Garlic extract diallyl sulde protects against dilated cardiomyopathy through inhibition of oxidative stress and apoptosis in mice

Background: Diallyl sulde (DAS) is an active ingredient in garlic that is induced when the garlic is chopped and ground. DAS has been found to act as a competitive inhibitor of CYP2E1, which is a member of the cytochrome P450 enzyme family and catalyses the metabolism of various substrates. CYP2E1 is upregulated in multiple heart diseases and causes damage mainly through the production of ROS. In mice, increased CYP2E1 expression induces cardiac myocyte apoptosis, and CYP2E1 knockdown can attenuate the pathological development of DCM. Nevertheless, Targeted inhibition of CYP2E1 for the treatment of dilated cardiomyopathy (DCM) remain limited. The aim of this study was to investigate the therapeutic effect of DAS on cardiomyopathy and its possible molecular mechanisms, and provide new clues and approaches for the clinical treatment of cardiomyopathy and heart failure. Methods: Echocardiography were performed to identify mouse heart function and structure. histological analysis and RT-PCR were conducted to investigate that improved Myocardial morphology and brosis and measurement of reactive oxygen species (ROS) and tunel Assay were used to detected DAS inhibit ROS production and myocyte apoptosis in cTnT R141W DCM mice. Western Blot were performed to investigate the mechanism of apoptosis pathway. Results: Diallyl sulde (DAS), a competitive inhibitor of CYP2E1, improves the typical DCM phenotype, including chamber dilation, wall thinning, brosis, poor myobril organization and decreased ventricular blood ejection, by inhibiting ROS production and myocyte apoptosis in cTnT R141W DCM mice. Conclusions: Our results suggest that inhibition of CYP2E1 might be a valuable therapeutic strategy to control the development of heart diseases associated with CYP2E1 overexpression. Moreover, the development of DAS analogues with superior inhibitory properties and lower substrate potential for CYP2E1 might be benecial for patients with heart disease.

CYP2E1 has been used as a therapeutic target in drug discovery [12]. Therefore, application of CYP2E1 inhibitors may be one of the feasible approaches for the treatment of cardiomyopathy. Diallyl sul de (DAS) is a competitive inhibitor of CYP2E1, which is isolated from garlic [12,13]. Garlic is chopped and ground to induce the production of a vacuolar alliinase enzyme that rapidly converts the most important initial sulphide in garlic, alliin, into allicin, which is extremely unstable and can be easily converted into oilsoluble sulphur compounds: diallyl disul de (DADS), diallyl sul de (DAS), diallyl trisul de (DATS). Of these, diallyl sul de is a competitive inhibitor of CYP2E1 and has low toxicity and rapid metabolism [14,15].
DAS treatment can recover ethanol-induced pathological changes in the liver by inhibiting CYP2E1mediated alcohol metabolism and subsequent oxidative stress [8,16]. Furthermore, DAS treatment can prevent or reduce myocardial systolic dysfunction induced by chronic ethanol exposure [17]. Studies of the effects of DAS have suggested that CYP2E1 inhibition might have potential effects on the development of diseases characterized by high CYP2E1 levels. Nevertheless, Targeted inhibition of CYP2E1 for the treatment of dilated cardiomyopathy (DCM) remain limited.
And in this study, we use the cTnT R141W transgenic mice, a mouse model with typical DCM phenotypes, which was established in our lab, as decreased survival rate, dilated chambers, thin walls, and cardiac dysfunction [4,18,19]. Accumulating evidence suggests an important role of oxidative stress in the pathophysiology of cardiac remodelling and HF [20,21]. We and others have shown that CYP2E1 is upregulated in human heart tissue with hypertrophic cardiomyopathy (HCM) and in animal heart tissue with DCHF [4,22,18,[23][24][25][26][27][28], including the cTnT R141W transgenic mice.
Accordingly, in the present study, we used the established engineered DCM mice models, to investigate the therapeutic effect of DAS on cardiomyopathy and its possible molecular mechanisms, and provide new clues and approaches for the clinical treatment of cardiomyopathy and heart failure.

Methods
Animals cTnT R141W transgenic mice were maintained in a C57BL/6J genetic background and developed characteristics of DCM by 4 months of age as in our previous report [19].
All mice were bred in an AAALAC-accredited facility, and the procedures were approved by the Animal Care and Use Committee at the Institute of Laboratory Animal Science, Peking Union Medical College (ZLF18004).

