Cabbage (Brassica oleracea var. capitata) Protects against H2O2-Induced Oxidative Stress by Preventing Mitochondrial Dysfunction in H9c2 Cardiomyoblasts

Oxidative stress plays an important role in the progression of cardiac diseases, including ischemia/reperfusion injury, myocardial infarction, and heart failure. Growing evidence indicates that cabbage has various pharmacological properties against a wide range of diseases, such as cardiovascular diseases, hepatic diseases, and cancer. However, little is known about its effects on oxidative stress in cardiomyocytes or the underlying mechanisms. Therefore, the present study examined the effects of cabbage extract on oxidative stress in H9c2 cardiomyoblasts. Cell viability, reactive oxygen species (ROS) production, apoptosis, mitochondrial functions, and expression levels of mitogen-activated protein kinase (MAPK) proteins were analyzed to elucidate the antioxidant effects of this extract. Cabbage extract protected against H2O2-induced cell death and did not elicit any cytotoxic effects. In addition, cabbage extract suppressed ROS production and increased expression of antioxidant proteins (SOD-1, catalase, and GPx). Cabbage extract also inhibited apoptotic responses and activation of MAPK proteins (ERK1/2, JNK, and p-38) in oxidative stress-exposed H9c2 cells. Notably, cabbage extract preserved mitochondrial functions upon oxidative stress. These findings reveal that cabbage extract protects against oxidative stress and suggest that it can be used as an alternative therapeutic strategy to prevent the oxidative stress in the heart.


Introduction
Oxidative stress arises due to a disturbance in the balance of oxidant and antioxidant systems in the body and is characterized by excessive reactive oxygen species (ROS) production [1]. ROS are oxygen-based chemically reactive species including superoxide (O 2 − ), the hydroxyl radical (⋅OH), and hydrogen peroxide (H 2 O 2 ). Among them, H 2 O 2 is a major ROS and an important precursor of other types of ROS [2]. Excessive accumulation of ROS due to oxidative stress can lead to apoptosis, lipid peroxidation, and mitochondrial dysfunction [3]. Therefore, continued oxidative stress causes the progression and development of diseases [4,5].
Importantly, oxidative stress plays a crucial role in the pathological progression of various heart diseases, including ischemic heart disease (IHD), also known as coronary artery disease, hypertension, and ischemia/reperfusion (I/R) injury [6][7][8]. Among them, IHD, which could cause myocardial infarction, is the leading cause of morbidity and mortality worldwide [9]. Until now, the effective treatment for IHD is surgical intervention. However, surgical restoration of blood flow to the ischemic region is paradoxically accompanied by cardiac injury called I/R injury [10]. Oxidative stress plays a crucial role in the pathophysiology of this I/R injury [11].
Overproduction of ROS, which mainly generated in the mitochondria, causes mitochondrial oxidative stress and further triggers mitochondrial dysfunction, including mitochondrial biogenesis, fatty acid metabolism, and antioxidant defense mechanisms. The heart, especially, as a high-energyconsuming organ, is susceptible to the mitochondrial dysfunction. Indeed, emerging evidences have linked mitochondrial dysfunction resulting from oxidative stress to many cardiac diseases, including myocardial infarction, cardiac hypertrophy, and heart failure [12]. Therefore, an antioxidant strategy should be considered as a possible approach to prevent the cardiac diseases. Cabbage (Brassica oleracea var. capitata) from the Brassicaceae family is an important vegetable worldwide. It grows extensively in more than 90 countries, such as coastal regions of southern and western Europe [13]. Cabbage is rich in various nutrients, including calcium, proteins, and vitamins C and E and contains various bioactive compounds with pharmacological properties, such as luteolin, myricetin, quercetin, and polyphenols [14]. Consequently, cabbage has been widely used in traditional medicine to treat various diseases. Specifically, it is used to alleviate symptoms associated with gastrointestinal disorders (gastritis, peptic ulcers, and irritable bowel syndrome) and idiopathic cephalalgia as well as treat injuries [15]. Growing evidence indicates that cabbage has pharmacological activities against various diseases, including liver cirrhosis, hepatitis, cancer, and hypocholesterolemia [16][17][18][19]. A recent study demonstrated that cabbage prevents pancreatitis and identified six bioactive compounds, including several flavonoids, by gas chromatography-mass spectrometry analysis [14]. In addition, another recent study reported that white cabbage essential oil contains organic polysulphides and has antioxidant and hepatoprotective properties in rats with carbon tetrachloride-induced liver damage [20]. However, the effects of cabbage on oxidative stress in cardiac cells have not been studied.
Present study demonstrated that cabbage protects against H 2 O 2 -induced oxidative stress in H9c2 cardiomyoblast by preventing mitochondrial dysfunction.

