Red clover flavonoids protect against oxidative stress-induced cardiotoxicity in vivo and in vitro

Abstract

Red clover flavonoids (RCF) which contain significant amounts of polyphenolic substances are known for their potential antioxidant properties. However, little is known about their effect on oxidative stress-induced myocardial injury. The objective of this study was to investigate the potential protective effects and mechanisms of RCF on isoproterenol (ISO)-induced myocardial injury in rats and on H₂O₂-induced apoptosis of H9c2 cardiomyocytes. An in vivo study revealed that RCF (200, 100 and 50 mg kg⁻¹, i.g., respectively) daily for 15 days can prevent ISO-induced myocardial damage, including a decrease of serum cardiac enzymes and improvement in heart vacuolation. RCF also improved the free radical scavenging and antioxidant potential, suggesting one possible mechanism of RCF-induced cardio-protection is mediated by blocking the oxidative stress. An in vitro investigation demonstrated that RCF pretreatment increased cell viability, decreased levels of LDH leakage and PI-positive cells compared with the H₂O₂ group. Moreover, RCF pretreatment inhibited cell apoptosis, as evidenced by improved mitochondrial membrane potential disruption, decreased caspase-3 level, as well as increased Bcl-2/Bax ratio. Further mechanism investigation revealed that RCF prevented H9c2 cardiomyocytes injury and apoptosis induced by MAPK pathways. These results suggest that RCF exerted cardioprotective effects against myocardial injury by inhibiting oxidative stress, cardiac myocyte apoptosis, and modulating MAPK pathways, indicating that RCF might be a potential agent in the treatment of heart disease.

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Introduction

Coronary heart disease is considered as one of the leading causes of mortality and morbidity worldwide.¹ It is widely accepted that cardiomyocyte apoptosis plays a key role in the pathogenesis of coronary heart disease.² Evidence also suggests that myocardial apoptosis is frequently associated with the production and release of reactive oxygen species (ROS).³ Hence, therapeutic strategies aim at preventing or regulating cardiomyocytes apoptosis and intracellular ROS levels may be an effective treatment of coronary heart disease.

In recent years, considerable efforts have been focused on the use of plant-derived natural antioxidants due to their presumed nutritional and therapeutic value. Red clover (Trifolium pretense L.), which belongs to the plant family Leguminosae, is recognized as an important crop in the world owing to its rapid growth and soil improving characteristics.⁴ Traditionally, red clover is mainly used as feed for livestock, but also has been used in traditional medicine to treat whooping cough, bronchitis and asthma.⁵ Red clover extracts are available nowadays as nutritional supplements in the form of tablets (Promensil), capsules, tea and liquid preparations.⁶ Recent research has shown that red clover extracts as a rich source of isoflavones contain significant high amounts of the four major isoflavones genistein, daidzein, biochanin A and formononetin, which has a remarkable high content of total polyphenols.⁷ Many polyphenol compounds are reported to have beneficial effects in the treatment of cardiovascular diseases likely through their potent antioxidant functions.^8–10 Therefore, whether red clover flavonoids (RCF) have the potential for cardiovascular therapy attracted our attention. Our previous study demonstrated that RCF could exert protective effects against H₂O₂-induced oxidative injury in endothelial cells, through its anti-oxidative and anti-apoptosis effects.¹¹ However, little information is available on the cardioprotective effects of RCF, particularly against oxidative stress-induced myocardial injury.

The objective of our present study was to investigate the potential effects of RCF against isoproterenol (ISO)-mediated myocardial injury in vivo and H₂O₂-induced cardiomyoblast H9c2 cell injuries in vitro. The underlying mechanisms were also elucidated by investigating the involvement of possible apoptosis-related pathways and mitogen-activated protein kinases (MAPKs).

Materials and methods

Animals

Male Sprague-Dawley rats, weighing 200–220 g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., Beijing, China. The animals were housed under standard laboratory conditions (25 ± 1 °C, 60% humidity, and 12 h photoperiod) and given standard rodent chow, and allowed free access to water. All of the procedures were approved by the Laboratory Animal Ethics Committee of the Institute of Medicinal Plant Development, Peking Union Medical College.

