Andrographolide Protects against Aortic Banding-Induced Experimental Cardiac Hypertrophy by Inhibiting MAPKs Signaling

Abstract

Despite therapeutic advances, heart failure-related mortality rates remain high. Therefore, understanding the pathophysiological mechanisms involved in the remodeling process is crucial for the development of new therapeutic strategies. Andrographolide (Andr), a botanical compound, has potent cardio-protective effects due to its ability to inhibit mitogen-activated protein kinases (MAPKs). Andr has also been shown to inhibit inflammation and apoptosis, which are factors related to cardiac hypertrophy. Our aim was to evaluate the effects of Andr on cardiac hypertrophy and MAPKs activation. Thus, mice were subjected to aortic banding (AB) with/without Andr administration (25 mg/kg/day, orally). Cardiac function was accessed by echocardiography and hemodynamic parameters. Our results showed that Andr administration for 7 weeks decreased cardiac dysfunction and attenuated cardiac hypertrophy and fibrosis in AB mice. Andr treatment induced a strong reduction in the transcription of both hypertrophy (ANP, BNP, and β-MHC) and fibrosis related genes (collagen I, collagen III, CTGF, and TGFβ). In addition, cardiomyocytes treated with Andr showed a reduced hypertrophic response to angiotensin II. Andr significantly inhibited MAPKs activation in both mouse hearts and cardiomyocytes. Treatment with a combination of MAPKs activators abolished the protective effects of Andr in cardiomyocytes. Furthermore, we found that Andr also inhibited the activation of cardiac fibroblasts via the MAPKs pathway, which was confirmed by the application of MAPKs inhibitors. In conclusion, Andr was found to confer a protective effect against experimental cardiac hypertrophy in mice, suggesting its potential as a novel therapeutic drug for pathological cardiac hypertrophy.

Keywords: MAPKs; andrographolide; cardiac hypertrophy; cardiomyocyte; fibroblast.

FIGURE 1

Andr improves cardiac function after chronic pressure overload in mice. (A–D) Echocardiography was performed at the end of the study (8 weeks) (n = 8). (A) Representative images. Andr attenuated the AB-induced increase in wall thickness [B, left ventricular (LV) posterior wall thickness at the end of systole (LVPWs) and, LV posterior wall thickness at the end of diastole (LVPWd)], and LV diameter (C), including LVESd and LVEDd as well as attenuated AB-induced changes in ejection fraction (EF) and fractional shortening (FS) (D). (E–H). Hemodynamics analysis was performed at the end of the study (8 weeks) (n = 8). HR, heart rate; ESP, end-systolic pressure; EDP, end-diastolic pressure; CO, cardiac output; dP/dt max, maximal rate of pressure development; dP/dt min, minimal rate of pressure decay. ^∗P < 0.05 compared with the corresponding sham group. ^#P < 0.05 vs. the veh-AB group. AB, aortic banding.

FIGURE 2

Andr attenuates cardiac hypertrophy after chronic pressure overload in mice. (A,B) Statistical results of the heart weight/body weight (HW/BW) ratio, and heart weight/tibial length (HW/TL) ratio (n = 8). (C) Gross hearts, and HE staining of hearts from sham and aortic banding (AB) mice at 8 weeks post-surgery. (D) Myocyte cross-sectional areas (CSAs) of the indicated groups (n = 200+). (E) Expression of transcripts for ANP, BNP, and β-myosin heavy polypeptide (MHC) induced by AB was determined by reverse transcription-polymerase chain reaction analysis (n = 6). The results are presented as a fold change, and the results are normalized to GAPDH gene expression. ^∗P < 0.05 as compared with the corresponding sham group. ^#P < 0.05 vs. the veh-AB group. AB, aortic banding.

FIGURE 3

Andr attenuated cardiac fibrosis after chronic pressure overload in mice. (A) Left ventricular histological sections from the indicated groups were stained with PSR (n = 6). (B) Fibrotic areas in the histological sections were quantified using an image-analysis system. (C) The mRNA expression of CTGF, collagen I, collagen III, fibronectin, and TGFβ1 in the myocardium was analyzed in the indicated groups using reverse transcription-polymerase chain reaction (n = 6). The results are presented as a fold change, and the results are normalized to GAPDH gene expression. ^∗P < 0.05 compared with the corresponding sham group. ^#P < 0.05 vs. the veh-AB group.

