Protective effects of purified safflower extract on myocardial ischemia in vivo and in vitro

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Phytomedicine 16 (2009) 694–702 www.elsevier.de/phymed

Protective effects of purified safflower extract on myocardial ischemia in vivo and in vitro Shu-Yan Hana,1, Hai-Xia Lia,1, Xu Maa, Ke Zhanga, Zhi-Zhong Mab, Peng-Fei Tua, a

State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University Health Science Center, No. 38 Xueyuan Road, Beijing 100191, PR China b Department of Intergration of Chinese and Western Medicine, School of Basic Medical Sciences, Peking University Health Science Center, No. 38 Xueyuan Road, Beijing 100191, PR China

Abstract Carthamus tinctorius L. (safflower) is one of the most commonly used Chinese herbal medicines to prevent and treat cardiac disease in clinical practice. However, the mechanisms responsible for such protective effects remain largely unknown. In this study, we investigated the anti-myocardial ischemia effects of a purified extract of C. tinctorius (ECT) both in vivo and in vitro. An animal model of myocardial ischemia injury was induced by left anterior descending coronary artery occlusion in adult rats. Pretreatment with ECT (100, 200, 400, 600 mg/kg body wt.) could protect the heart from ischemia injury by limiting infarct size and improving cardiac function. In the in vitro experiment, neonatal rat ventricular myocytes were incubated to test the direct cytoprotective effect of ECT against H2O2 exposure. Pretreatment with 100–400 mg/ml ECT prior to H2O2 exposure significantly increased cell viability as revealed by 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. ECT also markedly attenuated H2O2induced cardiomyocyte apoptosis, as detected by Annexin V and PI double labeling with flow cytometry. The intracellular level of reactive oxygen species (ROS) was shown by 20 ,70 -dichlorofluorescin diacetate (DCFH-DA), and ECT pretreatment significantly inhibited H2O2-induced ROS increase. We made a preliminary examination of the signaling cascade involved in ECT mediated anti-apoptotic effects. Phosphatidylinositol 3 kinase (PI3K) inhibitor (LY294002) blocked the cytoprotective effect conferred by ECT. Taken together, our findings provide the first evidence that the cardioprotective effects of ECT in myocardial ischemia operate partially through reducing oxidative stress induced damage and apoptosis. The protection is achieved by scavenging of ROS and mediating the PI3K signaling pathway. r 2009 Elsevier GmbH. All rights reserved. Keywords: Carthamus tinctorius L.; Safflower; Myocardial ischemia; H2O2; Apoptosis; ROS

Introduction Infarction induced by myocardial ischemia is one of leading causes of human death worldwide. Minimizing Corresponding author. Tel./fax: +86 10 8280 2750. 1

E-mail address: [email protected] (P.-F. Tu). These authors contributed equally to this work.

0944-7113/$ - see front matter r 2009 Elsevier GmbH. All rights reserved. doi:10.1016/j.phymed.2009.02.019

myocardial necrosis and improving heart function have been proved to be effective strategies to reduce the morbidity and mortality from myocardial infarction (Kloner and Rezkalla 2004). Levels of reactive oxygen species (ROS) are elevated during myocardial ischemia (Guzy et al. 2005; Lefer and Granger 2000), and induce a variety of cardiomyocyte abnormalities including cell death and apoptosis (Fu et al. 2007; Harsdorf et al.

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1999). Accordingly, antioxidants may decrease cellular injury and apoptosis through a radical-scavenging mechanism (Angeloni et al. 2007; Bognar et al. 2006). Hydrogen peroxide, the major source of endogenous ROS (Nohl et al. 2003), is generated during ischemia with or without reperfusion, and has been used extensively to induce oxidative stress in in vitro models (Chen et al. 2000). Carthamus tinctorius L. (safflower) has long been used as Chinese medicine in clinics to treat cardiovascular disease, and has demonstrated anti-myocardial ischemia effects (Li et al. 2006; Zheng et al. 2003). Wagner et al. (2006) developed the C. tinctorius monograph and gave it a comprehensive introduction. Safflower also possesses other pharmacological effects, including anticoagulant (Zang et al. 2002), antioxidant (Hiramatsu et al. 1998), neuroprotective (Wang et al. 2007a) and calcium antagonist (Meselhy et al. 1992) effects. The chemical constituents in safflower are reported to be flavonoids (Kazuma et al. 2000), lignans (Palter et al. 1972), triterpene alcohols (Akihisa et al. 1996), and polysaccharides (Hirokawa et al. 1997), among others. Safflower has also been reported to prevent electrophysiological abnormalities induced by hydrogen peroxide in guinea pig ventricular myocytes (Shan et al. 2004). Hydroxysafflor yellow A, a quinochalcone compound in safflower, could protect against hypoxia injuries of cardiomyocytes (Xue et al. 2007). Although considerable evidence demonstrates the cardiac protective effects of safflower, the underlying mechanisms remain largely unknown. In the present study, the rat model of myocardial ischemia was produced by occlusion of the left anterior descending (LAD) coronary artery. Our previous work showed the antioxidant property of the extract of C. tinctorius (ECT) in a cell-free system in vitro (unpublished data). The direct free radical donor, H2O2, is used to mimic the oxidative stress in myocardial ischemia in vitro. Thus, the purpose of this study was to investigate the cardioprotective effect of ECT in myocardial ischemia and to exploit its underlying mechanisms.