Chemicals and materials

Total flavonoid of red clover (ESI Fig. S1†) was provided from the Institute of Medicinal Plant Development (Beijing, China).¹¹ Quercetin (purity > 99%) was obtained from Shanghai Winherb Medical S & T Development (Shanghai, China). H₂O₂ was purchased from Beijing Chemical Works (Beijing, China). Cell culture products were purchased from Gibco BRL (NY, USA). The kits for determining malondialdehyde (MDA) contents, total creatine kinase (CK), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), total antioxidant capacity (T-AOC), glutathione peroxidase (GSH-Px), and superoxide dismutase (SOD) activity were obtained from Jiancheng Bioengineering Institute (Nanjing, China). Rat ROS ELISA kit, and Rat POD ELISA kit were from Rapid Bio Lab (Calabasas, CA, USA). Primary antibodies against Bcl-2, Bax, JNK, p-JNK, ERK, p-ERK, p38, p-p38 and Caspase-3 were obtained from Santa Cruz Biotechnology (CA, USA). β-Actin, and horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from CW Biotech (Beijing, China). All chemical reagents were at least of analytical grade.

Experimental protocols

A total of 105 SD rats were randomly assigned to seven groups: 1, control; 2, red clover flavonoids (RCF, 200 mg kg⁻¹) control; 3, isoproterenol treatment; 4, 5, and 6, isoproterenol combined with RCF (200, 100, and 50 mg kg⁻¹); 7, Di-ao-xin-xue-kang capsule (Di-ao, 218.75 mg kg⁻¹) as a positive control. Groups 1 and 3 were dosed intragastrically with the vehicle (1% Tween 80). Groups 2, 4, 5, and 6 were dosed intragastrically with RCF (200, 200, 100, and 50 mg kg⁻¹, respectively) for 15 days. Group 7 was dosed intragastrically with Di-ao (218.75 mg kg⁻¹) for 15 days. Within 2 h of RCF and Di-ao administration on days 14 and 15, rats in the groups 3 to 7 were injected with isoproterenol (4 mg kg⁻¹, i.H.), whereas the rats in groups 1 and 2 received saline.

Preparation of samples and measurement of biochemical variables

The experiment was stopped 12 h after the last administration of the drugs. Then the rats were bled to collect the serum for the determination of serum cardiac enzymes (CK, AST, and LDH) by kits. Subsequently, the rats were killed, and their hearts were removed rapidly. The left ventricle was excised for histo-pathological examination; and myocardial homogenates were prepared for the detection of MDA, T-AOC, GSH-Px, and SOD, by corresponding kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), which are based on the manufacturer's instructions. ROS and POD were detected by corresponding ELISA kits (Rapid Bio Lab, Calabasas, CA, USA).

Histo-pathological examination

The apex of the heart was fixed in 10% formalin, routinely processed, and embedded in paraffin. Paraffin sections (3 mm) were cut on glass slides, stained with hematoxylin and eosin (HE), and examined under a light microscope (CKX41, Olympus, Tokyo, Japan) by a pathologist blinded to the groups studied.⁹

Cell culture and treatment

H9c2 rat ventricular cardiomyocytes were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and cultured as previously described.⁹ For all experiments, cells were plated at an appropriate density according to the experimental design and were grown for 36 h before experimentation. H9c2 cells were pretreated with indicated concentrations (6.25, 12.5, 25 μg mL⁻¹) of RCF for 6 h and then treated with H₂O₂ (150 μM) for 6 h.

Cell viability analysis

Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT) assay as previously reported.⁹ Briefly, the cells were incubated with MTT solution (1 mg mL⁻¹ final concentration) at 37 °C for 4 h at the end of different treatments. The medium was then removed, and the formazan crystals were dissolved with dimethyl sulfoxide (DMSO). Absorbance was read at 570 nm on a microplate reader (SpectraFluor, Tecan, Sunrise, Austria).

Measurement of LDH, CAT, MDA levels and SOD activity

The supernate and cells were collected respectively after the different treatments for measuring LDH, CAT, and MDA levels as well as SOD activity using the corresponding detection kit according to the manufacturer's instructions.

Determination of intracellular ROS production

The cellular ROS levels were determined according to the manufacturer's protocol using total ROS detection kits according to the manufacturer's brochures (Invitrogen, California, USA). Cellular DCF fluorescence intensity was determined through the fluorescence microplate reader with excitation wavelength of 495 nm and emission wavelength of 529 nm. The ROS level was expressed as a percentage of the control.