Groups and Treatment
Four-month-old male and female cTnT R141W transgenic mice were randomly assigned to treatment groups, half male and half female. DAS (A35801, Sigma-Aldrich, Saint Louis, USA) was diluted in corn oil to a nal concentration of 80 mg/ml. In the treatment groups, cTnT R141W transgenic mice were administered DAS at a dose of 200 mg/kg (n = 12) or 400mg/kg (n = 10) via intraperitoneal injection twice weekly for 6 weeks. A group of cTnT R141W transgenic mice (n = 9) and Non-transgenic littermates (NTG, n = 10) were treated with corn oil as the placebo control and wild-type normal control, respectively.
As a positive control, a group of cTnT R141W transgenic mice were treated with Enalaprilat, an angiotensinconverting enzyme (ACE) inhibitor that has been widely used in the clinical treatment of DCM and HF [29,30], at a dose of 0.76 mg/kg (n = 9). The dose of DAS was selected based on other experimental studies [31,32]. The dose of Enalaprilat was calculated from the ratio of mice to human weight.

Echocardiography
Mouse heart function and structure were analysed by echocardiography once every two weeks during treatment, with a total of three assessments per mouse. Brie y, the mice were lightly anesthetized by

Histological Analysis
For light microscopy, heart tissues were rst xed in 4% formaldehyde and then processed according to standard pathology procedures [4]. Heart tissue sections were photographed after HE staining and Masson staining. For Masson trichrome staining, sections were incubated in celestine blue solution (Solarbio, G1345, Beijing, China) for 5 min, brie y washed with H 2 O, incubated in hemalun solution for 5 min, in H 2 O for 10 min, in Fuchsine acid / Ponceau Xylidine (0.5% Fuchsine acid, 1.5% Ponceau Xylidine, 1,75% glacial acetic acid) for 5 min, brie y washed with H 2 O, incubated in phosphomolybdic acid (1%) for 10 min, in aniline blue solution (2.5% anilin blue, 2.5% glacial acetic acid) for 5 min, brie y washed with H 2 O, incubated in acetic acid (1%) for 1 min, then brie y in an ascending isopropanol series followed by xylol, before they were embedded.
For transmission electron microscopy (TEM), heart tissues were xed in 2.5% glutaraldehyde that dissolved in phosphate buffer and processed according to standard procedures [4]. Cutting thin tissue sections on an ultramicrotome and mounted on a grid. Thin sections were further stained with an equal mixture of 4% uranyl acetate and acetone for 30 s and lead citrate for 2 min. Through JEM-1400 transmission electron microscope (JEOL Ltd, Tokyo, Japan) ultrastructure observation, verifying the effect of DAS on ultrastructure of cardiomyocytes in the cTnT R141W mice.

Measurement of ROS
After treatment, the mice were sacri ced, and total lysates were prepared as previously reported [4]. For H 2 O 2 assay, wash 10 mg tissue in cold PBS. Then homogenize tissue in 500 µL assay buffer with a Dounce homogenizer sitting on ice, with 10-15 passes. Centrifuge sample for 5 minutes at 4°C at top speed using a microcentrifuge to remove any insoluble material. Centrifuge at 13,000×g for 10 minutes. A microplate reader was used to measure the content of H 2 O 2 in the heart tissue of each group.
For lipid peroxidation assay, wash 10 mg heart tissue in cold PBS. Homogenize tissue in 303 µL Lysis Solution with a Dounce homogenizer sitting on ice, with 10-15 passes. Centrifuge at 13,000×g for 10 minutes to remove insoluble material. Add TBA reagent into the supernatant to Generate MDA-TBA adduct. Measure absorbance immediately at Ex/Em = 532/553 nm for uorometric assay.
For glutathione assay, Wash 10 mg heart tissue in cold PBS. Resuspend tissue in 400 µL of cold Mammalian Lysis Buffer. Homogenize heart tissue with 10-15 passes. Centrifuge sample for 15 minutes at 4°C at top speed using a cold microcentrifuge to remove any insoluble material. Collect supernatant and add 50 µL of GSH Assay Mixture (GAM) into each GSH standard and sample well to make the total assay volume 100 µL/well. Incubate for 25 minutes and monitor uorescence at Ex/Em = 490/520 nm with a uorescence microplate reader.