idine (5-BrdU), hydroxy peroxide (H2O2), 20 ,70 -dichlorodihydrofluorescein diacetate (DCFH-DA) were obtained from Sigma Chemical (St. Louis, MO, USA). LY294002 was obtained from Cell Signaling Technology (MA, USA). Annexin-FITC kit was obtained from Millipore company (PA, USA). The purity of all chemical reagents was at least analytical grade. The certificate code of the male Sprague-Dawley rats was SCXK 2002–2001. All protocols were performed in accordance with the Guidelines of the Peking University Animals Research Committee.

Materials and methods

Experiment 1: the protective effect of ECT on myocardial ischemia of rats

Preparation of C. tinctorius extract Dried flower petals were extracted three times under reflux in 50% ethanol. The ethanol volume was 8, 6, and 6 times of the plant material. The extract time was 2 h for the first and 1 h for the other extractions. The extracts were combined, filtered and evaporated in vacuo to obtain a condensed solution. Macroporous resin chromatography was used to purify the condensed extract solution. In brief, the procedure consisted of eluting the column with water, which was discarded, and then 70% ethanol, after which the eluent was collected. The 70% eluent portion was evaporated and sprayed to dryness for further research. Finally, the yield ratio of the extract of C. tinctorius (ECT) was 9%.

High Performance Liquid Chromatography (HPLC) profile of ECT The separation of ECT was achieved on Aichrom Bond-AQ CB18B column (250 mm  4.6 mm, 5 mm). The injection volume was 10 ml. The flow rate was set as 1.0 ml/min and the separation was performed at 30 1C. The mobile phase consisted of acetonitrile (solvent A) and 0.1% aqueous formic acid (solvent B), which were applied in the gradient elution as follows: 0–20 min, 2–19% A; 20–50 min, 19–25% A; 50–65 min, 25–37% A; 65–75 min, 37–40% A. Each run was equilibrated with 2% A for 5 min and the total run time was 80 min. Ultraviolet (UV) spectra were monitored at 275 nm.

Materials, animal and reagents Sodium pentobarbital and 2,3,5-triphenyltetrazolium chloride (TTC) were purchased from Beijing Chemical Reagent Company (Beijing, China). Dulbecco’s Modified Eagle medium (DMEM), trypsin and collagenase were purchased from Gibco BRL Co. (Gaithersburg, MD, USA). Fetal bovine serum was purchased from Invitrogen Inc. (MD, USA). MTT, 5-bromo-deoxyur-

Acute myocardial ischemia induced by LAD occlusion Male Sprague-Dawley (SD) rats weighing 250–270 g were randomized into seven groups of nine rats each. Group 1, subjected to LAD branch ligation, was treated with vehicle and groups 2–8 were treated with different doses (50, 100, 200, 400, 600 and 800 mg/kg body wt., respectively) of ECT. All rats were administered the respective dose twice daily for three consecutive days,

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and given the last dose 30 min before the coronary occlusion. Rats were anaesthetized with sodium pentobarbital (i.p., 50 mg/kg body wt.) and ventilated using an animal respirator (HX-300, Chengdu, China) with room air after tracheal intubation. A thoracotomy was performed, and the LAD was occluded with a 6-0 silk suture, after which the heart was returned immediately to the chest (Samsamshariat et al. 2005). After 24 h, the hearts were excised under anesthetic situation, sliced into 2–3 mm transverse sections, and immersed into 1% 2,3,5-triphenyl tetrazolium chloride (TTC) solution in PBS (pH 7.4) for 10 min at 37 1C. The non-infarct myocardium was stained red by dehydrogenase enzymes and the infarct area remained pale in color (Fishbein et al. 1981). The infarct size was represented as the ratio of the infarct area to the whole ventricular (Maczewski and Mackiewicz 2007).

