Sibjotang Protects against Cardiac Hypertrophy In Vitro and In Vivo

Abstract

Cardiac hypertrophy is developed by various diseases such as myocardial infarction, valve diseases, hypertension, and aortic stenosis. Sibjotang (, Shizaotang, SJT), a classic formula in Korean traditional medicine, has been shown to modulate the equilibrium of body fluids and blood pressure. This research study sought to explore the impact and underlying process of Sibjotang on cardiotoxicity induced by DOX in H9c2 cells. In vitro, H9c2 cells were induced by DOX (1 μM) in the presence or absence of SJT (1-5 μg/mL) and incubated for 24 h. In vivo, SJT was administrated to isoproterenol (ISO)-induced cardiac hypertrophy mice (n = 8) at 100 mg/kg/day concentrations. Immunofluorescence staining revealed that SJT mitigated the enlargement of H9c2 cells caused by DOX in a dose-dependent way. Using SJT as a pretreatment notably suppressed the rise in cardiac hypertrophic marker levels induced by DOX. SJT inhibited the DOX-induced ERK1/2 and p38 MAPK signaling pathways. In addition, SJT significantly decreased the expression of the hypertrophy-associated transcription factor GATA binding factor 4 (GATA 4) induced by DOX. SJT also decreased hypertrophy-associated calcineurin and NFAT protein levels. Pretreatment with SJT significantly attenuated DOX-induced apoptosis-associated proteins such as Bax, caspase-3, and caspase-9 without affecting cell viability. In addition, the results of the in vivo study indicated that SJT significantly reduced the left ventricle/body weight ratio level. Administration of SJT reduced the expression of hypertrophy markers, such as ANP and BNP. These results suggest that SJT attenuates cardiac hypertrophy and heart failure induced by DOX or ISO through the inhibition of the calcineurin/NFAT/GATA4 pathway. Therefore, SJT may be a potential treatment for the prevention and treatment of cardiac hypertrophy that leads to heart failure.

Keywords: H9c2 cell; apoptosis; heart diseases; heart failure; isoproterenol.

Figure 1

Three-dimensional HPLC chromatogram of SJT.

Figure 2

Effect of SJT on DOX–induced H9c2 cell death. (A) Cells were treated with concentrations (0–50 μg/mL) of SJT for 24 h. (B) H9c2 cells were exposed to doxorubicin (1 μM) while treated with or without SJT at concentrations of 1, 2, and 5 μg/mL for 24 h. Cell viability was measured by Cell Cytotoxicity Assay. The data are expressed as a percentage of basal value and are the means ± S.E of five independent experiments. *** p < 0.001, ** p < 0.01 vs. control, ### p < 0.001, # p < 0.05 vs. DOX.

Figure 3

Effect of SJT on DOX–increased cardiac hypertrophy in H9c2 cells. (A) H9c2 cells were pretreated with SJT (2, 5 μg/mL) for 30 min and then stimulated with doxorubicin (1 μM) for 24 h. Cells were stained with Alexa Fluor™ 488 Phalloidin F-actin. Cell size was quantified by measuring the surface area of the cells. (B) Effect of SJT on DOX–induced hypertrophic protein expression. The protein levels of MLC–2v and β–MHC were determined by Western blot analysis. (C) ANP, BNP, and β–MHC mRNA expressions were analyzed using Real–Time PCR. The data are expressed as a percentage of basal value and are the means ± S.E of three independent experiments. *** p <0.001 vs. control, ### p < 0.001, ## p < 0.01, # p < 0.05 vs. doxorubicin.

Figure 4

Effect of SJT on DOX–induced phosphorylation of p38 and ERK. H9c2 cells were pretreated with SJT (1–5 μg/mL) for 30 min and then treated with doxorubicin (1 μM) for 1 h. (A) p38, (B) ERK, and (C) JNK MAPK protein expressions were analyzed using Western blot analysis. p38, ERK, and JNK protein levels were used as loading controls. *** p < 0.001, * p < 0.05 vs. control, ### p < 0.001, ## p < 0.01, # p < 0.05 vs. DOX.

Figure 5

Effect of SJT on DOX–induced GATA–4 and Calcineurin. H9c2 cells were pretreated with SJT (1–5 μg/mL) for 30 min and then treated with DOX (1 μM) for 90 min and 24 h. (A) The protein levels of phosphorylated GATA–4 were determined by Western blot analysis. (B) Immunofluorescent images of p–GATA–4 nuclear translocation under the laser scanning confocal microscopy are shown (magnification. 400×). Nuclei were stained with DAPI (blue) and p–GATA–4 was stained with Alexa Fluor 488 (green) (immunofluorescence, 200×). (C) Calcineurin protein expression was analyzed using Western blot analysis. The results are expressed as the mean ± SE values of three experiments. *** p < 0.001, ** p < 0.01 vs. control, ### p < 0.001, ## p < 0.01 vs. DOX.

Figure 6

Effect of SJT on DOX–induced NFAT expression. H9c2 cells were pretreated with SJT (1–5 μg/mL) for 30 min and then treated with DOX (1 μM) for 90 min and 24 h. (A) NFAT–3 protein expressions were analyzed using Western blot analysis. (B) Effect of SJT on nuclear translocation of NFAT–3 was confirmed in H9c2 cells exposed to DOX. The results are expressed as the mean ± SE values of three experiments. *** p < 0.001, ** p < 0.01 vs. control, ### p < 0.001, ## p < 0.01, # p < 0.05 vs. DOX.

Figure 7

Effect of SJT on DOX–induced cardiac apoptosis. H9c2 cells were pretreated with SJT (1–5 μg/mL) for 30 min and then treated with DOX (1 μM) for 18 h and 12 h. (A) Caspase–3, (B) Caspase–9, (C) Bax, and (D) Bcl–2 protein expressions were analyzed using Western blot analysis. The results are expressed as the mean ± SE values of three experiments. *** p < 0.001, ** p < 0.01 vs. control, ## p < 0.01, # p < 0.05 vs. doxorubicin.

Figure 8

Effect of SJT on ISO–induced cardiac hypertrophy. Effect of SJT on heart size (A) and left ventricular/body weight (B) in ISO–induced ICR mice. (C) Images of the whole hearts of animal models. (D) Effect of SJT on cardiomyocyte hypertrophy markers in left ventricular tissues. Mice were infused with PBS (control), ISO, propranolol 30 mg/kg·day with ISO (PRO), and SJT 100 mg/kg·day with ISO (SJT–100). Data are expressed as mean ± SE. There are 6 experimental cases. * p < 0.05 vs. Control, ## p < 0.01, # p < 0.05 vs. ISO. BW, body weight; LVW, left ventricular weight; Iso, isoprenaline; PRO, propranolol.