Cucurbitacin I Protects H9c2 Cardiomyoblasts against H2O2-Induced Oxidative Stress via Protection of Mitochondrial Dysfunction

Cucurbitacin I, a triterpenoid natural compound, exhibits various pharmacological properties, including anticancer, anti-inflammatory, and hepatoprotective properties. However, antioxidant effects of cucurbitacin I in cardiac cells are currently unknown. In the present study, we assessed the preventive effects of cucurbitacin I against the oxidative stress in H9c2 cardiomyoblasts. To evaluate antioxidant effects of cucurbitacin I in H9c2 cardiomyoblasts, H2O2-treated H9c2 cells were pretreated with various concentrations of the cucurbitacin I. Cell viability, reactive oxygen species (ROS) production, and apoptosis were determined to elucidate the protective effects of cucurbitacin I against H2O2-induced oxidative stress in H9c2 cells. In addition, we assessed the mitochondrial functions and protein expression levels of mitogen-activated protein kinases (MAPKs). Cucurbitacin I prevented the cells against cell death and ROS production and elevated the antioxidant protein levels upon oxidative stress. Furthermore, cucurbitacin I preserved the mitochondrial functions and inhibited the apoptotic responses in H2O2-treated cells. Cucurbitacin I also suppressed the activation of MAPK proteins (extracellular signal-regulated kinase 1/2, c-Jun N-terminal kinase, and p38). Collectively, cucurbitacin I potentially protects the H9c2 cardiomyoblasts against oxidative stress and further suggests that it can be utilized as a therapeutic agent for the prevention of oxidative stress in cardiac injury.


Introduction
Ischemic heart disease (IHD), also known as coronary artery disease, is the most common type of cardiovascular disease, which occurred by reduced blood supply to the heart [1]. It is the most prevalent cause of death worldwide, especially in developed countries. Indeed, 110 million people are affected with ischemic heart disease and it resulted in 8.9 million deaths which make up 15.9% of all dead people [2,3]. Until now, one of the effective approaches for IHD is the surgical intervention which restores the blood flow to the ischemic region. However, restoration of blood flow paradoxically causes to cardiac tissue injury known as myocardial ischemia/reperfusion (I/R) injury [4,5]. Accumulating evidences indicate that major pathological events associated with I/R injury are oxidative stress, lipid peroxidation, intracellular calcium overload, and mitochondrial dysfunction [6]. Among them, oxidative stress which causes to accumulation of reactive oxygen species (ROS) plays a major role in the development of cardiac I/R injury [7]. Excessive ROS production leads to increased mitochondrial permeability and in turn induces apoptosis in cardiac cells, which further progresses to the chronic heart failure [8].
Recently, many natural compounds have been identified for their potential antioxidant properties by modulating the activity of antioxidant enzymes and survival signaling pathways in cardiac cells [9]. Of these, quercetin, a flavonoid found in wine, tea, and plants, has been intensively investigated for its antioxidant effect in H 2 O 2 -induced oxidative stress [10] and doxorubicin-induced cardiac injury in H9c2 cardiomyoblasts [11]. These studies demonstrated that the treatment of quercetin inhibited apoptosis, ROS production, and lipid peroxidation by modulating mitogen-activated protein kinase (MAPK) activity. Several studies also reported the beneficial actions of resveratrol, a polyphenol contained in grapes, red wine, and peanuts, in I/R-injured cardiomyocytes [12] and against the cardiotoxicity induced by various chemotherapeutic drugs, including doxorubicin and arsenic trioxide [13]. These cardioprotective roles of resveratrol are dependent upon activation of either AMPK or Sirt1 pathway [14,15].
Cucurbitacins are triterpenoids that originally isolated from the Cucurbitaceae family plants and other plant types, such as cabbage, cucumber, melon, and watermelon [16,17]. More than 40 members of cucurbitacin and their derivatives have been isolated, and cucurbitacin B, E, D, and I have been receiving special interests because of their relative abundance in various plants [16]. Cucurbitacins exhibit various biological and pharmacological activities, including antitumor, anti-inflammatory, hepatoprotective, and cytotoxic effects [18][19][20]. Particularly, most cucurbitacins, such as A, B, E, and I, affect the growth of various human cancer cell lines including breast, prostate, and brain cancer cells [21][22][23]. In addition, recent study also demonstrated that cucurbitacin B and I exert the preventive effects of adipocyte differentiation by modulating STAT3 signaling pathway [24].
In the present study, we first demonstrated that cucurbitacin I (Cu I) protects against H 2 O 2 -induced oxidative stress in H9c2 cardiomyoblasts and further determined that it could preserve the mitochondrial function and impaired the MAPK signaling for its underlying mechanisms.