Oxidative stress induced by exposure to H2O2 and drug treatment To determine the injury induced by H2O2, cardiomyocytes were exposed to different concentrations of H2O2 ranging from 50 to 400 mM and cultured for 6 and 24 h, respectively. To evaluate the protective effects of ECT, cells were pretreated for 6 h with 50–500 mg/ml of ECT in serum free medium, and washed twice with DHank’s prior to the addition of H2O2, preventing direct extracellular interactions between the compounds and H2O2. After that, cardiomyocytes were exposed to 200 mM H2O2 for 24 h, and control cells were also incubated under the same conditions. For further studies, LY294002, the PI3K inhibitor (Vlahos et al. 1994), was added to the culture medium 60 min before the addition of H2O2.

Cell viability assay Hemodynamic measurements Hemodynamic measurement was taken following a method described by Feng et al. (2001) before sacrificing the animals. In this experiment, a BL-420S version transducer (Chengdu, China) was used to measure the hemodynamic parameters. The right carotid artery of the anaesthetised rat was cannulated using a polyethylene catheter (PE-50, length 25 cm) filled with sterile saline containing sodium heparin (300 U/ml). The catheter was advanced into the left ventricle via the right carotid artery for measuring the 7dP/dt. The resting hemodynamic measurement was obtained over a 30 min steady-state period after placement of the catheter.

Experiment 2: the protective effect of ECT on neonatal rat cardiomyocytes Primary culture of neonatal rat cardiomyocytes Monolayer cultures of neonatal cardiac cells were performed according to the method of Fu et al. (2004). Briefly, the hearts from neonatal SD rats (born within 24 h) were dissected and digested with 0.125% trypsin and 0.05% of collagenase for 5–7 cycles. Supernatants from each cycle were pooled and centrifuged. In the end, cell pellet was resuspended in DMEM medium containing 10% fetal bovine serum, penicillin (100 U/ml) and streptomycin (100 U/ml). The cell suspension was incubated at 37 1C in a 5% CO2 incubator for 1.5 h in order to reduce the contamination of fibroblasts. The cell concentration was adjusted to 0.7  106 cells/ml. 0.1 mM BrdU was included in the medium for the first 48 h of culture in order to inhibit fibroblast growth. For subsequent experiments, cells at 3-day to 5-day culture were used and growth was arrested by serum starvation for 24 h.

Cellular damage elicited by H2O2 was monitored by MTT ([3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide], Sigma). During the process, MTT was added to the medium with a final concentration of 0.5 mg/ml, and cells were incubated for 4 h at 37 1C. The solution was then removed carefully, and DMSO was added. Absorbance was measured at 570 nm (Denizot and Lang 1986). Cell viability was calculated as: Absorbance of each injured group/Absorbance of normal group  100%.

Flow cytometry analysis for apoptosis Apoptotic cells were detected by both Annexin V and propidium iodide (PI) double labeling as described elsewhere (Zamal et al. 1996). The operation procedure was performed following the manufacturer’s instruction. Flow cytometry (Becton Dikinson, USA) was used to assess the apoptotic cells. The quantitation of apoptotic cells was calculated using CellQest software.

Fluorescent measurement of intracellular reactive oxygen species (ROS) Intracellular oxidant stress was measured using an intracellular peroxide-sensitive fluorescent probe, with 20 ,70 -dichlorofluorescin diacetate (DCFH-DA, 5 mM; Sigma) as described previously (Vanden Hoek et al. 1997). Following drug and H2O2 treatment, the cells were washed twice with D-Hank’s and incubated with 5 mM DCFH-DA for 30 min. The cells were analyzed by Becton Dickenson FACSCAN with excitation 495 nm/ emission 525 nm (Chen et al. 2002).

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ably improved in the dose range of 100–600 mg/kg body wt. ECT (Fig. 3). These results suggest that ECT could protect the heart from myocardial ischemia injury.

Statistics All results are expressed as mean7S.E.M. unless stated otherwise. The paired Student’s test and one-way ANOVA were used to evaluate the statistical significance of differences between paired observations. A value of po0.05 was considered to be significant in all cases.

ECT inhibited H2O2-induced damage in cardiomyocytes Exposure of isolated cardiomyocytes to H2O2 led to a dose-dependent decrease in cell viability as assessed by MTT assay. Data also indicated a time-related decrease in viability when cells were exposed to 50–400 mM H2O2 at time intervals from 6 to 24 h (Fig. 4A). As shown in Fig. 4B, pretreatment with ECT significantly increased

Results From the HPLC analytical results, we determined that flavonoids and other types of components in the ECT may synergistically contribute to the overall pharmacological effects (Fig. 1). The amounts of hydroxysafflor yellow A and kaemferol 3-O-rutinoside in the safflower extract were 12.2% and 0.71%, respectively.

