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Chinese medicinal formula Guan-Xin-Er-Hao protects the heart against oxidative stress induced by acute ischemic myocardial injury in rats.

Abstract

Guan-Xin-Er-Hao (GXEH) is a Chinese medicine formula for treating ischemic heart diseases (IHD) and has a favorable effect. Our aim was to examine whether or not acute oral GXEH could protect the heart against myocardial infarction and apoptosis in acute myocardial ischemic rats. If so, we would explain the antioxidative mechanism involved. The left anterior descending coronary artery was occluded to induce myocardial ischemia in hearts of Sprague-Dawley rats. At the end of the 3 h ischemic period (or 24 h for infarct size), we measured the myocardial infarct size, myocardial apoptosis and the activities of antioxidative enzymes. GXEH reduced infarct size, myocardial apoptosis and the serum level of malondialdehyde (MDA), increased the activities of total antioxidant capacity (T-AOC), superoxide dismutase (SOD) and GSH-peroxidase (GPX) activities and the serum level of glutathione (GSH). GXEH exerts significant cardioprotective effects against acute ischemic myocardial injury in rats, likely through its antioxidation and antilipid peroxidative properties, and thus may be used as a promising agent for both prophylaxis and treatment of IHD.

Crown Copyright [C] 2008 Published by Elsevier GmbH. All rights reserved.

Keywords: Chinese medicine formula; Guan-Xin-Er-Hao; Salvia miltiorrhiza; Ligusticum chuanxiong: Paeonia lactiflora; Carthamus tinctorius; Dalbergia odorifera; Ischemic heart disease; Oxidative stress

Introduction

Ischemic heart disease (IHD) is the leading cause of death in the world. Clinical study shows that most of the cases present with angina and myocardial infarction (MI) (Mario et al., 2006). Pathological studies show that ischemic apoptosis plays a critical role in acute MI (Baliga, 2001; Razavi et al., 2005; Yamamoto et al., 2002). However, oxidative stress has been proven to be a powerful inducer of programmed cell death (Kehrer, 2000; Kumar et al., 2002; Zhao, 2004). In recent years, researchers have found that many traditional plants and their extracts have antioxidative effects on IHD (Cai et al., 2004).

Chinese medicinal formula Guan-Xin-Er-Hao (GXEH) is widely used in China, Japan and Korea for the treatment of IHD and has produced a favorable effect (Xu et al., 2001; Zhu and Huo, 2002). GXEH contains Salvia miltiorrhiza Bge., Carthamus tinctorius L., Paeonia lactiflora Pall., Ligusticum chuanxiong Hort. and Dalbergia odorifera T.Chen. Its constitutive ratio of five herbs is 2:1:1:1:1 of the dry weight. Some studies have suggested that Salvia miltiorrhiza Bge. and Carthamus tinctorius L. possess antioxidative properties (Cai et al., 2004); other results have proved that the formula GXEH has antiischemia therapeutic effect (Zhao et al., 2007) and the phenomenon of scavenging active oxygen free radical (Wang et al., 2003).

However, there is little information on the antiox-idative mechanisms of GXEH. The object of the present research was to evaluate whether or not acute oral GXEH could protect the heart against myocardial infarction and apoptosis in acute MI rats. If so, we would explain the antioxidative mechanisms involved.

Materials and methods

Medicines

All the crude drugs of GXEH including 200 g of Salvia miltiorrhiza Bge., 100 g of Ligusticum chuanxiong Hort., 100 g of Paeonia lactiflora P., 100 g of Carthamus tinctorius L. and 100 g of Dalbergia odorifera T. Chen were purchased from West China Hospital (Chengdu, Sichuan). They were identified by the herbal medicine botanist Professor Hu Z.H., Department of Botanical Anatomy of Northwest University, where voucher specimens (No. 070421) had been kept.