Terminal Deoxynucleotidyl Transferase dUTP End
Labeling (TUNEL) Staining. Apoptosis in H9c2 cells was assessed by TUNEL assay. Briefly, cells were then treated with 500 μM H 2 O 2 alone or were pretreated with 0.1, 0.5, and 1 μM Cu I for 24 h followed by exposure to 500 μM H 2 O 2 for 24 h and were then fixed with 4% paraformaldehyde for 30 min at room temperature. TUNEL staining was performed using a Cell Death Detection kit (Roche Diagnostics, Manheim, Germany). The index of apoptosis was calculated using the formula ([number of TUNEL-positive cells/total number of cells] × 100%).
2.6. Mitochondrial Transmembrane Potential (MMP) Assessment. MMP was measured by staining with JC-1 (ThermoFisher Scientific Inc.). Briefly, 1 × 10 5 cells per well in 6-well plates were treated with 500 μM H 2 O 2 alone or were pretreated with 0.1, 0.5, and 1 μM Cu I for 24 h followed by treatment of 500 μM H 2 O 2 for 24 h and then incubated with 10 μg/mL JC-1 for 20 min at 37°C. JC-1labeled cells were observed under a fluorescence microscope (IX-81; Olympus Corp.). The fluorescence intensity of JC-1 was determined using a spectrophotometer (SpectraMax) with excitation and emission wavelengths of 550 nm and 600 nm, respectively, for red fluorescence, and 485 nm and 535 nm, respectively, for green fluorescence.
2.9. 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 < 0 05 was considered statistically significant.

Cytotoxicity of Cu I in H9c2
Cardiomyoblasts. To determine the cytotoxic effects of Cu I in H9c2 cardiomyoblasts, we evaluated the cell viability of cells treated with 0.1, 0.5, and 1 μM Cu I for 24 h and 48 h by MTT assay. Cell viability did not significantly decrease in all Cu I-treated groups compared with that in control cells (Figures 1(a) and 1(b)). ROS did not produce in cells treated with all Cu I-treated groups compared with that in control cells from the DCFH-DA staining assay (Figures 1(c) and 1(d)). Additionally, all Cu I-treated cells were shown that the protein expression of antioxidant proteins, including SOD-1, catalase, and GPx, did not change compared with those in control cells (Figures 1(e) and 1(f)). Finally, apoptosis did not induce in cells pretreated with all Cu I-treated cells compared with that in control cells from the TUNEL staining assay (Figure 1(g)) and Western blot analysis of apoptosis regulators, such as Bas and Bcl-2 (Figures 1(h) and 1(i)). Therefore, these results indicate that Cu I treatment has no cytotoxic effects on the H9c2 cardiomyoblasts.  (Figure 2(a)). These results indicate that Cu I prevents the mortality of H9c2 cardiomyoblasts induced by oxidative stress by H 2 O 2 without cytotoxic effects.  (Figures 2(b) and 2(c)).

Cu I Prevents the Accumulation of ROS Production in
Since antioxidant proteins, including SOD, catalase, and GPx, have preventive functions against oxidative stress, we further examined the expression levels of these proteins in cells pretreated with 0.1, 0.5, and 1 μM Cu I for 24 h followed by exposure to 500 μM H 2 O 2 for additional 24 h. H 2 O 2 treatment significantly decreased the expression levels of these proteins compared with those in control cells (0.43-fold, 0.45-fold, and 0.69-fold decreases in SOD1, catalase, and GPx expression versus control cells, resp.) (Figures 2(d) and 2(e)). As expected, pretreatment with Cu I significantly increased the expression of these proteins in a dose-dependent manner compared with those in H 2 O 2 -alonetreated cells (Figures 2(d) and 2(e)). Therefore, these results demonstrate that Cu I can effectively prevent accumulation of ROS production and restore the antioxidant protein levels in oxidative stress exposed-H9c2 cells.

Cu I Suppresses H 2 O 2 -Induced Apoptosis in H9c2
Cardiomyoblasts. To assess the preventive effects of Cu I on H 2 O 2 -induced apoptosis in H9c2 cells, TUNEL and Hoechst 33342 staining were performed using the cells pretreated with 0.1, 0.5, and 1 μM Cu I for 24 h followed by exposure to 500 μM H 2 O 2 for additional 24 h. TUNEL staining revealed that the percentage of TUNEL-positive cells in H 2 O 2 alone-treated cells was much higher than that in control cells (65% increase in TUNEL-positive cells versus control cells) (Figures 3(a) and 3(b)). However, the percentage of TUNEL-positive cells in Cu I-pretreated cells was significantly lower than that in H 2 O 2 alone-treated cells (33.8%,   (Figures 3(c) and 3(d)). In addition, the protein    (Figures 3(e) and 3(f)).  (Figures 4(a) and 4(b)). Similarly, the mRNA expression levels of mitochondrial biogenesis-related genes, including NRF-1, PPARα, ERRα, and PGC-1β, were significantly preserved by pretreatment with Cu I, while the decreased levels of these genes were shown in H 2 O 2 alone-treated cells (Figure 4(c)).