Hoechst 33342 and PI double staining

H9c2 cardiomyocytes were cultured in 24-well plates for 24 h. After treatment, cells were washed twice with phosphate-buffered saline (PBS) and incubated with 10 μg mL⁻¹ Hoechst 33342 dye for 15 min at 37 °C in the dark, and then 100 μg mL⁻¹ propidium iodide was added (PI, Sigma). The cells were then observed immediately, using a fluorescence microscopy (Leica, Heidelberg, Germany). The nuclear size and permeability of the cell membranes were calculated.¹²

Determination of mitochondrial transmembrane potential (ΔΨ_m)

5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1) (Sigma-Aldrich, St. Louis, USA) was used to analyze changes in mitochondrial transmembrane potential as previously reported.⁹ After removal of H₂O₂, the cells were suspended in warm medium at approximately 1 × 10⁶ cells per mL. Subsequently, 10 μL of 200 μM JC-1 (2 μM final concentration) was added to the medium, incubated for 30 min in the dark, and washed twice with PBS. The cells labeled with JC-1 were analyzed by BD FACSCalibur flow cytometry using 488 nm excitation and green (525 nm) or orange-red (575 nm) emission wavelengths with CellQuest software.

Western blot analysis

After treatment, H9c2 cells were harvested, washed with PBS, and lysed with cell lysis buffer containing 1% phenylmethylsulfonyl fluoride. Lysate preparation and Western blot analysis were performed as previously described.⁹

Statistical analysis

Data from at least three independent experiments were expressed as means ± SD. Statistical comparisons between different groups were measured by using one-way ANOVA followed by Student–Newman–Keuls test. The level of significance was set at p < 0.05.

Results

RCF prevented ISO-induced cardiac injury in rats

To assay the function of RCF on cardiac injury protection, we first evaluated the level of serum cardiac enzymes (CK, AST, and LDH) in different animal groups. As shown in Fig. 1A–C, pretreatment of RCF dose-dependently reduced ISO-induced increase of serum cardiac enzymes levels.

Fig. 1 Effects of RCF on ISO-induced myocardial injury in vivo. (A) The effects of RCF on LDH level; (B) the effects of RCF treatment on CK level; (C) the effects of RCF on AST level; (D) the effects of RCF on histological changes in rat hearts by HE staining (×200). Values (n = 10 per group) are expressed as means ± SD. ##p < 0.01 relative to the control group; *p < 0.05 relative to the ISO group; **p < 0.01 relative to the ISO group.

The morphological changes in the myocardial damage at the light microscopy level were detected by HE staining. No evident abnormalities were observed in the group with control and RCF alone group, while severe myocardial damages were found in ISO group, characterized by disorganization of myofibrillar arrays, cytoplasmic vacuolization, and intense infiltration with neutrophil granulocytes. RCF pretreatment groups markedly reduced the pathological changes by ISO (Fig. 1D).

RCF enhanced antioxidant capacity in cardiac tissues

To test if the protective effects of RCF against isoproterenol-induced heart dysfunction were due to its antioxidative activity, we measured the oxidative stress associated parameters in the heart. As shown in Fig. 2, ISO caused oxidative stress damage in rat heart as indicated by significantly decreases in SOD, POD, GSH-Px, and T-AOC activities and increases in lipid peroxidation (MDA) production and intracellular levels of ROS, whereas these changes were effectively improved by RCF pretreatment groups in a dose-dependent manner.

Fig. 2 Effects of RCF on antioxidative activities in rats. Rat received ISO treatment in the present or absent of RCF pre-treatment, the expression level of antioxidative components in the left ventricle were measured as described in Materials and Methods, and the values (n = 10 per group) are expressed as means ± SD. ##p < 0.01 relative to the control group; *p < 0.05 relative to the ISO group; **p < 0.01 relative to the ISO group.

RCF protected against H₂O₂-induced cytotoxicity in H9c2 cells

The protective effect of RCF against H₂O₂-induced cytotoxicity was detected by MTT assay. H9c2 cells were pretreated with RCF (6.25 to 25 μg mL⁻¹) for 6 h, followed by 150 μM H₂O₂ treatment for 6 h. As shown in Fig. 3A, RCF treatment dose-dependently attenuated H₂O₂-induced reduction in cell viability, and 25 μg mL⁻¹ RCF has the most significant protective effect. Therefore, 25 μg mL⁻¹ RCF was chosen for further experiments.

