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Archives of cardiovascular diseases
Volume 104, n° 5
pages 313-324 (mai 2011)
Doi : 10.1016/j.acvd.2011.02.004
Received : 13 April 2010 ;  accepted : 22 February 2011
Protective effects of salvianolate on microvascular flow in a porcine model of myocardial ischaemia and reperfusion
Effet protecteur du salvianolate sur la microcirculation dans un modèle d’ischémie myocardique reperfusion chez le porc
 

Beibei Han a, Xin Zhang b, Qingyong Zhang a, Gang Zhao a, Junbo Wei a, Shixin Ma a, Wei Zhu a, Meng Wei a,
a Division of Cardiology, Shanghai Sixth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China 
b Division of Cardiology, The First Affiliated Hospital of Baotou Medical College, Inner Mongolia Province, China 

Corresponding author. Fax: +86 21 64701932.
Summary
Background

Microvascular reflow is crucial for myocyte survival during ischaemia/reperfusion injury.

Aims

We aimed to assess if salvianolate, a highly purified aqueous extract from Radix salviae miltiorrhizae , could improve impaired microvascular reflow induced by ischaemia/reperfusion injury, using a porcine closed-chest model.

Methods

Left anterior descending coronary artery ligation was created by balloon occlusion for 2h followed by reperfusion for 14 days. Salvianolate was administrated intravenously for 7 days at low dose (5mg/kg/day), high dose (10mg/kg/day) or high dose combined with one 20mg intracoronary bolus injection just at the beginning of reperfusion. Control-group animals were only given the same volume of saline.

Results

After 14 days of reperfusion, animals treated with high-dose salvianolate showed improved myocardial perfusion assessed by real-time myocardial contrast echocardiography and coloured microspheres. The beneficial effect was further supported by increased capillary density and decreased infarct size. All these effects eventually resulted in well-preserved cardiac function detected by echocardiography. Moreover, we also demonstrated that salvianolate administration was associated with elevated superoxide dismutase activity, thioredoxin activity and glutathione concentration, and reduced malondialdehyde concentration, which, in turn, resulted in a significant decrease in terminal deoxynucleotide transferase-mediated dUTP nick end labelling-positive cells and an increased ratio of Bcl-2 to Bax expression.

Conclusion

Intravenous salvianolate at a dose of 10mg/kg/day for 7 days had significant beneficial effects on myocardial microvascular reflow, which were associated with decreased oxidative stress and apoptosis.

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Résumé
Justification

Le flux dans la microcirculation est déterminant pour la survie myocytaire pendant les lésions d’ischémie/reperfusion.

Buts

Nous avons pour objectifs d’évaluer si le salvianolate, un extrait hautement purifié provenant de miltiorrhizae salviae radix pouvait améliorer le flux dans la microcirculation, altéré par les lésions d’ischémie/reperfusion dans un modèle de porcs à thorax fermés.

Méthode

La ligature de l’artère interventriculaire antérieure a été créée par une occlusion au ballonnet d’une durée de deux heures, suivie d’une reperfusion d’une durée de 14jours. Le salvianolate a été administré par voie intraveineuse pendant septjours à faible dose (5mg/kg/jour), haute dose (10mg/kg/jour) ou de fortes doses associées à l’injection intracoronaire en bolus d’une dose de 20mg juste avant le début de la reperfusion. Il n’a été administré que du sérum salé dans le groupe d’animaux témoins.

Résultats

Après une reperfusion d’une durée de 14jours, les porcs traités par de fortes doses de salvianolate ont eu une amélioration de la perfusion myocardique évaluée par échographie de contraste myocardique et par microsphère colorée. L’effet bénéfique a été amélioré par l’augmentation de la densité capillaire et la diminution de la taille de l’infarctus. Tous ces effets observés ont été associés à une amélioration de la fonction ventriculaire gauche, détectée par échographie. De plus, il a été également démontré que l’administration de salvianolate était associée à une augmentation de l’activité superoxyde dismutase, de l’activité thioredoxine et de la concentration de glutathion ainsi qu’une réduction de la concentration de malondialdehyde, dont la conséquence a été une réduction significative de cellules spécifiques portant l’activité deoxynucléotide transférase, médiée par l’UTP et une augmentation du ratio de l’expression du rapport Bcl-2 sur Bax.

