TongGuanWan Alleviates Doxorubicin- and Isoproterenol-Induced Cardiac Hypertrophy and Fibrosis by Modulating Apoptotic and Fibrotic Pathways

Abstract

Heart failure, a major public health issue, often stems from prolonged stress or damage to the heart muscle, leading to cardiac hypertrophy. This can progress to heart failure and other cardiovascular problems. Doxorubicin (DOX), a common chemotherapy drug, and isoproterenol (ISO), a β-adrenergic agonist, both induce cardiac hypertrophy through different mechanisms. This study investigates TongGuanWan (TGW,), a traditional herbal remedy, for its effects on cardiac hypertrophy and fibrosis in DOX-induced H9c2 cells and ISO-induced mouse models. TGW was found to counteract DOX-induced increases in H9c2 cell surface area (n = 8, p < 0.01) and improve biomarkers like ANP (n = 3, p < 0.01)) and BNP (n = 3, p < 0.01). It inhibited the MAPK pathway (n = 4, p < 0.01) and GATA-4/calcineurin/NFAT-3 signaling, reduced inflammation by decreasing NF-κB p65 translocation, and enhanced apoptosis-related factors such as caspase-3 (n = 3, p < 0.01), caspase-9 (n = 3, p < 0.01), Bax (n = 3, p < 0.01), and Bcl-2 (n = 3, p < 0.01). Flow cytometry showed TGW reduced apoptotic cell populations. In vivo, TGW reduced heart (n = 8~10, p < 0.01), and left ventricle weights (n = 6~7), cardiac hypertrophy markers (n = 3, p < 0.01), and perivascular fibrosis in ISO-induced mice, with Western blot analysis confirming decreased levels of fibrosis-related factors like fibronectin, α-SMA (n = 3, p < 0.05), and collagen type I (n = 3, p < 0.05). These findings suggest TGW has potential as a therapeutic option for cardiac hypertrophy and fibrosis.

Keywords: TongGuanWan; apoptosis; cardiac hypertrophy; doxorubicin; fibrosis; heart failure.

Figure 1

TongGuanWan chemical component analysis. Extracted ion chromatograms (EICs) of UPLC/QE Orbitrap MS analysis of the positive and negative charged molecular ion show seven bioactive compounds.

Figure 2

Effects of TongGuanWan on DOX-induced H9c2 cell death and cell surface area size: (A) effects of TGW on changes in the viability of H9C2 cells induced by DOX. TGW was pretreated 30 min before DOX administration, and cell viability was assessed using an MTT assay 24 h after DOX treatment; (B) representative images of F-actin staining (green) and nucleus (blue) in H9c2 cells, used to assess changes in cell surface area size. Representative images and quantitative results demonstrating that TGW (1–10 μg/mL) inhibited DOX (5 μM; 24 h) induced H9c2 cells. All experiments were performed at least three times. Data represent mean ± standard deviation (SD). ** p < 0.01, vs. cont; # p < 0.05 and ## p < 0.01vs. DOX-treated cells.

Figure 3

Effects of TongGuanWan on cardiomyocyte hypertrophy markers in DOX-treated H9c2 cells: (A) cardiac ANP and BNP protein expression levels and (B) β-MHC and MLC-2v protein expression levels and experiments were performed independently three times; (C) cardiac signaling pathway responses to TGW determined by RT-qPCR assay. Data are presented as the mean ± SD. ** p < 0.01, vs. cont; # p < 0.05 and ## p < 0.01 vs. DOX-treated cells. TGW, TongGuanWan; ANP, atrial natriuretic peptide; BNP, brain/B-type natriuretic peptide.