Fig. 3 Effects of RCF on H₂O₂-induced H9c2 cell injury. Treatment of H9c2 cells with various concentrations (0, 6.25, 12.5, and 25 μg mL⁻¹) of RCF for 6 h followed by exposure to H₂O₂ for additional 6 h. (A) Cell viability was determined by MTT reduction assay. (B) The effect of RCF on the level of LDH in the H9c2 cells was measured using an LDH assay kit. (C) Hoechst 33342 and PI double staining were used in the qualitative and quantitative analyses of the apoptosis and necrosis cells. Data are presented as means ± SD (n = 3). ##p < 0.01 versus control; *p < 0.05 versus H₂O₂-treated cells; **p < 0.01 versus H₂O₂-treated cells.

LDH, which leaks from cells after plasma membrane disruption, can be used as an indicator of cell injury. As shown in Fig. 3B, a marked increase in LDH activity was observed after 6 h of exposure to 150 μM H₂O₂. However, when myocytes were pretreated with 25 μg mL⁻¹ RCF, LDH activity was significantly decreased.

To further understand the protective effect of RCF on H₂O₂-induced cell death, Hoechst 33342/PI staining was used in this study. Hoechst 33342 stained all cells (live and dead); while PI-positive cells with condensed blue nuclei were regarded as dead/dying cells. As shown in Fig. 3C, H9c2 cells treated with H₂O₂ clearly exhibited staining that was indicative of dead/dying cells. Interestingly, pretreatment with RCF significantly alleviated the PI-positive staining changes triggered by H₂O₂ treatment.

RCF inhibited intracellular ROS level in H₂O₂-treated H9c2 cells

ROS generation is one of the most common responses to cell injury.¹³ As shown in Fig. 4A, RCF pretreatment significantly attenuates H₂O₂-induced ROS release in H9c2 cells, and increased intracellular ROS levels were found in the H₂O₂-treated group compared with the control group. These results show that RCF protects the H₂O₂-induced H9c2 cell injury through the inhibitions of ROS production.

Fig. 4 Effects of RCF treatment on ROS level and antioxidative enzymes in H9c2 cells. (A) The effects of RCF on ROS level in H9c2 cells; (B) the effects of RCF on the expression levels of MDA; (C) the effects of RCF on the expression levels of SOD; (D) the effects of RCF on the expression levels of CAT in H9c2 cells. Data are presented as means ± SD from three independent experiments. ##p < 0.01 versus control; **p < 0.01 versus H₂O₂-treated cells.

RCF enhanced anti-oxidant enzyme activity in H₂O₂-treated H9c2 cells

Membrane lipid oxidation is one of the primary events in oxidative damage, which can be assessed by its degradation product MDA. Treatment of H9c2 cells with H₂O₂ caused a significant increase in intracellular MDA levels, whereas pretreatment with RCF markedly ameliorated the intracellular MDA levels (Fig. 4B). In addition, the activities of the endogenous antioxidative enzymes SOD and CAT in the H₂O₂-treated cells were decreased compared with the control, whereas RCF treatment effectively enhanced SOD and CAT activities (Fig. 4C and D). These results suggest that the antioxidative property of RCF helps protect cells against H₂O₂-induced cytotoxicity.

RCF decreased H₂O₂-induced apoptosis in H9c2 cells

The loss of mitochondrial membrane potential (ΔΨ_m) which is associated with oxidative stress-induced apoptosis, is considered as an important event during apoptosis.¹⁴ We assessed the possible effect of RCF on ΔΨ_m by JC-1 staining, which exhibited a potential-dependent accumulation in the mitochondria. Consistent with previous work,⁹ H₂O₂ treatment led to the dissipation of ΔΨ_m, indicated by the shift of fluorescence from the upper left to lower right in the panel. Cells pre-incubated with RCF showed strong protective effect against H₂O₂-induced loss of mitochondrial membrane potential (Fig. 5). This finding indicates that the anti-apoptotic effect of RCF occurs by preventing mitochondrial dysfunction.

Fig. 5 Effects of RCF on H₂O₂-induced H9c2 cell apoptosis. Flow cytometry analysis of mitochondrial membrane potential by JC-1 staining in H9c2 cells. Data are presented as means ± SD from three independent experiments. #p < 0.05 versus control; *p < 0.05 versus H₂O₂-treated cells.

RCF modulated the expressions of apoptosis-related protein in H₂O₂-treated H9c2 cells

To understand the mechanism by which RCF inhibits apoptosis in H9c2 cells, we then measured the effects of RCF on the expression of apoptosis-related proteins expression by Western blot analysis. As shown in Fig. 6A, H₂O₂ resulted in a decrease in the expression of the anti-apoptotic protein Bcl-2 as well as an increase in the pro-apoptotic protein Bax expression. Pretreatment with RCF increases the Bcl-2/Bax expression ratio compared with the H₂O₂ group (Fig. 6B). In addition, the expression level of caspase-3 which is one of the key processes involved in apoptosis was also inhibited by RCF pretreatment compared with H₂O₂ treatment (Fig. 6C).

