Hydroxysafflor yellow A attenuates left ventricular remodeling after pressure overload-induced cardiac hypertrophy in rats

Abstract

Context: Hydroxysafflor yellow A (HSYA), the main chemical component of the safflower yellow pigments, is used extensively in traditional Chinese medicine for the treatment of cerebrovascular and cardiovascular diseases.

Objective: The present study determined the effects of HSYA on left ventricular hypertrophy after pressure overload and investigated the underlying mechanisms.

Materials and methods: Cardiac hypertrophy was induced by the ligation of abdominal aorta in male Wistar rats. The rats were then divided into five groups and treated with captopril (100 mg/kg) or HSYA at different doses (0, 10, 20 and 40 mg/kg). Six weeks after treatment, the weight of left ventricle, LVMI (left ventricular mass index) and pathological changes were measured. MMP-2 (metalloproteinase 2) and MMP-9 (metalloproteinase 9) levels were determined by ELISA. Protein expressions of Bcl-2 and Bax were evaluated by immunohistochemistry.

Results: HSYA (20, 40 mg/kg) significantly attenuated the increase of LVMI (ventricular weight/body weight) by 13.04 and 30.43% respectively, when compared with the model group. This was associated with the amelioration of pathological lesion, such as cardiac muscle fibers were smaller and the nuclei of cardiomyocytes were lightly stained in animals treated with HSYA (20, 40 mg/kg). In addition, the administration of HSYA at doses of 20 and 40 mg/kg increased the Bcl-2/Bax ratio (1.17 ± 0.08 and 1.39 ± 0.07 versus 0.71 ± 0.06). In addition, the serum MMP-2 and MMP-9 levels were blocked by the treatment at doses of 20 and 40 mg/kg HSYA (MMP-2, 76.1 ± 9.2 and 65.6 ± 6.8 versus 82.9 ± 6.2, ng/ml; MMP-9, 66.6 ± 4.8 and 57.5 ± 5.0 versus 83.5 ± 6.0, ng/ml).

Conclusion: These findings indicated that HSYA has beneficial effects on hypertensive ventricular remodeling, which may involve mechanisms of inhibiting cell apoptosis and suppressing metalloproteinases expression.

• Anti-apoptosis
• metalloproteinases
• Hydroxysafflor Yellow A
• left cardiac remodeling
• left ventricular remodeling

Introduction

Left ventricular hypertrophy, occurring in many types of heart diseases is a compensatory response to abnormal changes in load, pressure and other stimuli. For the patients with cardiac hypertrophy caused by hypertension that produces mechanical stimulation to the heart, the incidence of arrhythmia, heart failure, coronary occlusion and sudden death rate are obviously increasing (Pearson et al., 1991). A direct relationship has been shown between the regression of cardiac hypertrophy and the decrease of cardiac mortality (Koren et al., 1991; Verdecchia et al., 2001). Therefore, the prevention of left ventricular hypertrophy is critical.

The flower of the safflower plant, namely Carthamus tinctorius Linn., is used extensively in traditional Chinese medicine for the treatment of cerebrovascular and cardiovascular diseases (Pu & Jin, 2005; Wei et al., 2005; Xiao, 2006). Hydroxysafflor yellow A (HSYA, Figure 1), the main chemical component of the safflower yellow pigments, has been shown to antagonize platelet activating factor receptor binding (Zang et al., 2002). Hypotensive and antithrombotic activities and inhibitory effects on platelet aggregation have also been reported (Tian et al., 2003). Moreover, HSYA has been shown to have cardioprotective effects after both acute and chronic myocardial ischemia in dogs and rats (Song et al., 2005; Xu et al., 2005; Zheng et al., 2005). However, there have been no reports on the effects of HSYA on left ventricular remodeling after pressure overload-induced cardiac hypertrophy, and the mechanisms of its cardioprotective effect remain poorly understood. In the present study, we used a left ventricular hypertrophy rat model to evaluate the effects of HSYA on hypertensive ventricular remodeling and the possible mechanisms involved.

Figure 1. The chemical structure of HSYA.

Materials and methods

Plant materials

HSYA, a water-soluble plant pigment, was provided by Yantai Luye Pharmaceutical Co. Ltd, It was isolated from the dried flower petals of Carthamus tinctorius. with a purity of >90% as determined by high performance liquid chromatography (HPLC) (Liu et al., 2004).

Animals and experimental proceeding

Eight-week-old male rats (body weight 160–190 g) were purchased from the Animal Experimental Division of Shandong Luye Pharmaceutical Co. Ltd. (SPF grade). They were kept in the room at the controlled temperature (22 ± 1 °C), humidity (50–70%) and 12 h light/dark cycle. The animals were allowed free access to food and water. All animal experiments were undertaken according to Institutional guidelines and approved by the Ethics Committee for Animal Care and Use.