The mixture of GXEH was soaked in distilled water (1:12, w/v) for 0.5h at room temperature with occasional stirring. They were prepared by boiling for 0.5 h, and the cooled decoction was filtered through two layers of cotton gauze. The residue was boiled again with distilled water (1:6, w/v) by the same procedure described above. The solution two times obtained was concentrated under underpressure at 65 [degrees]C, then lyo-philized and stored at 4 [degrees]C. The lyophilized powder was resolved to the scale with distilled water according to the standard of 1 g/ml (w/v) before experiment (Zhao et al., 2007).

Quantitative analysis of marker compounds

The high-performance liquid chromatography (HPLC) system included a Waters 2695 liquid chroma-tograph system consisting of a quaternary pump, an autosampler and a Waters 2996 photodiode array detector coupled with Empower chromatographic workstation. A Diamonsil [C.sub.18] column (4.6 mm x 150 mm, 5 [micro]m) was used for analysis. The mobile phase was methanol-aqueous acetic acid with gradient elution (0 min, 15:85; 15 min, 30:70; 40 min, 32:68). The detection wavelengths of the photodiode array detector were set at 280 nm, flow-rate was 1.0 ml/min. The freeze-dried powder of GXEH was further diluted with distilled water to obtain the working solution for HPLC. Ferulic acid served as the reference compound for Ligusticum chuanxiong Hort.; Tanshinol and protocatechualdehyde served as the reference compound for Salvia miltiorrhiza Bge.; Paeoniflorin served as the reference compound for Paeonia lactiflora P.; and hydroxysafflor yellow A served as the reference compound for Carthamus tinctorius L. The five reference components were purchased from the National Institute for Control of Pharmaceutical and Biological Products (Beijing, China) and their purity was > 99%. The contents of the five components were used for the quality control of GXEH.

According to the HPLC method used, we determine the main water-soluble components of GXEH. The result indicated that it contained several compounds, including tanshinol, protocatechualdehyde, peoniflorin, hydroxysafflor yellow A, ferulic acid and some unknown compounds as shown in Fig. 1. The content of each component in the GXEH formula (n = 3) and retention time (Rt, min) was as follows: tanshinol 0.704 [+ or -] 0.007 (mg/g, Rt = 5.804), protocatechualdehyde 0.015 [+ or -] 0.0005 (mg/g, Rt = 9.430), peoniflorin 3.715 [+ or -] 0.131 (mg/g, Rt = 17.820), hydroxysafflor yellow A 2.067 [+ or -] 0.017 (mg/g, Rt = 21.450) and ferulic acid 0.129 [+ or -] 0.001 (mg/g, Rt = 24.067). Each column represented the mean [+ or -] SD.

[FIGURE 1 OMITTED]

Surgical preparation of animals

The surgical procedure was performed according to a previous study (Ling and Lou, 2005) with minor modifications. Briefly, rats were anesthetized intraper-itoneally with pentobarbitone at a dose of 36 mg/kg and subcutaneous peripheral limb electrodes were inserted and the ECG was recorded for the entire duration of the experiment (ADInstruments, Australia). After performing a left thoracotomy, the incised area was extended using forceps and the pericardium was opened. After tracheal intubation, the rats were ventilated by a respirator (HX300, Chengdou, China) with room air with a tidal volume of 10 ml/min and a respiratory rate of 70 cycles/min. The heart was pushed out of the chest and the left anterior descending coronary artery was ligated using a 3/0 silk thread. Successful ligation was verified by the occurrence of arrhythmias and, visually, by the color change of the ischemic area. The heart was immediately returned to its anatomical position and the chest was closed while slight pressure was applied from outside so that air did not remain in the chest. Then the skin was sutured. Eighty male Sprague-Dawley rats (200-240 g) were randomly assigned to four groups: (1) sham-operated control group (sham MI): rats underwent the same surgical procedures except that the suture passing under the coronary artery was not tied. (2) Vehicle control group (MI [+ or -] Vehicle): rats were orally given vehicle (0.9% NaCl, 20 ml/kg). (3) Low dosage of GXEH group (MI [+ or -] GXEHL): rats were orally given GXEH extract (5 g/kg) 30 min before ischemia. (4) High dosage of GXEH group (MI [+ or -] GXEHH): rats were orally given GXEH extract (15 g/kg) 30 min before ischemia. At the end of the 3 h ischemic period (or 24 h for infarct size), the heart was quickly excised and the cardiac tissue was processed according to the procedures described below; the mortality of the operation was approximately 13%.