Preparation of Cabbage Extract.
Fresh cabbages were purchased from a local market in Korea, dried in an incubator at 60 ∘ C, and converted into a powder using an electric blender. The dried powder was extracted in 99% methanol (powder sample/99% methanol, 1:8) at 90 ∘ C for 3 h. The extracts were filtered and evaporated in a rotary evaporator (EYELA, Tokyo, Japan). The resultant yield of extract was 21.6% of dry weight. The extracts were stored in −80 ∘ C for further study. alone or were pretreated with different concentrations of cabbage extract (100, 200, and 300 g/ml). Thereafter, 0.5 mg/ml MTT was added to each well. After incubation at 37 ∘ C for 2 h, the supernatants were removed and the crystals were dissolved in 100 l DMSO. Absorbance was measured at 570 nm using a spectrophotometer (Spectra Max M5; Molecular Devices, Sunnyvale, CA, USA).

Measurement of ROS Production.
Intracellular ROS production was measured based on the fluorescence intensity of DCF-DA (2 , 7 -dichlorofluorescin-diacetate; Cat. No. D399, ThermoFisher Scientific Inc., Waltham, MA, USA). Briefly, cells in 6-well plates (1X10 5 cells/well) were treated with H 2 O 2 alone or were pretreated with different concentrations of cabbage extract (100, 200, and 300 g/ml) and then treated with 1 M DCF-DA for 30 min at 37 ∘ C. Cells were observed under a fluorescence microscope (IX-81; Olympus Corp., Shinjuku, Tokyo, Japan). The fluorescence intensity was determined using a spectrophotometer (Spectra Max) with excitation and emission wavelengths of 488 and 515 nm, respectively. T3168, ThermoFisher Scientific Inc.). Briefly, cells in 6-well plates (1X10 5 cells/well) were treated with H 2 O 2 alone or were pretreated with different concentrations of cabbage extract (100, 200, and 300 g/ml) and then incubated with 10 g/ml JC-1 for 20 min at 37 ∘ C. JC-1-labeled cells were observed under a fluorescence microscope (IX-81; Olympus Corp.). The fluorescence intensity of JC-1 was determined using a spectrophotometer (Spectra Max) with excitation and emission wavelengths of 550 nm excitation and 600 nm emission, respectively, for red fluorescence, and 485 nm excitation and 535 nm, respectively, for green fluorescence. No. #9664, Cell Signaling Tech.), and -actin (Cat. No. sc-47778, Santa Cruz Biotechnology). The membranes were then incubated with the appropriate horseradish peroxidaseconjugated secondary antibodies (Jackson ImmunoResearch Lab., Inc., West Grove, PA, USA) at room temperature for 1 h. Signals were detected using an Immobilon Western Chemiluminescence kit (Cat. No. WBKLS0100, Millipore Corp., Billerica, MA, USA) and a UVITEC Mini HD9 system (Cleaver Scientific Ltd., Warwickshire, UK). The intensity of each protein band was quantified using NIH Image J software.

Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR).
Total RNA was isolated from the cells treated with H 2 O 2 alone or were pretreated with different concentrations of cabbage extract (100, 200, and 300 g/ml) by using a Ribospin6 II kit (Cat. No. 304-150, GeneAll Biotechnology Co., LTD, Seoul, Korea). To examine the mRNA expression levels of mitochondrial biogenesis genes, 1 g total RNA from each group of cells was reverse transcribed into cDNA using ImProm II reverse transcriptase (Cat. No. A3802, Promega Co., Madison, WI, USA) with oligo-dT priming. qRT-PCR was conducted using a TaKaRa Thermal Cycler Dice Real-Time System (Takara Bio. Inc., Shiga, Japan) with SYBR Green (

Statistical Analysis.
Data were analyzed using a one-way analysis of variance (ANOVA) with the Bonferroni post hoc test using Prism 5.03 (GraphPad Software Inc., San Diego, CA, USA). All the results are expressed as mean ± SEM. p values < 0.05 were considered statistically significant.

