Sanguinarine inhibits angiotensin II-induced apoptosis in H9c2 cardiac cells via restoring reactive oxygen species-mediated decreases in the mitochondrial membrane potential

Cell apoptosis induced by Angiotensin II (Ang II) has a critical role in the development of cardiovascular diseases. The aim of the present study was to investigate whether sanguinarine (SAN), a drug which was proved to have anti-oxidant, anti-proliferative and immune enhancing effects, can abolish cell apoptosis induced by Ang II. In the present study, H9c2 cardiac cells were stimulated with 10 µM Ang II with or without SAN. The level of intracellular reactive oxygen species (ROS) generation was assessed using dichlorodihydrofluorescein diacetate, and changes of the mitochondrial membrane potential (MMP) were assessed using JC-1 staining. Furthermore, mRNA expression of NOX2 was determined by reverse transcription quantitative polymerase chain reaction, and apoptosis was detected by Annexin V/propidium iodide staining and flow cytometry. The expression of B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax) as well as cleaved (c)-caspase 3 and -9 were detected by western blot analysis, and the activity of caspase 3 and -9 was detected using an ELISA. The results of the present study showed that NOX2 expression and ROS generation induced by Ang II were inhibited by SAN, and the Ang 2-induced MMP loss was also ameliorated. Furthermore, Ang II-induced H9c2 cardiac cell apoptosis as well as c-caspase 3 and -9 levels were significantly reduced by SAN. Investigation of the possible pathway involved in the anti-apoptotic effect of SAN showed that the expression of Bcl-2 was decreased, while that of Bax was increased following stimulation with Ang II, which was reversed following treatment with SAN. In addition, Ang II enhanced the activity of caspase 9 and cleaved downstream caspases such as caspase-3, initiating the caspase cascade, while pre-treatment of H9c2 cardiac cells with SAN blocked these effects. In conclusion, the findings of the present study indicated that SAN inhibits the apoptosis of H9c2 cardiac cells induced by Ang II, most likely via restoring ROS-mediated decreases of the MMP.


Introduction
Apoptosis is a common cause of various diseases. It is one of the factors that is able to reduce cardiac contractility and can also lead to heart failure through multiple pathways. Certain studies have indicated that cardiomyocyte apoptosis promotes the transition from compensatory cardiac hypertrophy to heart failure in response to pressure overload. (1)(2)(3).
It is well established that the renin-angiotensin-aldosterone system (RAAS) is activated in cardiovascular diseases, including hypertension (4), atherosclerosis (5), left ventricular hypertrophy (LVH) (6) and heart failure (6). Angiotensin II (AngII) is the primary active peptide hormone of the RAAS, which impairs the homeostasis between the production and elimination of reactive oxygen species (ROS) through NADPH oxidase, cause a series of cascade reactions, and finally leads to cardiomyocyte hypertrophy and apoptosis (7,8). Ang II also induces H9c2 cardiac cell hypertrophy, oxidative stress, mitochondrial dysfunction and cell apoptosis through Ang II type 1 receptor activation (9,10).
Since the mitochondrial membrane potential (MMP) has central roles in cardiomyocyte apoptosis, Ang II also induces apoptosis via the mitochondrial-dependent apoptotic pathway (11); therefore, anti-oxidant supplementation may ameliorate apoptosis induced by Ang II, which may be an effective therapeutic method for cardiovascular disease.
Sanguinarine (SAN), derived from the root of Sanguinaria canadendid is a benzophenanthridin alkaloid (12). As a Traditional Chinese Medicine, SAN has been proved to have significant anti-bacterial, anti-oxidant, anti-proliferative, anti-tumor and immune enhancing effects (13). Previous studies indicated that SAN accelerated cell apoptosis and inhibited

Sanguinarine inhibits angiotensin II-induced apoptosis in H9c2 cardiac cells via restoring reactive oxygen species-mediated decreases in the mitochondrial membrane potential
cell proliferation in cancer cells (14,15). A recent study by our group demonstrated that SAN exerted a significant protective effect against pressure overload-induced cardiac remodeling via inhibition of nuclear factor-κB activation (16). However, whether SAN has protective effects against Ang II-induced H9c2 cardiac cell apoptosis has remained elusive. Therefore, the present study investigated the effect of SAN on the apoptosis, ROS generation and mitochondrial dysfunction of H9c2 cardiac cells induced by Ang II.