Determination of the infarct size

At the end of the 24 h of MI, 1 ml 2% Evans blue dye was injected into the left ventricular cavity. The heart tissue was washed with phosphate-buffer saline (PBS) three times and frozen at -20 [degrees]C and sliced into 1-mm-thick sections. Then sections were incubated for 15 min in 1% solution of 2,3,5-triphenyltetrazolium chloride (TTC) in phosphate buffer at pH 7.4 at 37 [degrees]C for pathological examinations. After that, the TTC-stained area (red staining), the TTC-stained negative area (infarct myocardium) and the Evans blue-stained area (area not at risk) were digitally captured and measured using ImagePro-Plus 5.0.1. The ischemic risk areas ratio (n = 6-7/group), which is the most reliable index for the determination of protective effect, was defined as the percentage of the total ischemic risk area over the total left ventricle areas measured.

Determination of myocardial cells apoptosis

The apoptotic myocytes were quantitatively detected by terminal deoxy-nucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL). TUNEL staining was performed using a Cell Death Detection Kit (Roche) according to the manufacturer's instructions. In brief, fixed tissue was embedded in a paraffin block and 5-[micro]m-thick slides were cut from each tissue block. After treating with proteinase K, the tissue sections were incubated with TUNEL reaction mixture and nonspecific binding sites were blocked. The slides were covered with the mounting medium containing DAPI to permit total nuclei counting. At least five slides per block were evaluated. An additional staining was performed with monoclonal anti-[alpha]-actinin. For each slide, 10 fields were randomly chosen using a defined rectangular field area (20 objective). The index of apoptosis was determined (i.e., number of positively stained apoptotic myocytes/ total number of myocytes counted x 100). Assays were performed in a blinded manner.

Antioxidant assay

For the assays of total antioxidant capacity (T-AOC), superoxide dismutase (SOD), malondialdehyde (MDA), glutathione (GSH) and GSH-peroxidase (GPX) activities, blood was sampled from the abdominal aorta and serum was obtained after centrifugation at 3000g for 10 min. The T-AOC, SOD, MDA, GPX and GSH levels were measured spectrophotometrically using diagnostic kits (Jiancheng, Nanjing, China) according to the manufacturer's instructions.

Statistical analysis

All data were expressed as mean [+ or -] standard deviation (SD). The database was set up with SPSS 15.0 software package (SPSS Inc. Chicago, USA). A Dunnett's t-test was used to compare the data obtained before and after occlusion. Differences among groups were analysed by one-way ANOVA. A probability of less than 0.05 was considered to be statistically significant.

Results

Effect of GXEH on infarct size

Photographs of myocardial infarct size in control and sample groups are shown in Fig. 2A. The area of necrosis (AN, white area), the area at risk (AAR, red staining and white area) and the area of left ventricle (LV) were measured. The results of GXEH on AAR/LV and AN/AAR are shown in Fig. 2B. There was no significant difference in AAR/LV among the groups. Administration of GXEH 15 g/kg significantly reduced the AN/AAR compared with the vehicle control group (18.76 [+ or -] 8.26% vs. 44.94[+ or -] 13.28%).