Cabbage Extracts Protect H9c2 Cardiomyocytes against
To determine the protective effects of cabbage extract against H 2 O 2 -induced injury in H9c2 cardiomyocytes, the MTT assay was performed to assess the viability of cells treated with 10, 50, 100, 200, 300, 500, 1000, and 2000 g/ml cabbage extract for 48 h alone. The results have shown that cell viability did not significantly change in the cells treated with the concentrations from 10 to 5000 g/ml cabbage extract; otherwise, the treatments of 1000 and 2000 g/ml cabbage extract caused reduction in the cell viability compared with that in control cells (Figures 1(a) and 1(b)). In addition, the cells treated with 100 to 300 g/ml cabbage extract have shown antioxidant proteins (SOD-1, catalase, and GPx) and apoptosis-related proteins (Bax, Bcl-2, and cleaved caspase 3) (Figures 1(c)-1(f)). Therefore, 100, 200, and 300 g/ml cabbage extract were chosen to elucidate the effects against oxidative stress on H9c2 cardiomyocytes. The viability of H 2 O 2 -treated cells was reduced to 42.6% compared with that of control cells. Pretreatment with 100, 200, and 300 g/ml cabbage extract for 24 h significantly restored the viability of H 2 O 2 -treated cells to 56%, 72%, and 93%, respectively, compared with that of control cells (Figure 1(b)). These results indicate that cabbage extract prevents death of H9c2 cardiomyocytes caused by H 2 O 2induced oxidative stress in a dose-dependent manner.

Cabbage Extract Inhibits H 2 O 2 -Induced ROS Production in H9c2
Cardiomyocytes. To determine whether cabbage extract can reduce ROS production, H9c2 cardiomyocytes were pretreated with cabbage extract (100, 200, and 300 g/ml) for 24 h and then treated with 500 M H 2 O 2 for an additional 24 h. ROS levels were assessed using DCFH-DA. H 2 O 2 treatment significantly increased intracellular ROS generation (140.6% versus control cells). However, pretreatment with cabbage extract dramatically decreased the ROS level in a dose-dependent manner (123.5%, 112.9%, and 102.1% in cells pretreated with 100, 200, and 300 g/ml cabbage extract versus control cells, respectively) (Figures 2(a) and 2(b)).
ROS-scavenging proteins, including SOD, catalase, and GPx, play important roles in the oxidant/antioxidant balance and prevent oxidative stress; therefore, their protein expression levels were analyzed by western blotting. H 2 O 2 treatment significantly decreased the expression levels of these proteins in a dose-dependent manner (0.53-, 0.45-, and 0.42-fold decreases in SOD1, catalase, and GPx expression versus control cells, respectively) (Figures 2(c) and 2(d)). As expected, pretreatment with cabbage extract dramatically restored these protein expression levels in a dose-dependent manner compared with those in H 2 O 2 alone-treated cells. Hence, cabbage extract effectively inhibits ROS production and restores expression of antioxidant proteins in H 2 O 2treated H9c2 cells.

Cabbage Extract Prevents Mitochondrial Dysfunction upon H 2 O 2 -Induced Oxidative Stress in H9c2 Cardiomyocytes.
To assess the prevention of oxidative stress by cabbage extract, the MMP and mitochondrial integrity in H 2 O 2 -treated H9c2 cells were analyzed. The percentage of cells labeled with the MMP sensor JC-1 was dramatically lower among H 2 O 2 alonetreated cells than among control cells (62.3% decrease among H 2 O 2 -treated cells versus control cells) (Figures 5(a) and 5(b)). As expected, the percentage of JC-1 labeled cells was significantly higher among cells pretreated with 100, 200, and 300 g/ml cabbage extract than among H 2 O 2 alonetreated cells (14.3%, 37.4%, and 46.7% increases versus H 2 O 2 alone-treated cells, respectively) ( Figures 5(a) and 5(b)). The expression levels of genes involved in mitochondrial biogenesis, including NRF-1, PPAR , ERR , and PGC-1 were determined. The expression levels of these genes were significantly reduced in H 2 O 2 -treated cells, otherwise, and were restored by pretreatment with cabbage extract (Figure 5(c)).