Cu I Blocks the Activation of MAPK Signaling Pathway in H 2 O 2 -Treated H9c2
Cardiomyoblasts. To determine the underlying mechanisms of the protective effects of Cu I against H 2 O 2 -induced oxidative stress in H9c2 cells, the expression levels of three MAPK proteins, including ERK1/ 2, JNK, and p38, were analyzed by Western blotting. The results showed that treatment with 0.1, 0.5, and 1 μM Cu I for 48 h did not significantly alter both phosphorylated and total forms of MAPK proteins (Figures 5(a) and 5(b)). In H 2 O 2 alone-treated cells, the phosphorylation of ERK1/2, JNK, and p38 was significantly increased compared with that in control cells (3.9-fold, 3.1-fold, and 1.3-fold increases in p-ERK1/2/ERK1/2, p-JNK/JNK, and p-p38/p38 versus control cells, resp.). Otherwise, pretreatment with Cu I attenuated phosphorylation of these proteins in a dose-dependent manner (

Discussion
Increasing evidence demonstrated that oxidative stress induced by excessive ROS production is involved in the pathogenesis of various heart diseases, including ischemic heart disease, myocardial infarction, and heart failure [25,26]. Since oxidative stress is mainly caused by imbalance between oxidants and antioxidants, antioxidant systems may play a crucial role in preventing cardiac injury, especially I/R injury. Furthermore, oxidative stress causes severe damage to the heart because heart is vulnerable to oxidative stress due to lower levels of antioxidant proteins than other organs [27]. Therefore, therapeutic strategy for preventing oxidative stress in cardiac cells is either supply of exogenous antioxidants or upregulation of endogenous antioxidants. Recently, naturally occurring bioactive compounds were intensively studied to find their antioxidant properties against the cardiac injury [9]. Cu I is one of the abundant members of cucurbitaceae family and exhibits cytotoxic and anticancer properties in various types of cancer cell lines [28][29][30]. Considering the pharmacological effects of Cu I against cardiac diseases, previous study showed that Cu I inhibit cardiomyocyte hypertrophy through inhibition of connective growth factor (CCN2) and transforming growth factor-(TGF-) β/SMAD signaling pathways [31]. Nevertheless, its antioxidant property in cardiac cells is still unknown. Hence, the present study sought to evaluate the antioxidant effects of Cu I in H 2 O 2treated H9c2 cardiomyoblasts.
In the present study, H 2 O 2 as a potent oxidant, which leads to reduced cell viability, antioxidant activity, and induced apoptosis [32], was used to induce the oxidative stress in H9c2 cardiomyoblasts. The present study demonstrated that pretreatment with Cu I increased the viability of H 2 O 2 -treated cells in a dose-dependent manner, while exposure to H 2 O 2 alone decreased the viability of H9c2 cells. In addition, inhibition of ROS production and increased expression of several antioxidant proteins (SOD-1, catalase, and GPx) were shown by pretreatment of Cu I in H 2 O 2induced H9c2 cells. Therefore, these results suggest that Cu I effectively protects the oxidative stress in H 2 O 2 -treated H9c2 cardiomyoblasts.
Mitochondria are major target of ROS which has the detrimental effects on the mitochondrial structure and function in cardiac injury [33,34]. Consequently, mitochondrial dysfunction triggers the apoptosis in cardiac cells under oxidative stress condition [26]. Of note, mitochondria are rich in the heart, as a high-energy demand organ, to maintain the cardiac functions [35]. Therefore, inhibiting the mitochondrial dysfunction may be an effective way to prevent the cardiac injury caused by oxidative stress. Here, we demonstrated that pretreatment with Cu I dramatically increased MMP and preserved the expression of mitochondrial biogenesis-related genes including NRF-1, PPARα, ERRα, and PGC-1-β in H 2 O 2 -exposed cells. In addition, Cu I inhibited the apoptotic responses and reduced the proapoptotic proteins (Bax and cleaved caspases 3) and increased the antiapoptotic protein (Bcl-2).
MAPK family implicated various cell functions, including proliferation, differentiation, and apoptosis [36]. When oxidative stress occurs, these MAPK proteins are activated and further stimulate the apoptotic responses [37]. Therefore, this study determined the protein expression of three major MAPK proteins, such as ERK1/2, JNK, and p38 to elucidate how Cu I protect against oxidative stress in H9c2 cardiomyoblasts. As expected, pretreatment of Cu I significantly suppressed activation of these MAPK proteins, while these were dramatically activated in H 2 O 2treated cells.

Conclusion
The present study demonstrated that Cu I effectively protects against oxidative stress responses, including cell viability, ROS production, mitochondrial dysfunction, and apoptosis in H 2 O 2 -treated H9c2 cardiomyoblasts. Cu I also blocks the activation of MAPK proteins, including ERK1/2, JNK, and p38. Therefore, we suggest that Cu I is a potent antioxidant drug to protect against oxidative stress in the heart.

Conflicts of Interest
The author declares that there is no conflict of interest regarding the publication of this article. The protein expression levels were quantified by scanning densitometry. β-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 using a one-way ANOVA followed by the Bonferroni post hoc test. # P < 0 05 and ### 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; Cu I: cucurbitacin I.