Materials and methods
Cell culture. Measurement of the mitochondrial transmembrane potential (MMP; ΔΨm). The change of the mitochondrial membrane potential (MMP) was assessed using the fluorescent dye 5,5' ,6,6'-tetrachloro-1,1' ,3,3'-tetraethylimidacarbocyanine iodide (JC-1; C2005; Beyotime Institute of Biotechnology, Shanghai, China). After the designated treatment of the cells, 100 µl serum-free DMEM basic (1X) and 5 µg/ml JC-1 was added to the cells, followed by incubation at 37˚C for 20 min. Following two washes with PBS, the FI of JC-1 monomer was analyzed by capturing excitation and emission spectra at 485 and 530 nm, respectively, with a Synergy HT. JC-1 multimer FI was analyzed via excitation and emission spectra at 528 and 590 nm, respectively. The mean values of the five wells were used to determine the percentage of FI levels according to the following formula: FI levels (%) = FI (treatment group)/FI (control group) x100%. In addition, cells labeled with JC-1 were observed using fluorescence microscopy. JC-1 fluorescence was measured using a single excitation wavelength (485 nm) with dual emission (shift from green at 530 nm to red at 590 nm). Caspase-3 and caspase-9 activity assay. ELISA kits were used to detect the activity of caspase-3 using the caspase-3 activity kit (C1115; Beyotime Institute of Biotechnology). Caspase-9 activity was assessed using the caspase-9 activity kit (H082; Nanjing Jiancheng Bioengineering, Nanjing, China). The two indicators were measured with the corresponding detection kit according to the manufacturer's instructions.

Reverse transcription quantitative polymerase chain reaction (RT-qPCR
Statistical analysis. Data are expressed as the mean ± standard error of the mean and analyzed using SPSS 19.0 (SPSS, Inc., Chicago, IL, USA). Comparisons between two groups were performed using an unpaired Student's t-test. Differences among groups were de termined by one-way analysis of variance followed by Student-Newman-Keuls tests. P<0.05 was considered to indicate a sta tistically significant difference between values.

SAN does not affect the viability of H9c2 cardiac cells.
The cytotoxicity of SAN and the ROS scavenger NAC were assessed in the presence or absence of Ang II by CCK-8 assay (Fig. 1). The viability of H9c2 cardiac cells treated with various concentrations of SAN with or without Ang II was lower than that of the control group, while cell viability remained >85% in all groups (Fig. 1A). Furthermore, the viability of the H9c2 cardiac cells in the Ang II and NAC + Ang II groups was the same as that in the control group (Fig. 1B). These results indicated that SAN and NAC exerted no cytotoxic effect on H9c2 cardiac cells.

SAN inhibits ROS generation induced by Ang II.
Previous studies showed that Ang II can generate excess intracellular ROS via stimulation of NADPH oxidase (8). In the present study, a marked increase in ROS was observed in H9c2 cardiac cells treated with Ang II, while SAN suppressed ROS generation in a dose-dependent manner ( Fig. 2A). Furthermore, NAC exerted a similar ROS-decreasing effect following co-treatment with Ang II (Fig. 2B). These results were further confirmed by fluorescence microscopy, which showed that the fluorescence of DCFH-DA observed in the Ang II-treated group was effectively suppressed following treatment with SAN or NAC (Fig. 3).