Rats were anaesthetized by intraperitoneal injecting of 3% pentobarbital sodium (30 mg/kg); the abdominal aorta was isolated above mesenteric artery through midline of left abdomen as an incision site and ligated with a 7-0 silk suture. Sham-operated animals also underwent all the above described surgical procedures except for the ligation. All rats were continuously injected with penicillin 100 000 U with normal diet for 3 days. Rats with abdominal aortic coarctation were randomized into five groups: model group, captopril group (100 mg/kg, Pingyuan Pharmaceutical Factory, Shandong, China), 10, 20 and 40 mg/kg doses of HSYA. Animals were treated by gavage with drugs or the same volume of distilled water (Sham-operated animals and model group animals) for 6 weeks.

Calculation of cardiac coefficient and histopathological examination

The weight of the left ventricle was measured after rinsing blood. LVMI (left ventricular mass index) was calculated as left ventricle/body weight. In addition, hearts were fixed in 10% formalin and embedded in paraffin. The paraffin-embedded tissues were sectioned and stained with hematoxylin--eosin (H&E). The pathological changes were examined under a light microscope by an independent observer who had no knowledge of this study.

Immunohistochemical staining

Immunohistochemical analyses of Bax and Bcl-2 (Abcam Company, Cambridge, MA) were performed on sections fixed with 4% paraformaldehyde using classical immunoperoxidase techniques. Three sections per animal and five fields per section were scanned and analyzed. Results were recorded by two investigators independently assessing the percentage of positive cells and the intensity of staining. The results were assessed by the Bcl-2/Bax ratio as in a previous study (Condorelli et al., 1999).

Determination of serum MMP-2 and MMP-9

Blood samples were centrifuged at 3000 rpm for 15 min at 4 °C and the plasma was immediately stored under 70 °C until being analyzed. Serum levels of MMP-2 and MMP-9 (Dalian Fanbang Company, China) were measured using ELISA kits according to manufacturer’s instructions.

Statistical analysis

Data were presented as mean ± SD and analyzed with one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test. Statistical analyses were performed by using the SPSS 11.5 (SPSS Inc., Chicago, IL), p < 0.05 was considered statistically significant.

Results

Determination of left ventricular hypertrophy

The initial body weight of each group of animals was similar. Six weeks after cardiac hypertrophy, the body weight of all animals was alike (data not shown). However, the ventricular weight/body weight in model animals was higher than that of Sham animals (p < 0.01), suggesting cardiac hypertrophy. In contrast, HSYA 20 and 40 mg/kg ameliorated this parameter (p < 0.05, p < 0.01, Figure 2).

Figure 2. Effects of HSYA on ventricular weight/body weight. Values are expressed as the mean ± SD. Significance was determined by ANOVA followed by Tukey’ s test. ##p < 0.01 compared with Sham group. *p < 0.05, **p < 0.01 compared with model group.

Effect of HSYA on the hypertrophy of cardiomyocytes

As shown in Figure 3, rats treated with aortic banding developed marked cardiac hypertrophy in the left ventricular myocardium. Cardiac muscle fibers were large and arranged in a loose pattern. The nuclei of cardiomyocytes were darkly stained. Treatment with HSYA at doses of 20 and 40 mg/kg significantly relieved the cardiac structural changes caused by ventricular hypertrophy.

Figure 3. Effect of HSYA on the hypertrophy of cardiomyocytes (H&E, ×400): (A) Sham-operated rats, (B) Model rats, (C) rats treated with captopril (100 mg/kg), (D) rats treated with HSYA 10 mg/kg, (E) rats treated with HSYA 20 mg/kg, and (F) rats treated with HSYA 40 mg/kg.

Bax and Bcl-2 protein expression

To determine whether HSYA regulates the expression of apoptosis-related proteins, Bax and Bcl-2 expressions were evaluated by immunohistochemistry. Figure 4 shows that the Bcl-2/Bax ratio decreased in the left ventricular myocardium (0.71 ± 0.06 versus 1.80 ± 0.09, p < 0.01) and this decrease was greatly decreased by the administration of HSYA at doses of 20 and 40 mg/kg (1.17 ± 0.08 and 1.39 ± 0.07, respectively. p < 0.01, p < 0.05, compared to model). These results suggested that HSYA was able to suppress the formation of cardiac hypertrophy by preventing the occurrence of apoptosis.

Figure 4. Effect of HSYA on Bax and Bcl-2 protein expression in left ventricular myocardium. Values are expressed as the mean ± SD. Significance was determined by ANOVA followed by Tukey’s test. ##p < 0.01 compared with Sham group. *p < 0.05, **p < 0.01 compared with model group.

