum (Procell) and 1% penicillin/streptomycin (Procell) at 37°C in a humidified environment with 5% CO₂. H9c2 cells cultured in DMEM were treated with 0.5, 1, and 2 μM quercetin, respectively. One hour after quercetin treatment, cells were treated with the hypertrophic agonist angiotensin II (Ang II; Solarbio, Beijing, China) at 0.1 μM. All cells were incubated for an additional 24 h before analysis.

Viability of H9c2 was determined by MTT assay. 20 μl MTT (5 mg/ml) was added to cultured cells in 96-well plate to incubated at 37°C for 4 h. Medium was replaced with 150 µl DMSO to dissolve the formazan crystals. Subsequently, the absorbance was measured using microplate reader (Thermo Scientific, Multiskan GO, United States) with wave length at 562 nm.

A small interfering RNA targeting SIRT3 (siRNA-SIRT3) and siRNA-NC were designed and synthesized by GenePharma (Shanghai, China). H9c2 cells were cultured in six-well plates (3×10⁵ cells/well) with Opti-MEM (Thermo Fisher [China], Shanghai, China) overnight before transfection to ensure 80% cell confluence. Diluted Lipofectamine 3,000 (Thermo Fisher [China], Shanghai, China) and diluted siRNA were mixed, and incubated at room temperature for 20 min before adding to the corresponding plates. H9c2 cells were cultured at 37°C for 48 h, followed by western blot detection of transfection efficiency.

Mitochondrial membrane potential was detected using a fluorescence tetraethylbenzimidazolylcarbocyanine iodide (JC-1) assay kit (Beyotime, C2006, Shanghai, China). Collected H9c2 cells were incubated with JC-1 staining mixture at 37°C for 20 min and washed with JC-1 staining buffer twice. ΔΨm was analyzed on a flow cytometer (BD Accuri C6 Plus, United States) with excitation and emission at 485 and 590 nm, respectively. The aggregate/monomer (red/green fluorescence) ratio was used to measure mitochondrial membrane depolarization.

Intracellular ATP levels were determined with an intracellular ATP assay kit (Beyotime, S0026, Shanghai, China) following the manufacturer’s instructions. H9c2 cells were lysed and centrifuged at 4°C for 10 min, and the resulting supernatant was used for further analysis. ATP detection reagent was added to each sample, and luminescence (RLU) was subsequently measured on a microplate reader (Bio-TEK, Synergy HTX, Vermont, United States). Finally, ATP levels were calculated according to a standard curve.

Total RNA was extracted from cultured H9c2 cells with TRIzol reagent (Invitrogen, Carlsbad, CA, United States) following the manufacturer’s protocol. Then, cDNA was synthesized and amplified by PCR with a Transcriptor Reverse Transcriptase kit (Roche, Basel, Switzerland). The mRNA levels of target genes were measured with ChamQ SYBR qPCR Master Mix (Vazyme, Nanjing, China) on the LightCyler 480 system (Roche, Basel, Switzerland). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression was used for normalization. The primers are listed in Supplementary Table S1.

The concentration of total protein extracted from heart tissues and H9c2 cells were assessed with the BCA Protein assay kit (Cwbio, Jiangsu, China) and adjusted to 20 µg/µl. Then, 30 µg total protein per samples was separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred onto PVDF membranes. After blocking, the PVDF membranes were incubated with primary antibodies (Anti-Collagen I Rabbit pAb, GB11022, 1:1,000, Servicebio, Wuhan, China; Anti-alpha smooth muscle Actin Rabbit pAb, GB11044, 1:1,000, Servicebio, Wuhan, China; Anti-SIRT3 Rabbit pAb, GB11354, 1:1,000, Servicebio, Wuhan, China; PARP1 Rabbit pAb, A0942, 1:1,000, Abclonal, Wuhan, China; Anti-PAR mouse pAb, ab14459, 1:1,000, Abcam, Cambridge, United Kingdom) at 4°C overnight. After a washing step, the blots were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG or goat anti-mouse IgG (1:5,000) at room temperature for 2 h. Finally, immunoreactive bands were quantified by ImageJ (National Institutes of Health, Bethesda, Maryland, United States). GAPDH expression was used for normalization. Western blot was performed in three biological replicates.

The level of NAD⁺ in H9c2 cells was determined using NAD⁺/NADH assay kit (Beyotime, S0175, Shanghai, China) with WST-8 according to the manufacturer’s instructions. In brief, 1 × 10⁶ cells were collected and lysed with 200 ul cold lysis buffffer to obtain sample. Afer reagent preparation, alcohol dehydrogenase working solution was added to 20 ul sample, and the suspension was incubated at 37°C for 10 min to obtain total NAD and NADH, respectively. Based on the relationship between NADH and the absorbance value measured at 450 nm, a standard curve was generated. Subsequently, the total NAD and NADH concentration were estimated according to the standard curve, and NAD⁺ concentration was calculated by subtracting NADH from total NAD.

Statistical analysis was performed using SPSS 26.0. Data are mean ± standard deviation (SD). Statistical differences among groups were determined by one-way analysis of variance (ANOVA) followed by least significant difference-t test (LSD-t). Differences were considered statistically significant at p < 0.05.

As shown in Figures 1A,B, SHRs had significantly higher SBP and DBP at each time point compared with WKY rats, and SBP and DBP levels were homogenous among SHRs before treatment. As expected, from the second week of treatment, both SBP and DBP in SHRs were significantly decreased by quercetin, suggesting an anti-hypertensive effect for quercetin.