Conclusion

L’administration intraveineuse de salvianolate à la dose de 10mg/kg/jour pendant septjours s’accompagne d’effets bénéfiques sur le flux dans la microcirculation coronaire, associée à une diminution du stress oxydatif et de l’apoptose.

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Keywords : Salvianolate, Ischaemia/reperfusion, Microvascular flow, Oxidative stress, Apoptosis

Mots clés : Salvianolate, Lésion d’ischémie/reperfusion, Flux dans la microcirculation, Stress oxydatif, Apoptose


Abbreviations

LAD : left anterior descending coronary artery
LV : left ventricular
MBF : myocardial blood flow
MCE : myocardial contrast echocardiography
vWF : von Willebrand factor
TUNEL : terminal deoxynucleotide transferase-mediated dUTP nick end labelling

Background

Reperfusion therapy is the most important therapeutic strategy for improving the prognosis of patients with acute myocardial infarction. However, although Thrombolysis in Myocardial Infarction (TIMI) grade 3 coronary flow is restored, MCE studies showed that about 20–35% of patients had severely reduced tissue-level perfusion [1]. This inadequate tissue perfusion was closely associated with progressive LV remodelling, leading to severe congestive heart failure, and hence, high mortality [2]. Radix salviae miltiorrhizae , known as ‘Danshen’, is an herb that is widely used in the treatment of cardiovascular diseases in China. The aqueous extract from Danshen has shown beneficial effects in terms of limiting the size of an infarct caused by myocardial ischaemia/reperfusion injury [3, 4]. However, it is not clear whether the aqueous extract from Danshen can improve myocardial microvascular reflow in the setting of ischaemia/reperfusion injury. Salvianolate, as a highly purified aqueous extract from Danshen, contains mainly magnesium lithospermate B (≥85%), rosmarinic acid (≥10.1%) and lithospermic acid (≥1.9%) [5]. In the present study, we aimed to determine whether salvianolate could protect microvascular reflow against ischaemia/reperfusion injury, by using a closed-chest porcine model that can minimize the inflammatory responses induced by open-chest surgery, thus mimicking the clinical situation.

Methods
Animal procedures

The protocol was approved by the Animal Care and Use Committee at Shanghai Jiaotong University and conformed to the Chinese Medical Association guidelines for the use of animals in research.

On day 1, minipigs (23–30kg) were sedated with ketamine (15mg/kg intramuscularly) and anaesthetized with an intravenous infusion of sodium pentobarbital (10mg/kg initially, then 1mg/kg as needed), then intubated and mechanically ventilated. Through a right femoral arterial sheath, a 6-F guiding catheter (Amplatz Left 1) was placed into the ostium of the left main coronary artery. The LAD was occluded for 2h using an intracoronary balloon catheter (15×2.5mm balloon) at the site distal to the second diagonal branch. After reperfusion for 2h, intravenous anaesthesia was stopped. Sedation with midazolam (1mg/kg intramuscularly, as needed) was continued for 24h, during which the right femoral artery and vein remained cannulated for MCE and microsphere injection at 24h of reperfusion. Blood pressure and heart rate were continuously monitored. During coronary intervention, pigs were heparinized (5000U bolus followed by 100U/kg/hour) and then oral aspirin (300mg) was given daily until euthanization.

On day 8 and day 15 (7 and 14 days after reperfusion, respectively), under sedation with midazolam, the carotid artery was cannulated for microsphere injection, while the carotid vein was used for MCE contrast injection.

