Diallyl sulfide protects against dilated cardiomyopathy via inhibition of oxidative stress and apoptosis in mice

Abstract

Cytochrome P450 family 2 subfamily E member 1 (CYP2E1) is a member of the cytochrome P450 enzyme family and catalyzes the metabolism of various substrates. CYP2E1 is upregulated in multiple heart diseases and causes damage mainly via the production of reactive oxygen species (ROS). In mice, increased CYP2E1 expression induces cardiac myocyte apoptosis, and knockdown of endogenous CYP2E1 can attenuate the pathological development of dilated cardiomyopathy (DCM). Nevertheless, targeted inhibition of CYP2E1 via the administration of drugs for the treatment of DCM remains elusive. Therefore, the present study aimed to investigate whether diallyl sulfide (DAS), a competitive inhibitor of CYP2E1, can be used to inhibit the development of the pathological process of DCM and identify its possible mechanism. Here, cTnT^R141W transgenic mice, which developed typical DCM phenotypes, were used. Following treatment with DAS for 6 weeks, echocardiography, histological analysis and molecular marker detection were conducted to investigate the DAS‑induced improvement on myocardial function and morphology. Biochemical analysis, western blotting and TUNEL assays were used to detected ROS production and myocyte apoptosis. It was found that DAS improved the typical DCM phenotypes, including chamber dilation, wall thinning, fibrosis, poor myofibril organization and decreased ventricular blood ejection, as determined using echocardiographic and histopathological analyses. Furthermore, the regulatory mechanisms, including inhibition both of the oxidative stress levels and the mitochondria‑dependent apoptosis pathways, were involved in the effects of DAS. In particular, DAS showed advantages in terms of improved chamber dilation and dysfunction in model mice, and the improvement occurred in the early stage of the treatment compared with enalaprilat, an angiotensin‑converting enzyme inhibitor that has been widely used in the clinical treatment of DCM and HF. The current results demonstrated that DAS could protect against DCM via inhibition of oxidative stress and apoptosis. These findings also suggest that inhibition of CYP2E1 may be a valuable therapeutic strategy to control the development of heart diseases, especially those associated with CYP2E1 upregulation. Moreover, the development of DAS analogues with lower cytotoxicity and metabolic rate for CYP2E1 may be beneficial.

Keywords: cytochrome P450 family 2 subfamily E member 1; diallyl sulfide; dilated cardiomyopathy; inhibitor of CYP2E1; oxidative stress.

Figure 1.

Echocardiographic analysis of cardiac morphology and function. Mice in the five groups were analyzed after 6 weeks of treatment: NTG (wild-type control), cTnT^R141W (placebo control), treatment with 400 mg/kg DAS, treatment with 200 mg/kg DAS and treatment with enalaprilat (commercially available drug control). (A) Representative M-mode echocardiographic images of the LV long axis. (B) LVESD. (C) LVEDD. (D) LVPWS. (E) LVPWD. (F) LVAWS. (G) LVAWD. (H) LVEF. (I) LVFS. n=7. *P<0.05, ***P<0.001 vs. NTG group; ^#P<0.05, ^##P<0.01, ^###P<0.001 vs. cTnT^R141W group; ^$P<0.05 vs. 400 mg/kg DAS group. NTG, non-transgenic; LV, left ventricular; LVESD, left ventricular end-systole diameter; LVEDD, left ventricular end-diastole diameter; LVPWS, left ventricular ventricle posterior wall at end systole; LVPWD, left ventricular posterior wall at end diastole; LVAWS, left ventricular anterior wall at end systole; LVAWD, left ventricular anterior wall at end diastole; LVEF, left ventricular ejection fraction; LVFS, left ventricular fractional shortening; DAS, diallyl sulfide.

Figure 2.

Pathological histology observation. (A) CYP2E1 and cTnT expression in the heart tissues of mice in the NTG, cTnT^R141W, 400, 200 mg/kg DAS and enalaprilat groups was detected via western blotting. (B) CYP2E1 and (C) cTnT were semi-quantitatively analyzed, using GAPDH for normalization (n=3). (D) Ratio of HW to BW (n=6). (E) H&E staining patterns of whole-heart longitudinal sections. (F) Magnification of H&E-stained sections of the left ventricle (magnification, ×400; scale bar, 20 µm). (G) Magnification of Masson's trichrome-stained left ventricle sections (magnification, ×400, scale bar, 20 µm). Myocytes are stained red; collagenous tissue is stained blue. (H) Ultrastructure observation via transmission electron microscopy (scale bar, 0.5 µm). (I) Quantitative analysis of Masson staining (n=3). (J) Col3α1 expression was detected and (K) analyzed (n=3). *P<0.05, ***P<0.001 vs. NTG group; ^#P<0.05, ^##P<0.01, ^###P<0.001 vs. cTnT^R141W mice. cTnT, cardiac troponin T; HW, heart weight; BW, body weight; Col3α1, procollagen type III α1; NTG, non-transgenic; DAS, diallyl sulfide; CYP2E1, cytochrome P450 family 2 subfamily E member 1.

Figure 3.

Determination of oxidative stress levels. Levels of (A) H₂O₂, (B) MDA and (C) GSH in the heart tissues of mice in the NTG, cTnT^R141W, 400, 200 mg/kg DAS and enalaprilat groups were determined using colorimetric assays (n=3). *P<0.05, ***P<0.001 vs. NTG group; ^##P<0.01, ^###P<0.001 vs. cTnT^R141W mice. MDA, malondialdehyde; GSH, glutathione; NTG, non-transgenic; DAS, diallyl sulfide.

Figure 4.

Analysis of the mitochondria-dependent apoptosis pathways. (A) Cyt-c release and activation of caspase 9 and caspase 3 in the heart tissues of mice in the NTG, cTnT^R141W, 400, 200 mg/kg DAS and enalaprilat groups were detected via western blotting. (B) Cyt-c in the cyto and mito was semi-quantitatively analyzed using β-tubulin or VDAC1 for normalization. (C) Procaspase 9 and (D) procaspase 3 and active caspases 3 and 9 were semi-quantitatively analyzed using β-tubulin for normalization (n=3). (E) Cardiac myocyte apoptosis was detected using a TUNEL assay, and the arrows indicate TUNEL-positive cells (magnification, ×400). (F) Number of positive cells was counted, and the proportion of apoptotic cells among the total cells in each image was calculated (n=3). *P<0.05, **P<0.01, ***P<0.001 vs. NTG group; ^#P<0.05, ^##P<0.01, ^###P <0.001 vs. cTnT^R141W mice. Cyt-c, Cytochrome c; cyto, cytoplasm; mito, mitochondria; VDAC1, voltage dependent anion channel 1; NTG, non-transgenic; DAS, diallyl sulfide.