[FIGURE 2 OMITTED]

Effect of GXEH on TUNEL-positive cells

Representative photomicrographs of in situ detection of DNA fragments in heart tissue from rats subjected to sham ischemia or 3 h of ischemia receiving vehicle or GXEH are shown in Fig. 3A. Total nuclei were labeled with DAPI (blue), and apoptotic nuclei were detected by TUNEL staining (green). The summary of percent TUNEL-positive myocytes is shown in Fig. 3B. Heart tissue from sham-operated rats exhibited very low levels of staining for TUNEL. The percentage of TUNEL-positive cells was 54.44 [+ or -]14.96% in the vehicle control group. Treatment of GXEH at doses of 5 and 15 g/kg resulted in a reduction in the percentage of TUNEL-positive cells, 45.83 [+ or -] 9.78% and 36.83 [+ or -] 5.32%, respectively. There were significant differences in the percentage between the control rats and those treated with GXEHL and GXEHH (p < 0.01).

[FIGURE 3 OMITTED]

Effect of antioxidant assay

The serum levels of T-AOC, SOD, GPX and GSH were significantly increased (15.64 [+ or -] 2.42, 189.95 [+ or -] 20.05, 417.88 [+ or -] 30.86 U/ml and 210.32[+ or -]32.67mg/l, respectively), while the serum MDA level was significantly decreased (6.33 [+ or -] 2.41 U/ml), compared with the normal controls and sham-operated rats (Fig. 4). GXEH at doses of 5 and 15 g/kg significantly reduced the serum levels of T-AOC (to 25.86 [+ or -] 4.88 and 27.19 [+ or -] 6.75 U/ml, respectively), SOD (to 222.56 [+ or -] 27.94 and 234.42 [+ or -] 31.49 U/ml, respectively) and GSH (to 271.58 [+ or -] 61.77 and 271.37 [+ or -] 49.04 mg/l, respectively) (all p < 0.05, Figs. 4A, C and E). In addition, GXEH at a dose of 15 mg/kg increased the serum GPX level (to 450.04 [+ or -] 16.19 U/ml, p < 0.05) (Fig. 4D), but at a dose of 5 mg/kg decreased the serum MDA level (to 4.35 [+ or -] 1.06 U/ml, p < 0.05) (Fig. 4B).

[FIGURE 4 OMITTED]

Discussion

The present study demonstrated that GXEH effectively reduces infarct size in acute myocardial ischemic rats. Further studies found that GXEH reduces the TUNEL-positive cells and the amounts of MDA, but enhances T-AOC, SOD, GPX activities and GSH content.

Traditionally, cardiac myocyte death during ischemic injury has been thought to occur exclusively by necrosis (Madias, 1988). However, this view has been challenged by recent accumulating studies (Baliga, 2001; Elsasser et al., 2001; Razavi et al., 2005; Yamamoto et al., 2002) documenting that large numbers of myocytes undergo apoptosis in response to prolonged ischemia in animal models of myocardial ischemia and in humans. One of the most important biochemical hallmarks of apoptosis in many cells is nuclear DNA fragmentation. The TUNEL system is designed for the detection of apoptotic cells via this hallmark. Zhao et al. (2007) have reported that GXEH is effective in treating myocardial apoptosis in a myocardial ischemia-reperfusion (I/R) rat model, and this study showed that the effects in acute myocardial ischemic rats were similar to previous studies. The strongest nuclear green fluorescence was observed in the GXEH group (Fig. 3). The ability of GXEH to reduce green fluorescence from the TUNEL assay indicates the cardioprotective potential of GXEH after MI.

Evidences gathered from systematic reviews show that oxidative stress could powerfully induce programmed cell death (Kehrer, 2000; Kumar et al., 2002; Zhao, 2004). Reactive oxygen species (ROS), which possess highly reactive and toxic properties, can be generated as a result of ischemia and exacerbate the degree of myocardial damage (Ferrari et al., 2002; Wattanapi-tayakul and Bauer, 2001). In response to this, animals have developed a natural defensive system to cope with these unwanted and toxic species. Such defense mechanisms include superoxide dismutase (SOD), glutathione peroxidase (GPX) and others (Gilberto et al., 2008). In the present study, our study shows that acute oral GXEH possess antioxidative properties; it is similar to the result of Wang et al. (2003).