Determination of serum MMP2 and MMP9

Figure 5 shows the serum MMP2 and MMP9 values recorded in anesthetized animals. After aortic banding, the serum levels of MMP2 and MMP9 were increased when compared with the Sham group. However, chronic continuous treatment with HSYA (20 and 40 mg/kg) could significantly improve these parameters (p < 0.05, p < 0.01).

Figure 5. Effects of HSYA on serum levels of MMP2 and MMP9. Values are expressed as the mean ± SD. Significance was determined by ANOVA followed by Tukey’ s test. ##p < 0.01 compared with Sham group. *p < 0.05, **p < 0.01 compared with model group.

Discussion

HSYA has various pharmaceutical properties such as anti-thrombosis and inhibition of platelet aggregation. It has been proven safe and effective in the treatment of cardiovascular and cerebrovascular diseases. In the present study, we focused on demonstrating the possible molecular mechanisms underlying the effect of HSYA to protect overload-induced cardiac hypertrophy. The results indicated that HSYA could prevent left ventricular remodeling after pressure overload-induced cardiac hypertrophy in rats, attenuating ventricular weight/body weight and ameliorating cardiomyocytes hypertrophy. On the basis of the obtained results that HSYA protected cardiac hypertrophy induced by aortic banding, further investigation was performed with focusing on the possible mechanisms involved in the cardioprotective of HSYA.

It has been reported that apoptosis can be induced in cardiomyocytes by various factors including pressure overload (Choi et al., 2009; Sano et al., 2007). Many genes have been reported to be linked with the regulation of programmed cell death under physiological and pathological conditions, in which Bcl-2 and Bax genes are suggested to play a major role in determining cell's survival or death after apoptotic stimuli (

 Google Scholar

Liu et al.,

 Google Scholar

2007 ). Moreover,

 Google Scholar

Bax in aortic-banded group was overexpressed when Bcl-2 expression was not apparent, indicating that apoptosis occurs during the development of myocardial hypertrophy (Choi et al., 2009). Thus, the effects of

 Google Scholar

HSYA on

 Google Scholar

the

 Google Scholar

expression of Bcl-2 and Bax-proteins were detected by immunohistochemical staining. The results

 Google Scholar

indicated that Bcl2/Bax in HSYA-treated rats was significantly increased compared with aortic-banded group after chronic pressure overload, indicating that anti-apoptosis of HSYA may play a major role in the beneficial effects of HSYA on cardiac hypertrophy in rats.

Matrix metalloproteinases (MMPs) belong to a family of structurally related zinc-containing endopeptidases, which are important in the degradation of extracellular matrix (ECM) components (Creemers et al., 2001). Many studies have verified the role of abnormal ECM metabolism in hypertension ( Derosa et al.,

 Google Scholar

2006; Zervoudaki et al.,

 Google Scholar

2003). Broad-spectrum pharmacological inhibition of MMPs significantly attenuates myocardial remodeling associated with chronic volume overload, hypertension, myocardial infarction and other conditions in animal models ( Chancey et al.,

 Google Scholar

2002). MMP-2 and MMP-9 play important roles in cardiac remodeling from direct genetic evidence ( Kim et al.,

 Google Scholar

2000). Hence, the regulation of MMPs activities is a potential target for therapy in the left ventricular remodeling and heart failure. In our study, HSYA could also significantly inhibit the development of cardiac hypertrophy and remodeling during 6 weeks of abdominal aortic coarctation in rats. Notably, these effects were along with the decrease of MMP2 and MMP9 levels. Taken together, these findings suggested that HSYA might be an important MMPs regulatory factor, and further experiments will be needed to clarify this question.

 Google Scholar

It is well known that several multi-target interference approaches have been widely used in traditional Chinese medicine (TCM) for a very long time and their efficiencies have also been proved (Li et al., 2007; Zhou & Li, 2007). This study demonstrated that HSYA attenuates left ventricle hypertrophy, remodeling after pressure overload by regulating the expression of apoptosis-related proteins and MMPs. To our knowledge, the present study demonstrated for the first time that HSYA protected overload-induced cardiac hypertrophy. Practically, these insights could encourage future analysis as to whether long-term pharmacological application of HSYA may offer some therapeutic opportunities for protection against heart diseases and exploring the underlying mechanisms.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.

Acknowledgements

The authors are grateful to Prof. Xuguo Liang (Yantai Yuhuangding Hospital) and Prof. Zhifeng Liu (Luye pharmaceutical Ltd. Company, China) for their assistance and invaluable suggestions.