Figure 4

The effects of TongGuanWan on calcineurin-NFAT and -GATA4 signaling pathway signaling protein expression levels in H9c2 cells. The protein levels of calcineurin (A), NFATc3 (B), GATA4 (C), and phosphorylated GATA4 were determined by Western blot analysis. GATA4 mRNA levels were analyzed using real-time PCR. (D) Immunofluorescent images of p-GATA-4 nuclear translocation under the laser scanning confocal microscopy are show. Nuclei were stained with DAPI (blue) and p-GATA-4 was stained with Alexa Fluor 488 (green) (immunofluorescence, 200x). The results are expressed as the mean ± SD values of three experiments. ** p < 0.01, vs. cont; ## p < 0.01 vs. DOX-treated cells.

Figure 5

The effects of TongGuanWan on the MAPK/NF-κB signaling pathway in H9c2 cells under DOX conditions: (A) protein expression of MAPK (JNK, ERK, and p38) signaling pathway according to Western blot analysis; (B) expression and translocation of NF-kB p65 were evaluated by measuring protein in cytoplasmic and nuclear fractions; (C) representative images of immunofluorescence staining for NF-κB p65 in H9c2 cells. Immunofluorescence staining used anti-NF-κB p65 (green) and DAPI (blue, nuclei). The white arrow indicates that NF-κB has moved to the nucleus (immunofluorescence, 200×). The results are expressed as the mean ± SD values of three experiments. ** p < 0.01, vs. cont; ## p < 0.01, vs. DOX-treated cells.

Figure 6

Effects of TongGuanWan on DOX-induced cell apoptosis in H9c2 cells. Western blot analysis results showing the expression levels of apoptosis-related proteins including (A) caspase-3, caspase-9, (B) Bax and Bcl-2 in DOX-induced H9c2 cells. (C) mRNA levels of Bax, Bcl-2, and caspase-3 were quantified using real-time PCR. (D) Cellular apoptosis was evaluated using annexin V/PI dual staining followed by flow cytometric analysis. All experiments were conducted in triplicate. Data are presented as mean ± SD. ** p < 0.01, vs. cont; # p < 0.05 and ## p < 0.01, vs. DOX-treated cells.

Figure 7

Effects of TGW on cardiac fibrosis markers in H9c2 cells subjected to DOX treatment. Levels of fibronectin, collagen I, α-SMA, TGF-β1, and p-smad3 was determined using Western blot analysis. Data represent mean ± SD. * p < 0.05 and ** p < 0.01, vs. control; # p < 0.05 and ## p < 0.01, vs. DOX-treated cells.

Figure 8

Effects of TongGuanWan on isoproterenol-induced cardiac hypertrophy. TGW administration was started 1 week after infusion of ISO (7 days) in ICR mice. Effects of TGW on heart weight/body weight (A) and left ventricular/body weight (B) in ISO-induced ICR mice; (C) images of the whole hearts of ICR mice; (D) WGA staining was performed to evaluate the cell size in heart sections. Green color represents the cardiomyocyte boundary; (E) effects of TGW on cardiomyocyte hypertrophy markers in left ventricular tissues. Mice were infused with saline (control), ISO, propranolol (PC), TGW 100 mg/kg·day with ISO (TGW-100), or TGH 200 mg/kg·day with ISO (TGW-200) for 1 week. Data are expressed as mean ± SD. Experimental cases numbered 6~9. * p < 0.05 and ** p < 0.01 vs. Cont; # p < 0.05 and ## p < 0.01 vs. ISO group. HW, heart weight; BW, body weight; LV, left ventricular weight; Iso, isoprenaline; Con, control.

Figure 9

Effects of TongGuanWan on cardiac fibrosis: (A) picro-sirius red staining for cardiac fibrosis (red) in a section of the heart; (B) representative blots of fibronectin, collagen I, α-SMA, transforming growth factor-β1, and p-Smad3 in the heart tissues of mice in the indicated groups; (C) the mRNA expression of markers of fibrosis (Fibronectin and collagen I) was determined by RT-qPCR. Levels of fibronectin, collagen I, α-SMA, TGF-β1, and p-Smad3 was determined using Western blot analysis. Data represent mean ± SD. * p < 0.05 and ** p < 0.01, vs. control; # p < 0.05 and ## p < 0.01, vs. ISO group. Iso, isoprenaline; Con, control.