Next, we evaluated the efficacy of quercetin to attenuate cardiac hypertrophy in SHRs. As shown in Figure 2A, heart weight to body weight ratio (HW/BW) was higher in SHRs than WKY rats, which was related to increased LVPWd and IVSd, and decreased LVIDd in SHRs as revealed by the M-mode echocardiography. Whereas these pathological phenotype were significantly prevented in quercetin-treated rats (Figures 2B,C). In addition, SHRs developed increased LVEF and LVFS, which were also reversed to normal levels by quercetin administration (Figures 2D,E). Moreover, histological analysis showed elevated collagen volume fraction and cardiomyocyte cross-sectional area in SHRs compared with WKY rats, accompanied by increased collagen I and α-smooth muscle actin (α-SMA). However, quercetin treatment rescued these changes (Figures 2F–J). Since previous studies suggested a protective role of SIRT3 in cardiac hypertrophy (Pillai et al., 2015), we assessed SIRT3 levels in rats by western blot. SIRT3 levels were markedly reduced in SHRs, but significantly increased by quercetin treatment (Figures 2K,L). These results demonstrated that quercetin might attenuate cardiac hypertrophy by activating SIRT3-mediated signaling pathway in vivo.

Previous data demonstrated a close link between mitochondrial dysfunction and pressure overload-induced cardiac hypertrophy (Kumar et al., 2019). In order to assess the effect of quercetin on mitochondrial integrity and function in SHRs, we observed mitochondrial ultrastructure using TEM. Mitochondria in the hearts of WKY rats had regular morphology, with abundant cristae. Meanwhile, SHRs hearts showed defective mitochondrial organization, as indicated by swollen mitochondria with distorted cristae, even accompanied by mitochondrial membrane damage. However, quercetin treatment rescued these ultrastructure changes, with a relatively improved mitochondrial phenotype (Figure 3A). Based on the mitochondrial function in ROS generation, we detected mitochondrial superoxide with MitoSOX™ Red staining. As shown in Figure 3B, superoxide generation was significant increased in SHRs, which was decreased by quercetin treatment. In agreement, oxidative stress was exacerbated as determined by increased MDA and decreased T-SOD and GSH-PX in SHRs, whereas quercetin treatment significantly ameliorated oxidative stress (Figure 3C). These data confirmed that quercetin alleviated mitochondrial damage and oxidative stress in vivo.

Before evaluating anti-hypertrophic effects of quercetin in vivo, we tested the cytotoxicity of quercetin by MTT assay. The MTT results showed that Ang II reduced viability of H9c2 cells by 46% compared with the control, while no significant reduction in cell viability was observed after quercetin intervention, indicating that there were no cytotoxic effects (Figure 4A). Subsequently, we investigated the hypertrophic response in Ang II-induced H9c2 cells by using hypertrophic markers, including atrial natriuretic factor (ANF) and β-myosin heavy chain (β-MHC), as indicators of cardiomyocyte hypertrophy. As shown in Figures 4B,C, Ang II-induced H9c2 cells showed increased mRNA levels of ANF and β-MHC compared with control cells, which were markedly reduced after quercetin treatment in a dose dependent manner.

To get further support for the protective effect of quercetin on mitochondrial function, we measured ΔΨm using JC-1. As shown in Figure 4D, the aggregate/monomer (red/green fluorescence intensity) ratio represented mitochondrial membrane potential depolarization. Quercetin attenuated mitochondrial membrane potential depolarization and restored the decreased ΔΨm in Ang II-induced H9c2 cells in a dose dependent manner (Figure 4E). Moreover, SIRT3 expression was decreased by Ang II, whereas quercetin significantly restored SIRT3 levels, especially in the high-dose group (Figures 4F,G). Therefore, 2 μM quercetin was chosen as the optimal dose for subsequent experiments. These data confirmed that quercetin inhibited the cardiac hypertrophic response and protected mitochondrial function in vitro, likely via SIRT3 activation.

Due to the vital roles of SIRT3 in cardiac mitochondrial dysfunction and oxidative stress (Wei et al., 2017), we focused on SIRT3-mediated pathway in quercetin pharmacology using siRNA-SIRT3 to knock down SIRT3. In this study, western blot showed that SIRT3 was effectively knocked down after siRNA transfection (Figures 5A,B). As shown in Figures 5C,D, Ang II induced mitochondrial membrane depolarization and altered the mitochondrial membrane potential in H9c2 cells, which was markedly reversed by quercetin treatment. However, these effects were partly abolished in siRNA-SIRT3 cells but not in siRNA-NC cells. Similar effects were found in the ATP assay (Figure 5E). Since SIRT3 is an NAD⁺-dependent enzyme, its activity is dependent on NAD⁺ level, thus we examined the NAD⁺ level in H9c2 cells. As shown in Figure 5F, quercetin alleviated Ang II-induced NAD⁺ level decrease, which was markedly reduced after SIRT3 knockdown. These results suggested the involvement of SIRT3 activation in quercetin pharmacology in mitochondrial protection.

PARP-1 is an important effector suppressed by SIRT3 overexpression in hypertrophic cardiomyocytes (Feng et al., 2020), its activity is represented by PAR expression. In the present study, quercetin dramatically inhibited PARP-1 and PAR upregulation in Ang II-induced H9c2 cells, whereas this effect was reduced after SIRT3 knockdown (Figures 5G–J). Taken together, these findings demonstrated that quercetin played a protective role in mitochondrial function by regulating SIRT3/PARP-1 signaling pathway.