FIGURE 4

Andr suppresses Ang II-induced cardiomyocyte hypertrophy. Cardiomyocytes were stimulated with Ang II (1 μM) and treated with different concentrations of Andr (0, 12.5, 25, or 50 μM). (A) Cell viability was accessed by the cell counting kit-8 assay (n = 5). (B) The mRNA levels of ANP, BNP, and β-MHC in cardiomyocytes in the indicated groups (n = 6). (C,D) Immunofluorescence staining of α-actinin and the cell surface area of cardiomyocytes in the indicated groups (n = 5 samples, and n = 100+ cells per group). ^∗P < 0.05 compared with the control group. #P < 0.05 vs. the Ang II group. (E) Cardiomyocytes were stimulated with Ang II (1 μM) and treated with Andr (50 μM) for 0, 6, 12, and 24 h. The mRNA levels of ANP, BNP, and β-MHC in cardiomyocytes in the indicated groups (n = 6). The results are presented as a fold change, and the results are normalized to GAPDH gene expression. ^∗P < 0.05 compared with the corresponding 0 h group. ^#P < 0.05 vs. the corresponding Ang II group.

FIGURE 5

Andr blocks MAPKs signaling in vivo and in vitro. (A) Representative blots of phosphorylated (P-) and total (T-) ERK1/2, JNK, and P38 in the heart tissues of mice in the indicated groups (n = 6). (B) Comparison of expression among the indicated groups. ^∗P < 0.05 compared with the corresponding sham group. #P < 0.05 vs. the veh-AB group. (C) Representative blots of phosphorylated (P-) and total (T-) ERK1/2, JNK, and P38 in the cardiomyocytes in the indicated groups (n = 6). (D) Comparison of expression among the indicated groups. ^∗P < 0.05 compared with the corresponding PBS group. ^#P < 0.05 vs. the vehicle-Ang II group.

FIGURE 6

Andr-mediated cardioprotection depends on the inhibition of MAPKs in cardiomyocytes. Cardiomyocytes were treated with ERK1/2 inhibitor (SCH7729, 5 μM), JNK inhibitor (SP600125, 10 μM), and/or P38 inhibitor (SB209063, 10 μM) as well as stimulated with Ang II (1 μM) and treated with Andr (50 μM). (A) Cell viability was accessed by the cell counting kit-8 assay (n = 5). (B–E) Immunofluorescence staining of α-actinin and the cell surface area of cardiomyocytes in the indicated groups (n = 5 samples and n = 100+ cells per group). (F–J) The mRNA levels of ANP and β-MHC in cardiomyocytes in the indicated groups (n = 6). The results are presented as a fold change, and the results are normalized to GAPDH gene expression. ^∗P < 0.05 compared with the control group. ^#P < 0.05 vs. the Ang II group.

FIGURE 7

Andr reduces cardiac fibroblast activation via MAPKs. (A–F) Cardiac fibroblasts were treated with different concentrations of Andr (0, 12.5, 25, or 50 μM) and/or stimulated with Ang II (1 μM). (A) Cell viability was accessed by the cell counting kit-8 assay (n = 5). (B) Cell proliferation was accessed by the cell counting kit-8 assay (n = 5). (C) Immunofluorescence staining of α-SMA in the indicated groups. (D) The mRNA levels of collagen I, collagen III, and CTGF in cardiac fibroblasts in the indicated groups (n = 6). (E) Representative blots of phosphorylated (P-) and total (T-) ERK1/2, JNK, P38, and smad4 in the cardiac fibroblasts in the indicated groups (n = 6). (F) Comparison of expression among the indicated groups. ^∗P < 0.05 compared with the corresponding PBS group. #P < 0.05 vs. the vehicle-Ang II group. (G–J) Cardiac fibroblasts were treated with ERK1/2 inhibitor (SCH7729, 5 μM), JNK inhibitor (SP600125, 10 μM), and/or P38 inhibitor (SB209063, 10 μM) as well as stimulated with Ang II (1 μM) and treated with Andr (50 μM). (G) Cell viability was accessed by the cell counting kit-8 assay (n = 5). (H) Cell proliferation was accessed by the cell counting kit-8 assay (n = 5). (I) Immunofluorescence staining of α-SMA in the indicated groups. (J,K) The mRNA levels of collagen I, collagen III, and CTGF in cardiac fibroblasts in the indicated groups (n = 6). The results are presented as a fold change, and the results are normalized to GAPDH gene expression. ^∗P < 0.05 compared with the control group. ^#P < 0.05 vs. the Ang II group.