A B
monomers was increased and that of JC-1 multimers was decreased in the Ang II-treated group compared with that in the control group. When H9c2 cardiac cells were co-treated with SAN at various concentrations, the decreased MMP as well as the amount of JC-1 monomers and -multimers were restored to normal levels in a dose-dependent manner (Fig. 5A). In the positive control group, in analogy to the effect of SAN, NAC reversed the MMP loss induced by Ang II (Fig. 5B). These results were further confirmed using fluorescence microscopy. After the indicated treatments of H9c2 cardiac cells for 24 h, cells were stained with JC-1 and red/green image densities were measured. The green fluorescence density, indicating JC-1 monomers the red fluorescence density indicating multimers. A collapse of the MMP, was markedly increased in the Ang II group, while treatment with SAN or NAC was able to shift the green fluorescence to red fluorescence and therefore a restored MMP (Fig. 6).

SAN attenuates Ang II-induced apoptosis.
Previous studies have proved that SAN is able to regulate cell apoptosis (14). Thus, the present study investigated the anti-apoptotic effect of SAN in Ang II-treated H9c2 cardiac cells. Compared with the control group, Ang II significantly promoted cell apoptosis, as shown by the Annexin V/PI staining: The early apoptotic rate was significantly enhanced (25.3%) compared to that in the control group (1.8%), while SAN and NAC attenuated the level of apoptosis, with the early apoptotic rate decreased to 14.5 and 14.4%, respectively (Fig. 7).

SAN ameliorates the expression of apoptosis family proteins.
The lysate of cells of the experimental groups was assessed regarding the expression of c-caspase 3, c-caspase 9 and Bcl-2 family proteins. The expression of c-caspase3 and c-caspase 9 was increased in the Ang II group, while the expression of the anti-apoptotic protein Bcl-2 was significantly decreased. Furthermore, the expression levels of pro-apoptotic protein Bax were increased. These results are in line with the finding that Ang II induced H9c2 cardiac cell apoptosis. Treatment with SAN or NAC was able to significantly reduce the Ang II-induced expression of c-caspase 3 and -9 as well as Bax, and to decrease the expression of Bcl-1 (Fig. 8). The results therefore indicated that SAN is able to block apoptosis of cardiac cells by interfering with the mitochondrial-mediated apoptosis signaling pathway.

SAN inhibits caspase-3 and caspase-9 activation induced by
Ang II. Caspase-3 and caspase-9 activity were significantly increased in Ang II-treated H9c2 cardiac cells, while SAN and

Ab Bb
NAC significantly inhibited Ang II-induced caspase activation (Fig. 9). This result further confirmed the mechanism of action of SAN as an inhibitor of the mitochondrial-mediated apoptotic pathway to inhibit apoptosis of cardiac cells.

Discussion
The present study demonstrated that Ang II increased ROS generation, initiating a signaling cascade contributing to cardiac cell apoptosis caused by Ang II, which is likely to be due to restoration of ROS-mediated decreases of the MMP (Fig. 10). Increasing evidence suggests that Ang II treatment significantly increases NADPH oxidase activity via the Ang II receptor, type 1, and subsequently leads to ROS generation, which can cause damage to mitochondria and lipid peroxidation (17,18). A study by Chu et al (19) showed that Ang II

Aa Ba
Bb Ab Figure 9. Effects of SAN on the activities of (A) caspase-3 and (B) caspase-9. After stimulation by Ang II (10 µM), the activity of caspase-3 and caspase-9 was significantly increased, while the activity of caspase-3 and caspase-9 was significantly decreased after SAN (0.5 µM) or NAC (1 mM) treatment. Values are expressed as the mean ± standard error of the mean for three independent experiments, # P<0.01 vs. control; * P<0.01 vs. Ang II group. SAN, sanguinarine; Ang, angiotensin; NAC, N-acetylcysteine.