Fig. 6 Effects of RCF pretreatment on apoptotic-related protein expressions in H₂O₂-treated H9c2 cells. Cells were pretreated with RCF for 6 h followed by 6 h treatment with 150 μM H₂O₂. Proteins related to the apoptotic signaling were analyzed using Western blot. (A) Western blot analysis of Bcl-2, Bax and Caspase-3; (B) Bcl-2/Bax ratio; (C) Caspase-3/β-actin ratio; (D) Western blot analysis of P-JNK1/JNK1; (E) Western blot analysis of P-ERK/ERK; (F) Western blot analysis of P-P38/P38. β-Actin expression was examined as the protein loading control. Data are presented as means ± SD from three independent experiments. ##p < 0.01 versus control; *p < 0.05 versus H₂O₂-treated cells; **p < 0.01 versus H₂O₂-treated cells.

The MAPK signaling pathways, including the ERK, p38 kinase, and JNK signaling pathways, play a major role in apoptosis signaling. Therefore, to further determine whether the MAPK signaling pathway is involved in the anti-apoptotic effects of RCF, the expression levels of MAP kinase proteins in H9c2 cells were also evaluated through Western blot. H₂O₂ treatment increased the MAPK phosphorylation. However, RCF pretreatment remarkably decreased the levels of p-JNK/JNK, p-ERK/ERK, and p-p38/p38 compared with the H₂O₂ treatment group (Fig. 6D–F). All these results indicate that the MAPKs signaling pathway was involved in the anti-apoptotic effect of RCF.

RCF regulated COX-2 expression in H₂O₂-treated H9c2 cells

It has been reported that ROS and the MAPK pathways can regulate the expression of COX-2.^15–18 Thus, we examined the expression level of COX-2. Western blot results showed that COX-2 expression level in the H₂O₂ treatment group was significantly higher than that in control group. In RCF pretreatment group, the level of COX-2 was decreased significantly compared with that in the H₂O₂ treatment group (Fig. 7).

Fig. 7 Effects of RCF pretreatment on COX-2 expression in H₂O₂-treated H9c2 cells. β-Actin expression was examined as the protein loading control. Data are presented as means ± SD from three independent experiments. ##p < 0.01 versus control; **p < 0.01 versus H₂O₂-treated cells.

Discussion

Natural flavones found in plants are used as antioxidants for the preventing and treating of cardiovascular diseases for many years.⁸ Red clover flavonoids (RCF) extract, as a rich source of isoflavones, with the high content of polyphenols, showed effective antioxidant and radical scavenging activity.^4,7 The aim of this study was to evaluate the cardioprotective effects of RCF in vitro and in vivo and to clarify the underlying mechanisms.

In the present study, we demonstrated that RCF possesses significant cardio-protective effects both in vivo and in vitro. We observed that isoproterenol (ISO) treatment resulted in severe heart injury, with widespread subendocardial necrosis, serum enzyme leakage, and formation of lipid oxide product, which were in agreement with previous reports.⁹ Interestingly, compared with the experimental model of ISO-induced myocardial injury in rats, RCF administration concentration-dependently reduces the level of serum cardiac enzymes as well as ameliorates histological damage in the heart, indicating that RCF exerts a pronounced protective effect against myocardial injury. In addition, RCF enhances activities in antioxidant enzymes (SOD, GSH-Px, and POD) and decreases the contents of MDA and ROS in heart tissues, suggesting that RCF can alleviate ISO-induced heart injury by enhancing antioxidative enzymatic activity, thus reducing intracellular ROS levels. Consistent with the in vivo results, using H₂O₂ exposure to generate ROS and cause H9c2 cell damage, RCF pretreatment was also observed to neutralize the reduction in cell viability, SOD and CAT activity, and increased LDH release and MDA content induced by H₂O₂ exposure. All these results directly reveal that the cardioprotective effects of RCF are associated with its antioxidant activity.