TUNEL Assay
For apoptotic cell staining, para n slices of heart tissues were stained with an ApopTag Plus Peroxidase In Situ Apoptosis Kit (S7101, Millipore, Billerica MA, USA) following the manufacturer's procedures. In brie y, hearts were stained in duplicate using the Apoptag kit previously optimized for formalin-xed, para n embedded whole tissue sections. Formalin-xed, para n-embedded tissue slides were rehydrated using xylene to alcohol washings, followed by a hydrogen peroxide-methanol quench. The samples were treated with 25 µg/mL of proteinase K at 37° C for 8 minutes. After washing and incubation with equilibration buffer for 5 minutes, Tdt was diluted 1:3.9 with reaction buffer and incubated on the heart sections for 1 hour at 37°. After applying stop solution for 15 minutes and washing, the samples were incubated with antidigoxigenin peroxidase conjugate at 37° C for 30 minutes.
Slides were developed with a 1:20 dilution of diaminobenzidine (3,3'-diaminobenzidine) substrate, counterstained with methyl green, dehydrated, and coverslipped. In each group of three mice, three discrete slices were selected from each mouse and the proportion of apoptotic cardiomyocytes was calculated.

Statistical Analysis
The data were analysed by two-tailed unpaired t-tests and one-way ANOVA followed by Tukey's post hoc analysis. Data shown are the mean ± s.d with P < 0.05 considered statistically signi cant.

DAS Improves Cardiac Morphology Breakage and Dysfunction in cTnT R141W DCM Mice
The cTnT R141W transgenic mice displayed typical DCM phenotypes, with dilated chambers, thin walls, and cardiac dysfunction, which were successfully established in our previous study. In cTnT R141W DCM mice, CYP2E1 expression is induced in heart tissues, and knockdown of endogenous CYP2E1 level signi cantly prevents the development of DCM [4,23].
To test the protective effect of DAS, an inhibitor of CYP2E1, on the development of DCM, we analysed cardiac morphology and function with M-mode echocardiography in the cTnT R141W DCM mice during treatment. And four-month-old cTnT R141W transgenic mice, typically develop DCM from this time of age, were randomly assigned to three treatment groups, including. DAS low-dose group (200 mg/kg, n = 12), DAS high-dose group (400 mg/kg, n = 10), positive drug control group (Enalaprilat, an angiotensinconverting enzyme (ACE) inhibitor that has been widely used in the clinical treatment of DCM and HF, 0.76 mg/kg, n = 9). The dose of DAS was selected based on other experimental studies [31,32]. The dose of Enalaprilat was calculated from the ratio of mice to human weight. Non-transgenic littermates (NTG, n = 10) and a group of cTnT R141W transgenic mice (Placebo, n = 9) were treated with corn oil as the wild-type normal control and placebo control. Therefore, there are all ve groups, including NTG, cTnT R141W , DAS treatment (400mg/kg), DAS treatment (200mg/kg), Enalaprilat treatment in this study.
The DAS treatment improved cardiac morphology breakage and dysfunction in a dose-dependent manner; furthermore, signi cant improvements were observed in the early stages of treatment compared with the Enalaprilat group (Fig. 1).
After 6 weeks of processing, compared with the cTnT R141W group (placebo), LVESD decreased by 22.4% in the 400 mg/kg group (Fig. 1E and Table S3, n = 7, P < 0.001), while the Enalaprilat group (positive control) fell by only 11.1% (Fig. 1E and Table S3, n = 7, P < 0.05). LVPWS also increased signi cantly in the 400 mg/kg group compared with the placebo group ( Fig. 1F and Table S3, n = 7, P < 0.05), but LVPWS between the 400 mg/kg group and the Enalaprilat group was no signi cant difference. There was a 50.2% increase in LVEF in the 400 mg/kg group ( Fig. 1G and Table S3, n = 7, P < 0.001) compared with the cTnT R141W group, but did not increase signi cantly in the Enalaprilat group ( Fig. 1G and Table S3, n = 7, P > 0.05), And all three parameters given no signi cance between the 200 mg/kg group and the placebo group.
Compared with Enalaprilat treatment, treatment with 400 mg/kg DAS resulted in a greater improvement of DCM dysfunction (Fig. 1H-1K), as evidenced by the changes in LVPWS and LVAWD ( Fig. 1I and 1K). Furthermore, this improvement occurred earlier in both DAS groups than in the Enalaprilat group according to the signi cance analysis.