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was able to stimulate intracellular Ca 2+ accumulation, which altered the MMP and caused a release of cytochrome C from the mitochondria to the cytoplasm and subsequent apoptotic cascades in neonatal rat ventricular myocytes. Chang et al (20) demonstrated that caspase 3 and -9 were increased in H9c2 cardiac cells stimulated by Ang II, which was confirmed by the present study. Therefore, it was hypothesized that Ang II causes mitochondrial damage and MMP loss via ROS generation and subsequent activation of apoptosis, and the present study was designed to investigate whether SAN was able to decrease ROS generation in H9c2 cardiac cells and ameliorate MMP loss and apoptosis caused by Ang II. Numerous studies have focused on SAN as an anti-oxidant and anti-inflammatory drug (21)(22)(23). Ahmad et al (14) showed that low-dose SAN (1 µM) treatment of A431 cells resulted in a significantly decreased cell viability and an enhanced apoptotic index, while this treatment had no effect on NHEK normal keratinocyte cells, which exclusively showed necrotic staining at the high doses of 2-5 µM. In the present study, in order to avoid H9c2 cardiac cell necrosis, a maximum SAN concentration of 0.5 µM was used, and after treatment for 12 h, the viability of H9c2 cardiac cells treated with different concentrations of SAN with or without Ang II was lower than that in the control group, while always remaining >85%; this indicated that SAN had no significant toxicity to H9c2 cardiac cells.
The chemical reactivity of SAN is based on the nucleophilic character of its iminium moiety, which may participate in oxidant scavenging and/or enzyme inhibition (24). SAN was shown to inhibit phorbol myristate-induced oxidative burst (25), and the most important enzyme in oxidative burst is the NADPH oxidase complex (26). SAN may have exerted its anti-oxidative function by impairing the activity of the NADPH enzyme, which is supported by a study by Qin et al (26), which demonstrated that SAN is an enzyme inhibitor rather than an ROS scavenger. NADPH oxidases are transmembrane enzymes designated to produce superoxide by transferring an electron from NADPH to molecular oxygen. NOX2 is a major NADPH oxidase isoform expressed in cardiac cells (27,28), and it is well established that several actions of NOX2 NADPH in cardiac remodeling are activated through activation by Ang II, and alongside tumor necrosis factor alpha, it constitutes the main source of ROS generation (29,30). NOX2-derived ROS has a major role in the regulation of cardiac hypertrophy, apoptosis, fibrosis and mitochondrial dysfunction (26,31,32). As NOX2 NADPH oxidase has an important role in ROS generation induced by Ang II, it was speculated that SAN impaired the generation of ROS through decreasing the expression of NOX2 NADPH oxidase. These hypotheses were confirmed by the results of the present study, showing that SAN (0.5 µM) inhibited NOX2 NADPH oxidase activity in a time-dependent manner and that ROS generation induced by Ang II was also inhibited by SAN in a dose-dependent manner.
In the present study, ROS were significantly elevated and the MMP was declined in the Ang II-treated group. A high level of ROS production may cause mitochondrial oxidative attack as well as accumulating damage to mitochondrial DNA and proteins, which further stimulates ROS generation (ROS-induced ROS release) (33). These interactions cause mutual damage and lead to H9c2 cardiac cell apoptosis, which, however, was demonstrated to be inhibited by pre-treating the H9c2 cardiac cells with SAN. NOX2 levels induced by Ang II were also inhibited by SAN, and Ang II-induced H9c2 cardiac cell apoptosis as well as c-caspase 3 and -9 expression were significantly reduced by SAN. Furthermore, the present study investigated the possible molecular pathway underlying the anti-apoptotic effect of SAN. The results showed that the expression of Bcl-2 was decreased, while Bax increased following treatment with Ang II, which was rescued by treatment with SAN. In addition, Ang II enhanced the activation of caspase 9 and caspase 3, while pre-treatment of H9c2 cardiac cells with SAN blocked these effects. Pre-treatment with NAC as a positive reference exerted similar effects to those of SAN on protein expression, changes in the MMP and the apoptotic index in response to Ang II.
In conclusion, the present study provided novel insight into the cardioprotective effect of SAN as well as the underlying molecular mechanisms. SAN inhibits ROS generation, MMP loss and apoptosis of H9c2 cardiac cells induced by Ang II, possibly via inhibiting NOX2 and the mitochondrial-mediated apoptotic pathway. Although the precise mechanism remains to be fully elucidated, the present study may contribute to the selection of SAN as a candidate drug for the treatment or prevention of cardiovascular diseases.