Cardiomyocytes apoptosis is also one of the major contributors to the development of myocardial injury.¹⁹ In the present study, the anti-apoptotic effects of RCF were confirmed by the results of JC-1 staining. The results strongly demonstrated that H₂O₂ led to mitochondrial depolarization and dissipation of ΔΨ_m, while RCF pretreatment significantly stabilized the H₂O₂-induced mitochondrial dysfunction. Inhibition of caspase activity has been shown to attenuate both myocardial injury and apoptosis in cardiomyocytes.²⁰ Caspase-3 is thought to be a major executioner caspase during apoptosis.²¹ In our experiments, we found that caspase-3 level was up-regulated by H₂O₂ treatment and RCF pretreatment significantly inhibited the expression level, indicating that the anti-apoptotic effect of RCF may be mediated by caspase-3. The Bcl-2 family members are known to play important roles in caspase activation.²² Previous studies have also indicated that the ratio of Bcl-2 and Bax may decide the cellular threshold for apoptosis.²³ Our results showed that RCF pretreatment increased the expression of Bcl-2, as it decreased the expression of pro-apoptotic Bax. Based on these results, we speculated that RCF might exert protective effects through modulating the balance between anti-apoptotic protein Bcl-2 and pro-apoptotic protein Bax and thereby maintaining mitochondrial function.

Mitogen-activated protein kinases (MAPKs) pathways (i.e., JNK, ERK1/2, and p38 MAPK) are reported to regulate apoptosis and other cellular programs.²⁴ Activation of the MAPK pathways have been associated with ROS-induced apoptosis.²⁵ Previous study demonstrated that H₂O₂ treatment induced ERK, JNK and p38 activation in vascular endothelial cells.²⁶ In this regard, we evaluated the role of MAPK pathways to thoroughly examine the mechanisms by which RCF modulates H₂O₂-induced apoptosis in H9c2 cells. As expected, our results showed that RCF pretreatment inhibited the activation of the MAPK pathway. Therefore, our results suggest that the anti-apoptotic effect of RCF is possibly related to modulation of MAPK signaling pathways.

Inflammation is another important event that can be evoked by increased oxidative stress during cardiovascular disease.¹⁵ COX-2, an inducible isoform of cyclooxygenases, has been shown to be a key mediator of acute and chronic inflammation.²⁷ Our results showed that increased COX-2 expression by H₂O₂ is attenuated by pretreatment with RCF. This result has provided us the initial confirmation about the protective effect of RCF against the oxidative stress-induced myocardial inflammation.

The major constituents of RCF are calycosin, genistein, kaempferol, formononetin, and biochanin A (ESI Fig. S1†). Many researches demonstrated that these constituents have promising anti-oxidative and anti-apoptotic properties. For example, genistein, as an isoflavone and a rich constituent of red clover flavonoids, has been reported to prevent ISO-treated H9c2 cardiomyocyte apoptosis through regulation of MAPK pathways.²⁸ Calycosin exerts neuroprotective effect against cerebral ischemia/reperfusion injury via its antioxidant effects.²⁹ Kaempferol could protect oxidative stress-induced heart injuries such as myocardial ischemia/reperfusion³⁰ and doxorubicin-induced cardiotoxicity.³¹ Research also indicates that formononetin has a protective potential against myocardial infarction injury by elevating expression of endogenous antioxidant defence enzymes, thus attenuating cardiomyocyte apoptosis.³² These results may be responsible for the cardioprotective effects of RCF. However, further studies on the active compounds and their biochemical mechanisms of RCF cytoprotection against oxidative stress-induced cardiovascular diseases will be necessary.

In conclusion, our study demonstrated that RCF showed cardioprotective effects against ISO-induced myocardial injury by reducing intracellular enzyme leakage, maintaining the antioxidant enzyme levels and preventing histological damage. Moreover, RCF can protect against H₂O₂-induced apoptotic death in H9c2 cells via its effects on the inhibition of oxidative stress, apoptotic pathway and mediation of MAPK signaling pathway. Our findings indicate that RCF might be a promising agent for the treatment of ROS-induced heart injuries such as myocardial ischemia/reperfusion injuries.

Statement of conflict of interest

The authors declare that there are no conflicts of interest.

Acknowledgements

This study was supported by the Major Scientific and Technological Special Project for “Significant New Drug Formulation” (grant no. 2012ZX09501001004 and 2012ZX09301002-001), the National Natural Sciences Foundation of China (Grant no. 81374010), and the Program for Innovative Research Team in IMPLAD (Grant no. IT1301).

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ra08407a

‡ Min Wang and Jian-yong Si contributed equally to this work.

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