After 14 days of reperfusion, the LAD was reoccluded and 2% Evans Blue dye was injected into the LAD via a carotid venous sheath, to identify the risk area. The animals were then euthanized and the heart was rapidly removed and cut into five short-axis slices, perpendicular to the ventricular septum from the base to the apex of the heart (6–8mm thick; labelled as S1 to S5, respectively). All five slices were weighed and recorded.

Study groups

After LAD occlusion, pigs were randomized into the following groups: Group I (saline control), normal saline without salvianolate; Group II, 5mg/kg salvianolate diluted with 100mL saline and given intravenously at a rate of 100mL/hour, starting 30min before reperfusion; the same dose was repeated once a day for 7 days; Group III, as for Group II but using 10mg/kg salvianolate; Group IV, in addition to the same intravenous dose as Group III, 20mg salvianolate in 2mL saline given through an over-the-wire balloon catheter just after reperfusion.

Drug under investigation

Salvianolate is derived from the dried root of cultivated Radix salviae miltiorrhizae and manufactured by Greenvalley Pharmacia (Shanghai, China), using the technique described previously [6]. The manufacture and clinical application of salvianolate was granted by the State Food and Drug Administration of China in 2005. The major active component, magnesium lithospermate B (content>85%), is detected for quality control during production. The other components of salvianolate consist of different phenol salts, including magnesium lithospermate, dipotassium lithospermate, sodium rosmarinate, potassium Danshensu, dipotassium isolithospermate B and magnesium salvianolate G.

Salvianolate is recommended for use at a dose of 200mg/day (3.3mg/kg/day for an adult weighing 60kg) in the treatment of chronic stable ischaemic heart disease [7]. The equivalent dose for a minipig is approximately 5mg/kg according to the exchange coefficient determined by surface area. In the present study, we selected an equivalent standard dosage (5mg/kg/day) and a higher dosage (10mg/kg/day) for investigation.

Myocardial contrast echocardiography examination

In order to detect MBF, real-time MCE was performed with a Sequoia 512 machine (Acuson, Mountain View, CA, USA) at different time points, including baseline, 2h after LAD occlusion, and 2h, 24h, 7 days and 14 days after reperfusion, using the methodology described previously [8]. Briefly, after gain settings were optimized, real-time perfusion images were acquired from the short-axis papillary muscle view (mechanical index of 0.17), while SonoVue (Bracco Diagnostics, Inc., Geneva, Switzerland) was infused at a rate of 1mL/minute via a femoral or carotid venous sheath. After high-energy flash frames (mechanical index of 1.9) lasting for 10 cardiac cycles were manually triggered to destroy the microbubbles within the myocardium, continuous refilling sequences of 15 cardiac cycles were obtained and recorded on magnetic optical disks.

The risk area was identified as the region of opacification defect during LAD occlusion. The perfusion defect area was manually traced for the final three end-diastolic images of the 15-cycle refilling sequence. The risk area was expressed as percentage of left ventricle and the reperfusion defect area was expressed as percentage of risk area.

For quantitative analysis, the sample volume was placed in the risk area (region of interest) avoiding both the epicardium and the endocardium. Using Cardio U/S Quantification software (Version 1.4; Siemens Healthcare, Erlangen, Germany), quantitative perfusion data were obtained by fitting intensity data to an exponential function: y=A(1−e−βt)+C, where y is the signal intensity at any given time; A is the plateau signal intensity that reflects the microvascular cross-sectional area; β is the rate of signal intensity rise that reflects myocardial microbubble velocity; t is the time after the high-energy flash frames; and C is the intercept at the origin reflecting the background intensity level. The product (A×β) correlates with MBF. Because the value of A can be affected by various factors, including gain and attenuation, it was normalized to the A value derived from the adjacent LV cavity [9].

