Forsythiaside A inhibits hydrogen peroxide-induced inflammation, oxidative stress, and apoptosis of cardiomyocytes

Purpose: To investigate the effect of forsythiaside A on heart failure. Methods: An in vitro cell model of myocardial injury was established by incubating H9c2 primary cardiomyocytes with hydrogen peroxide (H2O2). Apoptosis was measured by flow cytometry. Expression of inflammatory factors, including tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6), was determined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and enzymelinked immunosorbent assay (ELISA). Oxidative stress was evaluated by measuring malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) levels by ELISA. Results: Incubation with H2O2 increased H9c2 cell apoptosis (p < 0.001). Treatment with forsythiaside A reduced Bax expression and enhanced Bcl-2 expression which suppressed apoptosis of H2O2induced H9c2 cells. Forsythiaside A also attenuated the H2O2-induced increase in TNF-α and IL-6 expressions in H9c2 cells (p < 0.001). The H2O2-induced increase in MDA and decrease in SOD and GSH-Px in H9c2 cells were reversed by treatment with forsythiaside A. IκBα protein expression was downregulated, whereas p65 phosphorylation (p-p65), p-IκBα, nuclear factor erythropoietin-2-related factor 2 (Nrf2), and heme oxygenase 1 (HO-1) were upregulated in H2O2-induced H9c2 cells. Forsythiaside A increased IκBα, Nrf2, and HO-1 expression and decreased p-p65 and p-IκBα expression in H2O2-induced H9c2 cells. Conclusion: Forsythiaside A exerts anti-inflammatory, anti-oxidant, and anti-apoptotic effects against H2O2-induced H9c2 cells through inactivation of NF-κB pathway and activation of Nrf2/HO-1 pathway. These results support the potential clinical application of forsythiaside A for the treatment of heart failure.


INTRODUCTION
Heart failure occurs when the heart is unable to pump blood adequately to maintain the body's needs [1]. Heart failure results in ventricular filling or impaired ejection and leads to multifactorial disorders [1,2]. Although heart failure associates with high morbidity and mortality, there is currently no ideal treatment regimen for the disease [1].
Oxidative stress and inflammation have attracted extensive attention as important pathophysiological contributors to heart failure syndrome [3]. Excessive and unregulated production of reactive oxygen and reactive nitrogen species in the heart promotes oxidative stress and myocardial cell damage [4]. Moreover, oxidative stress induces inflammation, which contributes to heart failure [4]. Therefore, antioxidants and anti-inflammatory drugs are regarded as potential therapies for heart failure.
Forsythiaside A is the main bioactive component extracted from Forsythia suspensa (Thunb.) Vahl and it has hepatoprotective, neuroprotective, antiviral, anti-inflammatory, and antioxidant properties [5]. Forsythiaside A protected mice against ovalbumin-induced asthma through its anti-inflammatory properties [6]. Forsythiaside A also effectively inhibited hydrogen peroxide (H2O2)-induced oxidative stress and apoptosis in PC12 cells [7]. However, there are limited studies on the effect of forsythiaside A on heart failure.
In this study, H2O2-induced myocardial injury in H9c2 primary cardiomyocytes was established as an in vitro cell model of heart failure. The effects of forsythiaside A on inflammation, oxidative stress, and apoptosis of H2O2-induced H9c2 cells were investigated.

Cell culture and treatment
The primary cardiomyocyte cell line H9c2 was purchased from the Chinese Academy of Sciences (Shanghai, China) and was cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum and 1% penicillin/streptomycin (Gibco, Carlsbad, CA, USA) in a 37 °C incubator. The cells were treated with 2.5, 5, or 10 μg/mL forsythiaside A (Sigma-Aldrich, St. Louis, MO, USA) for 6 h and then incubated with 150 μmol/L H2O2 for another 24 h.

Flow cytometry
H9c2 cells were harvested and resuspended in the 1X binding buffer from the Annexin V/FITC kit (Beyotime, Shanghai, China). Then, 5 μL of Annexin V/FITC and 10 μL propidium iodide were added to probe the H9c2 cells. The cells were then analyzed using a FACScan flow cytometer (Becton-Dickinson, Mountain View, CA, USA) to evaluate apoptosis.

Quantitative reverse transcription PCR (qRT-PCR)
Total RNA was extracted from H9c2 cells with the RNAeasy Mini Kit (QIAGEN, Gaithersburg, MD), and the RNA was reverse-transcribed into cDNA with the QuantiTect Reverse Transcription Kit (QIAGEN). The QuantiTect SYBR Green PCR Kit (QIAGEN) was used to measure TNF-α and IL-6 expression with GAPDH as the internal control. The qRT-PCR conditions were 95 °C for 10 min, 40 cycles of 95 °C for 10 s, and 60 °C for 60 s. Primers are shown in Table 1.

Statistical analysis
Experiments were performed in triplicate at the least and data were presented as mean ± standard deviation. Statistics between two groups were analyzed using the Student's t-test in GraphPad Prism 7 (GraphPad Inc., San Diego, CA, USA). One-way Analysis of Variance was used to analyze differences between multiple groups. A p value < 0.05 was considered statistically significant.

Forsythiaside A attenuated H2O2-induced apoptosis in H9c2 cells
To establish an in vitro model of heart failure, H9c2 cells were incubated with H2O2 and then analyzed by flow cytometry, which showed that apoptosis of H9c2 cells increased upon incubation with H2O2 (Figure 1 A). Western blot and qRT-PCR analyses showed that Bax expression increased and Bcl-2 expression decreased upon incubation with H2O2 ( Figure  1B). However, pretreatment of H9c2 cells with forsythiaside A reduced H2O2-induced cell apoptosis in a dose-dependent manner (Figure 1  A). Moreover, the increased Bax and decreased Bcl-2 expression in H2O2-induced H9c2 cells was reversed upon pretreatment with forsythiaside A ( Figure 1B). These data suggest that forsythiaside A has an anti-apoptotic effect on H2O2-induced H9c2 cells.