Infarct size (%)

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Effects of ECT on infarct size and hemodynamic As shown in Fig. 2, the infarct size in the control group reached 32.8671.87%, while the ECT-treated group showed a dose-dependent decrease in infarct size. Compared with the control group, results for the groups treated with 100, 200, 400, and 600 mg/kg body wt. ECT were statistically significant (po0.05 vs. control). Consistent with the occurrence of infarct size, coronary artery occlusion led to a marked hemodynamic impairment and left ventricular deterioration from myocardial ischemia. However, the cardiac function was remark-

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Fig. 2. Effect of different doses of C. tinctorius extract (ECT) on myocardial ischemia induced by left anterior descending (LAD) coronary artery occlusion for 24 h in rats (n ¼ 9). Infarct size was evidenced by 2,3,5-triphenyl tetrazolium chloride (TTC) staining. Data are expressed as means7S.E.M. (*po0.05 vs. control group).

VWD1 A, Wavelength=275 nm (E:\? ? \HPLC\WLI-6\HONGHUA\07092506.D) mAU

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Fig. 3. Effect of different doses of C. tinctorius extract (ECT) on myocardial ischemia induced by left anterior descending (LAD) coronary artery occlusion for 24 h in rats (n ¼ 9). Cardiac function as monitored by maximal rate of increase and decrease of left ventricular pressure (7dP/dtmax). Data are expressed as means7S.E.M. (*po0.05, **po0.01 vs. control group).

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the viability of H2O2-exposed cardiomyocytes in the range of 100–400 mg/ml. Based on this result, ECT 100 mg/ml was chosen as the minimal effective dose to protect cells from oxidative stress injuries.

ECT inhibited H2O2-induced apoptosis in cardiomyocytes Exposure to 200 mM H2O2 resulted in cell apoptosis by the annexin V/propidium iodide staining in the present study. The result indicated that cells treated with 200 mM H2O2 for 24 h were mainly apoptotic in neonatal rat cardiomyocytes as demonstrated previously (Chen et al. 2000). However, pretreatment with ECT 100 mg/ml significantly reduced H2O2-induced apoptotic cells (17.1173.30 vs 32.1073.69, po0.05) (Figs. 5A and B).

Influence of PI3K inhibitor on the cardioprotective effects of ECT Taking into account that ECT increased cardiomyocyte survival and decreased the apoptotic rate induced by H2O2, we studied the signaling pathway involved in the anti-apoptotic effect of ECT. Cells were pretreated with Phosphatidylinositol 3 kinase (PI3K) inhibitor (LY294002) and maintained in the presence or absence of ECT. PI3K is important for survival of many cell types. Indeed, H2O2 treatment activated PI3K in cardiac myocytes (Aikawa et al. 1998; Wang et al. 2007b). In the present research, inhibition of PI3K using LY294002 (10 mM) reduced the protective effect of ECT as evidenced by either MTT assay (Fig. 4C) or apoptotic analysis (Fig. 5B). This result demonstrated that the

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Fig. 4. Effects of ECT on cardiomyocytes viability determined by MTT assay. (A) Cells treated with different dose of H2O2 for 6 h and 24 h, respectively. Each line represents mean7S.E.M. of three separate experiments (n ¼ 6 wells in each individual experiment, *po0.05 vs. normal group). (B) Cells exposed to 200 mM H2O2 for 24 h with or without pretreatment with ECT. (C) Cells were supplemented with 10 mM LY294002 for 60 min before the addition of ECT, and then followed the same procedure as B. (mmpo0.01 vs. normal group, *po0.05 vs. H2O2 treated alone group, Kpo0.01 vs. ECT plus H2O2 treated cells). Each bar represents mean7S.E.M. of three separate experiments.

cytoprotective effects of ECT may mediate through PI3K pathway.

ECT inhibited reactive oxygen species (ROS) generation ROS is a key component in activating the apoptotic pathway in cardiovascular system (Fu et al. 2007;

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Fig. 5. Inhibition of H2O2-induced cardiomyocytes apoptosis by ECT. (A) Cells were stained with annexin V/propidium iodide, and determined by flow cytometry. (B) One group of cells were pretreated with 100 mg/ml ECT for 6 h before exposing to 200 mM H2O2 for 24 h. The other group of cells were supplemented with 10 mM LY294002 for 60 min before the addition of ECT, and then followed the same procedure as previous described. (mmpo0.01 vs. normal group, *po0.05 vs. H2O2 treated alone group, Kpo0.01 vs. ECT plus H2O2 treated cells). Each bar represents mean7S.E.M. of three separate experiments.