The measure of total antioxidant capacity (T-AOC) is considered the cumulative action of all the antioxidants present in serum, thus providing an integrated parameter rather than the simple sum of measurable antioxidants (Ghiselli et al., 2000). The capacity of known and unknown antioxidants and their synergistic interaction is therefore assessed, thus giving an insight into the delicate balance in vivo between oxidants and antioxidants. Our present study showed that T-AOC of the vehicle control group was a significant decrease compared to the sham-operated control group, and demonstrated that MI increased oxidative stress. The results also demonstrated that GXEH increases the serum level of T-AOC (Fig. 4A). SOD and GPX are two critical enzymes of the known antioxidant, which may contribute to the increase of T-AOC.

SOD is a very important enzyme; an increase in its activity has been reported to be beneficial to the cellular capability of scavenging/quenching free radicals (Chen et al., 1996). Malondialdehyde (MDA), the degradation product of the oxygen-derived free radicals and lipid oxidation, reflected the damage caused by ROS (Das, 1999; Marnett, 1999). The studies on the antioxidant system showed that the changes in the activity of SOD and the level of MDA were always negatively correlated (Zheng et al., 2008). In the present study, GXEH also increased the serum levels of SOD when administered at doses of 5 and 15 g/kg but low MDA production (results are shown in Fig. 4B and C), which implies that the formula may affect the level of endogenous antioxidants or oxidative stress or both. One of the possible explanations is that elevated activities of SOD scavenged excessive ROS and attenuated the lipid peroxidation.

GSH is an important cellular reductant that offers protections agaionst free radicals, peroxide and toxic compounds against free radicals, peroxide and toxic compounds (Sakurai et al., 1999; Wojtczak and Sly-shenkov, 2003). It is reformed from GSSG, and GPX is a key catalyzer (Kayanoki et al., 1996; Shidoji et al., GPX levels might signify that the level of oxidative stress was not severe enough for these compounds to be involved in the present study. However, the results showed that administration of GXEH caused a significant increase in the GSH and GPX levels, which might be due to the treatment group containing compounds present in the GXEH (Fig. 4D and E).

The quality control of traditional Chinese medicine (TCM) is necessary for carrying out a standardized pharmacology research. So far, it is widely accepted that multiple constituents are responsible for the therapeutic effects of TCM, and water-soluble components are attracting more attention recently since water decoctions of GXEH are commonly used in clinics in China. In the freeze-dried powder of water extract (stabile to preserve), the reference compounds are danshensu, proto-catechualdehyde, paeoniflorin, hydroxysafflor yellow A and ferulic acid (the chromatogram is shown in Fig. 1). The content is similar to our previous study (Zhao et al., 2007), and its stability also ensures our further research.

In conclusion, GXEH exerts significant cardioprotective effects against myocardial cells apoptosis in MI rats, likely through its antioxidant and antilipid peroxidation property, and thus may be used as an effective and promising agent for both prophylaxis and treatment of IHD.

Acknowledgements

This work was supported by a grant (No. 30325045) from National Science Fund for Distinguished Young Scholars of China and partly supported by the Natural Science Foundation of China (No. 30572339).

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F. Qin (a), Y.-X. Liu (b), H.-W. Zhao (b), X. Huang (a), *, P. Ren (a), Z.-Y. Zhu (b)

(a) Laboratory of Ethnopharmacology and Institute of Integrated Chinese and Western Medicine, Xiangya Hospital, Central South University, Changsha 410008, PR China

(b) Department of Integrated Chinese and Western Medicine, West China Hospital, Sichuan University, Chengdu 610041, PR China

* Corresponding author. Tel.: +86 731 4327222; fax: +86 731 4328386.

E-mail address: tcmhuangx59@163.com (X. Huang).
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Author:Qin, F.; Liu, Y.-X.; Zhao, H.-W.; Huang, X.; Ren, P.; Zhu, Z.-Y.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:9CHIN
Date:Mar 1, 2009
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