Forsythiaside A attenuated H2O2-induced inflammation in H9c2 cells
TNF-α and IL-6 mRNA and protein expression was upregulated in H9c2 cells upon incubation with H2O2, however treatment with forsythiaside A attenuated this H2O2-induced increase in TNFα and IL-6 expression (Figure 2 A and B) indicating that forsythiaside A plays an antiinflammatory role in H2O2-induced H9c2 cells.

Forsythiaside A attenuated H2O2-induced oxidative stress in H9c2 cells
Upon incubation with H2O2, the MDA level increased and the SOD and GSH-Px levels decreased in H9c2 cells, however the increase in MDA and the decreases in SOD and GSH-Px were reversed by treatment with forsythiaside A in a dose-dependent manner ( Figure 3). These data demonstrate the anti-oxidant effect of forsythiaside A in H2O2-induced H9c2 cells.    (Figure 4), however incubation with H2O2 induced p-p65 expression, and this H2O2-induced increase of p-p65 in H9c2 cells was attenuated upon incubation with forsythiaside A (Figure 4). In addition, p-IκBα expression increased and IκBα expression decreased upon incubation with H2O2, and these H2O2-induced changes in gene expression were reversed upon incubation with forsythiaside A (Figure 4). These data indicate that forsythiaside A suppressed activation of NF-κB in H2O2-induced H9c2 cells. Protein expression of Nrf2 and HO-1 was upregulated in H2O2-stimulated H9c2 cells that were treated with forsythiaside A (Figure 4).

DISCUSSION
Oxidative and inflammatory biomarkers are upregulated in chronic heart failure suggesting their potential in the prognosis of heart failure [4]. Strategies to suppress oxidative stress and inflammation have been shown to ameliorate myocardial injury in heart failure [8]. Forsythiaside A has attracted increasing attention due to its anti-inflammatory and antioxidant properties [5]. The protective effects of forsythiaside A on heart failure was investigated in this study.
Apoptosis of cardiomyocytes occurs in a variety of cardiovascular diseases. It has been reported that apoptotic cells around infarct tissue play a role in heart remodeling and that inhibition of cell apoptosis significantly improves left ventricular remodeling and chronic heart failure [9]. Therefore, oxidative stress-induced apoptosis is considered a therapeutic target for heart failure [9]. In this study, incubation with H2O2 induced apoptosis, enhanced Bax expression, and reduced Bcl-2 expression in H9c2 cells. A previous study showed that forsythiaside A protected NIH/3T3 fibroblast cells against ultraviolet radiation-induced cell apoptosis [10]. The results of this study confirmed that pretreatment with forsythiaside A decreased Bax expression and increased Bcl-2 expression to suppress apoptosis of H2O2-induced H9c2 cells.
Heart failure is a systemic pro-inflammatory state characterized by high levels of inflammatory cytokines, such as TNF-α and IL-6 [11]. Excessive levels of TNF-α and IL-6 are regarded as poor prognostic markers in heart failure [12]. Forsythiaside A was shown to reduce levels of TNF-α and IL-6 to ameliorate CuSO4-induced inflammation in zebrafish [13]. Forsythiaside A repressed the H2O2-induced increases in TNF-α and IL-6 expression in H9c2 cells in a dosedependent manner. Prolonged activation of the NF-κB pathway stimulates chronic inflammation with enhanced expression of TNF-α and IL-6 resulting in endoplasmic reticulum stress and cell death, thereby promoting heart failure progression [14]. Protein expression of p-p65 was upregulated in H2O2-stimulated H9c2 cells suggesting that the NF-κB pathway was activated. Forsythiaside A was shown to decrease NF-κB expression to alleviate virusinduced lung tissue damage [15]. In this study, forsythiaside A reduced p-p65 expression in H2O2-induced H9c2 cells. Therefore, we propose that forsythiaside A exerts its anti-inflammatory effect against heart failure through inactivation of the NF-κB pathway.
Oxidative stress, which is implicated in the pathophysiology of heart failure, induces cell apoptosis and inflammation of cardiomyocytes [16]. The level of MDA increased and levels of SOD and GSH-Px decreased in H2O2-induced H9c2 cells suggesting that H2O2 induced oxidative stress in H9c2 cells. Forsythiaside A attenuated the H2O2-induced increase in MDA and decreases in SOD and GSH-Px in H9c2 cells in a dose-dependent manner, indicating that forsythiaside A has an anti-oxidant effect against heart failure. Nrf2 is transferred into the nucleus to bind with anti-oxidant response elements of target genes, upregulates HO-1 to reduce oxidative stress, and protects against cardiovascular diseases, including heart failure [17]. Forsythiaside A was shown to increase levels of Nrf2 and HO-1, which exerted antioxidant effects in lipopolysaccharide-stimulated microglial cells [18]. In this study, forsythiaside A also promoted protein expression of Nrf2 and HO-1 in H2O2-induced H9c2 cells, which most likely exert anti-oxidant effects against heart failure.

CONCLUSION
Forsythiaside A reduces apoptosis of H2O2stimulated H9c2 cells, downregulates TNF-α and IL-6 expressions via inactivation of NF-κB pathway, and suppresses oxidative stress via activation of Nrf2/HO-1 pathway. These results indicate that forsythiaside A may be useful for the management of heart failure.