Semenza 2000). We hypothesized that inhibiting ROS generation might play a role in the cardioprotective effects of ECT. Intracellular ROS levels were measured by fluorescent probe DCFH-DA. As shown in Fig. 6, a significant increase in DCF fluorescence (n ¼ 6) was observed when cells were exposed to H2O2, which was attenuated by pretreatment with 100 mg/ml ECT (po0.01). The result suggested that ECT attenuated intracellular oxidative stress induced by exogenous H 2O 2.

Discussion Ample evidence demonstrated that safflower or its components possess protective effects against myocardial or cerebral ischemia (Li et al. 2006; Wang et al. 2007a; Zheng et al. 2003). The present results suggest that pre-treatment with ECT has anti-myocardial ischemia effects. This study yielded four major findings: (1) ECT improved cardiac function and decreased

myocardial infarct size induced by left anterior descending coronary artery occlusion in adult rats; (2) ECT inhibited H2O2-induced cell damage and apoptosis in cultures of neonatal rat ventricular myocytes; (3) ECT inhibited H2O2-induced intracellular ROS; and (4) the cytoprotective effects of ECT were achieved partially through the PI3K pathway. We examined the cardiac protection of ECT in the rat acute myocardial ischemia model. That pretreatment with ECT at 100, 200, 400, and 600 mg/kg body wt. reduced cardiac damage was determined using TTC staining and assessing left ventricular pressure (7dP/ dtmax). To investigate the mechanisms involved in the protective effects of ECT, we tested the direct cytoprotective effect of ECT on neonatal rat cardiomyocytes. In our study, the results showed that 100–400 mM H2O2 decreased the viability of neonatal rat ventricular myocytes in a dose- and time-dependent manner. However, pretreatment with 100–400 mg/ml ECT could inhibit the injury induced by H2O2. In addition, exposure to 200 mM H2O2 induced cell apoptosis and

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2000), which plays an important role in ischemic heart diseases and inevitably leads to heart failure (Akazawa et al. 2003; Williams 1999). The present study revealed that H2O2 exposure induced oxidative stress characterized by increased intracellular ROS in cardiomyocytes. Moreover, treatment with ECT, an extract with antioxidant properties and identified as a potent inhibitor of ROS production, could block these cellular events. Collectively, these findings suggested that ECT could protect cells from apoptosis by decreasing oxidant generation. Supporting evidence for an anti-apoptotic role for PI3K is provided by the observations in which PI3K exhibits over-expression (Matsui et al. 1999; Wu et al. 2000). In cardiomyocytes, PI3K elicits a survival signaling following exposure to H2O2, which leads to the inhibition of apoptosis (Wang et al. 2007b). Therefore, inhibition of PI3K promotes H2O2-induced apoptosis. Indeed, when LY294002, the PI3K inhibitor, was preloaded with myocytes, the protective effects of ECT were abolished. In conclusion, pretreatment with ECT could limit infarct size and improve cardiac function through reducing damage and apoptosis of cardiomyocytes induced by oxidative stress. Many factors beyond oxidative stress may, however, play a role in the pathological process of myocardial ischemia injury. The cardiac protections of safflower are due to not only its antioxidant activity, but also to the platelet aggregation inhibiting effect (Wu et al. 2007) and calcium antagonist property (Meselhy et al. 1992), contributed by different compounds, which synergistically increase the tolerance to myocardial ischemia injuries.

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Fig. 6. Effect of ECT on H2O2-induced DCF-detectable reactive oxygen species by flow cytometry. Cardiomyocytes were loaded with 5 mM DCFH-DA for 30 min in the absence or presence of H2O2 and ECT for the indicated situations. DCF fluorescence was measured by flow cytometry of 10,000 cells, and histograms of DCF fluorescence in all the cases yielded a single population. The DCF positive cells were plotted. (mmpo0.01 vs. normal group, *po0.05 vs. H2O2 treated group). Each bar represents mean7S.E.M. of three separate experiments.

ECT could attenuate cardiomyocyte apoptotic death. Therefore, the anti-myocardial ischemia effects of ECT exhibited in the rat model were achieved partially through improving cardiomyocytes viability and inhibiting cell apoptosis. Very little is known, however, about the antiapoptotic mechanisms of ECT in cardiomyocytes. Cumulative evidence suggests that reactive oxygen species produced during oxidative stress response could trigger myocyte apoptosis (Fu et al. 2007; Semenza

Acknowledgments This work was supported by the National Science Fund for Distinguished Young Scholars (Grant No. 30525043).

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