Discussion
Over the past several decades, experimental and clinical studies have implicated oxidative stress mediated by excessive ROS in a variety of cardiomyopathies, such as I/R injury, myocardial infarction, and heart failure [3,21,22]. The heart is susceptible to oxidative stress because it contains lower levels of antioxidant proteins than other organs [23]. This decreased antioxidant capacity of cardiac cells is a major contributor to oxidative stress, which causes progression of pathological heart conditions. Therefore, a therapeutic strategy that can prevent oxidative stress in the heart is to supply exogenous antioxidants or to upregulate endogenous antioxidants. Intensive efforts have been made to identify exogenous antioxidants that can prevent oxidative stress in the heart and to elucidate their underlying mechanisms. Recently, the antioxidant activities of naturally occurring, plant-derived natural compounds have been intensively studied due to their safety and efficacy against oxidative stress-induced cardiovascular diseases [24]. Various herbal plants have been identified as new agents in the treatment of oxidative stress [25][26][27][28]. Furthermore, several natural antioxidants, including vitamin E, flavonoids, and polyphenols, have been exploited [29]. Curcumin has been intensively investigated due to its potent antioxidant properties against oxidative stress in the heart as well as its inhibitory effects on various heart diseases [30,31]. The present study sought to determine the  -Actin was used as the loading control. Western blot analysis was performed in triplicate with three independent samples. Data are expressed as fold changes ± SEM versus control cells. Significance was analyzed by a one-way ANOVA followed by the Bonferroni post hoc test. ### P < 0.001 versus control cells. * P < 0.05, * * P < 0.01, and * * * P < 0.001 versus H 2 O 2 alone-treated cells. Cont, control; Cab, cabbage extract. Scale bar, 100 m.  . Western blot analysis was performed in triplicate with three independent samples. Data are expressed as fold changes ± SEM versus control cells. Significance was analyzed by a one-way ANOVA followed by the Bonferroni post hoc test. ## P < 0.01 and ### P < 0.001 versus control cells. * P < 0.05 and * * P < 0.01 versus H 2 O 2 alone-treated cells. Cont, control; Cab, cabbage extract. Scale bar, 100 m.
antioxidant effects of cabbage extract in H 2 O 2 -exposed H9c2 cardiomyoblasts.
Since H 2 O 2 causes oxidative stress and thereby markedly decreases viability and induces apoptosis in H9c2 cells [32,33], this study confirmed that the viability of H9c2 cells was decreased following exposure to H 2 O 2 for 24 h.
However, pretreatment with cabbage extract increased the viability of H 2 O 2 -treated cells in a dose-dependent manner. Furthermore, pretreatment with cabbage extract inhibited intracellular ROS production and increased expression of antioxidant proteins, including SOD-1, catalase, and GPx, while H 2 O 2 treatment resulted in accumulation of ROS and Evidence-Based Complementary and Alternative Medicine 9 reduced levels of these antioxidant proteins in H9c2 cells. These results suggest that cabbage extract has protective effects against oxidative stress in H 2 O 2 -treated H9c2 cells.
Accumulation of ROS can be a crucial mediator of apoptotic cell death [34]. Apoptosis of cardiac cells, which do not proliferate once differentiated, markedly triggers heart dysfunction [35,36]. Therefore, this study determined whether cabbage extract could prevent apoptotic responses and the related signaling pathway in H 2 O 2 -treated cells. TUNEL and Hoechst 33342 staining revealed that H 2 O 2 treatment triggered fragmentation and condensation of DNA in nuclei, typical features of apoptosis, whereas pretreatment with cabbage extract significantly attenuated these apoptotic changes in H 2 O 2 -treated cells. In addition, cabbage extract reduced levels of proapoptotic proteins, such as Bax and cleaved caspase 3, and increased the level of Bcl-2, an antiapoptotic protein.
The MAPK signaling pathways play crucial roles in the regulation of cell survival, apoptosis, and inflammatory responses in various pathological conditions [37,38]. Activation of these MAPK signaling pathways upon oxidative stress can stimulate apoptosis [21,39]. Therefore, the present study analyzed the expression of three major MAPK proteins (ERK1/2, JNK, and p38) to elucidate the preventive effects of cabbage extract against oxidative stress in H9c2 cells. Here, I demonstrated that activation of these proteins by H 2 O 2 treatment was significantly suppressed by pretreatment with cabbage extract.
Importantly, previous study demonstrated that the heart has a high-energy demand and mitochondria therefore play a pivotal role in maintaining heart functions, such as energy production and cardiac contractile function [40]. Furthermore, overproduction of ROS due to oxidative stress can damage mitochondria, and, in turn, mitochondrial dysfunction can mediate apoptosis and is one of the major contributors to cardiac diseases [41]. Thus, prevention of mitochondrial dysfunction may be a therapeutic strategy to inhibit cardiac injury caused by oxidative stress. Regarding this, I sought to elucidate the preventive effects of cabbage extract against mitochondrial dysfunction upon oxidative stress for the underlying mechanism. As expected, pretreatment with cabbage extract dramatically restored the mitochondrial function against oxidative stress by performing several mitochondrial functional assays, including MMP assay, and expression of mitochondrial biogenesis genes.
This study provides new evidence that cabbage extract protects against oxidative stress in H9c2 cardiomyoblast by inhibiting ROS production and apoptosis and by preserving mitochondrial functions. Additionally, the present study demonstrates that cabbage suppresses activation of proapoptotic MAPK proteins in H9c2 cells exposed to oxidative stress. I propose that cabbage is a potential antioxidant-agent to protect against oxidative stress.

Conflicts of Interest
The author declares that there are no conflicts of interest.