DAS Inhibits Cardiac Pathological Changes in cTnT R141W DCM Mice
After DAS treatment, hearts from all ve groups were sampled for gross morphology examination and pathological examination.
First of all, we have analysed the expression of CYP2E1 protein by western blot, and which was dose dependent with the dose treated by DAS. The expression of CYP2E1 protein in 200 mg/kg and 400 mg/kg DAS groups decreased by 35.5% ( Fig. 2A-2B, n = 3, P < 0.01) and 51.8% ( Fig. 2A-2B, n = 3, P < 0.001). While the expression of CYP2E1 protein fell by 35.9% in the Enalaprilat group ( Fig. 2A-2B, n = 3, P < 0.01). Furthermore, we have analysed the expression of cTnT protein, too. The mutant form of cTnT is the human mutant form which was introduced by the transgenic method, that is, the cTnT band at the higher molecular weight in Fig. 2A, and the location of endogenous cTnT proteins in mouse myocardium was slightly lower than that of exogenous mutants. The expression of the cTnT given no difference between three therapeutic groups and the model group (Fig. 2C) The increased heart to body weight ratio of cTnT R141W mice was reversed by DAS treatment to nearly the normal level found in the 400 mg/kg group and 200 mg/kg group ( Fig. 2A, n = 6, P < 0.05). DAS treatment signi cantly improved the chamber dilation, wall thinning, myocyte disarray in cTnT R141W mice, as analysed by hematoxylin-eosin (H&E) (Fig. 2E-2F). By contrast, Enalaprilat treatment failed to inhibit chamber dilation in cTnT R141W mice ( Fig. 2D-2E, n = 6, P > 0.05). Through transmission electron microscopy (TEM) ultrastructure observation, DAS treatment also clearly improved the poor myo bril organization, including diffusion, damage and lysis, in cTnT R141W mice (Fig. 2H). Quantitative analysis of the Masson stain and the RT-PCR of col3α1 (Fig. 2I-2K) showed that collagen deposition in the interstitial space of cTnT R141W mice was reduced by DAS treatment (Fig. 2F-2G, n = 3, P < 0.001).
Although Enalaprilat treatment clearly improved most of the cardiac pathological changes, especially changes in microstructure and ultrastructure, DAS treatment exhibited superior effects in controlling dilation of the heart chamber in cTnT R141W mice.

DAS Reduces Oxidative Stress in cTnT R141W DCM Mice
CYP2E1 catalyses the production of ROS even in the absence of substrate, leading to oxidative stress. We measured hydrogen peroxide (H 2 O 2 ), malondialdehyde (MDA) and glutathione (GSH) as indicators of oxidative stress levels in all ve groups.
Thus, inhibition of the expression of CYP2E1 by DAS resulted in reversion of the levels of H 2 O 2 , MDA and GSH in the heart to nearly the normal levels found in the 400 mg/kg group and 200 mg/kg group.
However, Enalaprilat failed to control oxidative stress in the hearts of cTnT R141W DCM mice.

DAS Inhibits Mitochondrial Pathways of Apoptosis in cTnT R141W DCM Mice
The induction of CYP2E1 causes cytochrome c release and activation of the mitochondrial apoptosis pathway in the heart in cTnT R141W DCM mice [4]. In both DAS groups, the increased release of cytochrome c in cTnT R141W DCM mice was reversed to normal levels ( Fig. 4A-4B, n = 3, P < 0.05). The activation of caspases 9 and 3 in the heart was inhibited by DAS treatment in cTnT R141W mice in a dosedependent manner (Fig. 4A, 4C-4D). The animation of caspase 9 was fell by 66.6% (Fig. 4C, n = 3, P < 0.001) and 66.9% (Fig. 4C, n = 3, P < 0.001) in the 400 mg/kg and 200 mg/kg groups, respectively, compared with the placebo control cTnT R141W group. The animation of caspase 3 was fell by 65.1% (Fig. 4D, n = 3, P < 0.001) and 62.7% (Fig. 4D, n = 3, P < 0.001) in the 400 mg/kg and 200 mg/kg groups, respectively, compared with the placebo control cTnT R141W group.
The release of cytochrome c from the mitochondria to the cytoplasm triggers the apoptosis of cardiac myocytes in cTnT R141W mice. There were 69.3% and 64.7% decreases in apoptosis in the 400 mg/kg ( Fig. 4E-4F, n = 3, P < 0.05) and 200 mg/kg groups ( Fig. 4E-4F, n = 3, P < 0.05), respectively, compared with the placebo control cTnT R141W group (Fig. 4E-4F). Interestingly, Enalaprilat exhibited stronger effects than DAS on apoptosis inhibition, but DAS and Enalaprilat had equivalent inhibitory effects on the animation of the mitochondrial apoptosis pathway.

Discussion
CYP2E1 is an inducible gene that is upregulated under multiple conditions, such as fasting, nutrition intake, and a wide variety of pathophysiological states [2,[6][7][8][9][10]33]. In addition to metabolizing endogenous substrates and xenobiotics, CYP2E1 is the main source of cellular ROS, and its NADPH oxidase activity is higher than that of other CYP family members [34,35].
We previously demonstrated that expression of CYP2E1 increased in multiple mouse models of heart disease, including models of DCM and HCM, and Myc is upregulated and binds to the CYP2E1 promoter to activate its transcription [4,23,18]. The increase in CYP2E1 induces cardiac myocyte apoptosis through mitochondrial pathways, and knockdown of CYP2E1 expression by siRNA can attenuate the pathological development of DCM in cTnT R141W mice [4].
DAS is an organosulfur compound derived from the metabolism of allicin and has anti-cancer properties [36,37]. DAS inhibits the activity of CYP2E1 and thus has attracted attention as a potential therapeutic or prophylactic agent [38,39]. DAS treatment can attenuate the pathogenesis of diseases associated with CYP2E1 overexpression in animal models, such as alcoholic liver disease, nonalcoholic steatohepatitis, diabetes, and alcoholic cardiomyopathy [12,[40][41][42][43].
DCM is one of the main causes of HF and the leading global indication for heart transplantation.
In the present study, we found that DAS treatment improved the DCM phenotypes of chamber dilation, wall thinning, myocyte disarray, brosis, poor myo bril organization and decreased ventricular blood ejection in cTnT R141W DCM mice ( Fig. 1 and Fig. 2). Furthermore, DAS treatment inhibited ROS production and decreased cytochrome c release, caspase 9-dependent caspase 3 activation, and thus apoptosis of myocytes in cTnT R141W DCM mice (Fig. 3 and Fig. 4).
Meanwhile, we compared the DAS treatment groups and Enalaprilat treatment systematically. Enalaprilat is an angiotensin-converting enzyme (ACE) inhibitor that has been widely used in the clinical treatment of HF, including in patients with DCM [29,30,[45][46][47]. The characteristic of DAS is its action speci city, its boundedness is rapid metabolism and cellular toxicity, so DAS can be chemically modi ed to create a relatively better inhibitor and weaker substrate of CYP2E1. Compared with Enalaprilat treatment, treatment with 400 mg/kg DAS resulted in a greater improvement of DCM dysfunction, as evidenced by the changes in LVFS and LVEF. Furthermore, this improvement occurred earlier in both DAS groups than in the Enalaprilat group according to the signi cance analysis after 2 weeks of treatment. DAS treatment exhibited superior effects in controlling dilation of the heart chamber in cTnT R141W mice, while Enalaprilat treatment improved most of the cardiac pathological changes clearly, especially changes in microstructure and ultrastructure. Inhibition of the expression of CYP2E1 by DAS resulted in reversion of the levels of H 2 O 2 , MDA and GSH in the heart were close to the normal levels of 400 mg/kg group and 200 mg/kg group. However, Enalaprilat failed to control oxidative stress in the hearts of cTnT R141W DCM mice. Interestingly, Enalaprilat exhibited stronger effects than DAS on apoptosis inhibition, but DAS and Enalaprilat had equivalent inhibitory effects on the animation of the mitochondrial apoptosis pathway.
The values of the three ROS indicators we measure in this paper are 5-10-fold different from those in the literature, which may be attribute to the difference from the previous methods and kits, which resulting in the difference of order of magnitude in values. However, the treatment on each group is consistent, so the conclusion obtained from this result is still meaningful.
Therefore, DAS showed stronger effects in terms of improved chamber dilation and increased ventricular blood ejection, and the improvement occurred earlier in both the DAS treatment group, while the persistence is not good in the low-dose of DAS group. Furthermore, the ROS-reducing effects of DAS were superior to those of Enalaprilat. Future studies will explore the range of diseases to which DAS can be applied and its side effects and disadvantages.

Conclusion
Diallyl sul de (DAS), a competitive inhibitor of CYP2E1, improves the typical DCM phenotype by inhibiting ROS production and myocyte apoptosis in cTnT R141W DCM mice. Our results suggest that inhibition of CYP2E1 might be a valuable therapeutic strategy to control the development of heart diseases associated with CYP2E1 overexpression. Moreover, the development of DAS analogues with superior inhibitory properties and lower substrate potential for CYP2E1 might be bene cial for patients with heart disease.

Declarations Ethical Approval
All mice were bred in an AAALAC-accredited facility, and the procedures were approved by the Animal Care and Use Committee at the Institute of Laboratory Animal Science, Peking Union Medical College (ZLF18004).

Consent for Publication
Not applicable. All authors agree to publication, and there are no permissions needed.

Availability of data and materials
The datasets used or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.     Cardiac myocyte apoptosis was detected by TUNEL assay, and the arrows indicate TUNEL-positive cells