Measurement of myocardial blood flow

MBF was also quantified using 15μm coloured microspheres (Dye-Trak, Triton Technology, Inc., South Easton, MA, USA) as described previously [10]. Via the femoral or carotid artery sheath, a pigtail catheter was introduced into LV cavity. After being thoroughly vibrated for optimal mixing, an adequate amount of microspheres [11] was injected into the LV cavity for 10s. A 10mL bolus of warm saline was immediately injected to wash out the catheter. A reference arterial blood sample was continuously aspirated from 10s before microsphere injection until 60s after the injection, at a rate of 5.8mL/minute. The microsphere injection was repeated at different time points with different colours. After the animal was euthanized after 14 days reperfusion, the anterior wall on the S3 heart tissue slice was used for quantification analysis. Microspheres were recovered by a sedimentation process. The microsphere dye was then extracted and MBF (mL/minute/g) was calculated.

Evaluation of the effects of myocardial contrast echocardiography and myocardial blood flow examination on haemodynamics

To ensure that MCE and microsphere examination had no side effects on microcirculation, a pilot study was done on four normal minipigs without any intervention. MCE and microsphere injection was done at baseline, and 2 and 4h after anaesthesia on days 1, 2, 8 and 15; time course changes in microcirculation and cardiac function were quantified.

Myocardial function measurement by transthoracic echocardiography

While the animal was sedated for MCE examination at each time point, as described above, transthoracic echocardiography was performed, using a Sequoia 512 machine with a 1.75–3.5MHz transducer. LV dimensions and wall thickness were measured from the parasternal short-axis view at papillary muscle level. Wall thickening and fractional area change were calculated and expressed as percentages [12]. LV volume was measured from the apical four-chamber view and LV ejection fraction was determined by Simpson’s method.

Histological assessment

For infarct size quantification, only three slices (S1, S3 and S5) were incubated in 1% triphenyltetrazolium chloride at 37°C for 15minutes. The infarct size (stained in the pale area) and risk area (delineated with Evans Blue dye) were measured by planimetry (ImageJ 1.36, National Institutes of Health, Bethesda, MD, USA) at both the apical and basal sides of the three slices. The infarct areas or risk areas for slices S1, S3 and S5 were the average of the apical and basal values of each slice; the values for slice S2 were the average of the basal side of S3 and the apical side of S1; and the values for slice S4 were the average of the basal side of S5 and the apical side of S3. Summation of the infarct weights of all slices was obtained and the infarct size was expressed as a percentage of the total weight of the left ventricle and as the total weight of the risk area.

The heart tissue slices S2 and S4 were used for immunohistochemistry, protein expression and oxidative stress measurement. For the histochemistry examination, the tissue was fixed with 4% paraformaldehyde, paraffin embedded and then sliced at a thickness of 5μm. Endothelial cells were stained using rabbit antihuman vWF antibody (Dako Denmark A/S, Glostrup, Denmark). Capillary density was calculated in 20 random high-power fields (400×) and expressed as the number per mm2.

Terminal deoxynucleotide transferase-mediated dUTP nick end labelling and endothelial cell staining

TUNEL staining was performed using a commercially available kit (Nanjing KeyGen Biotech. Co. Ltd., Nanjing, China). The cell nuclei were labelled with 4’6-diamidino-2-phenylindole (DAPI), while endothelial cells were labelled with antibody against vWF. For each animal, at least 10 randomly selected high-magnification fields (400×) from five different sections were analysed. The apoptotic index was calculated as the ratio of TUNEL-positive cells to total cells, while the endothelial cell apoptotic index was calculated as the ratio of TUNEL-positive endothelial cells to total endothelial cells.

Assessment of tissue concentrations of superoxide dismutase, glutathione, malondialdehyde and nitric oxide

The tissue levels of superoxide dismutase enzyme activity and tissue concentrations of glutathione, malondialdehyde and nitric oxide were all determined spectrophotometrically using appropriate kits purchased from the Jiancheng Bioengineering Institute (Nanjing, China), according to the manufacturer’s instructions. The protein concentration of heart tissue homogenates was determined with the bicinchoninic acid (BCA) assay (BioTime Inc., Haimen, China).

Thioredoxin activity assessment

Using the same method as described previously [13, 14], the activity of thioredoxin was determined for heart tissue from the infarct area using an insulin reduction assay, where insulin is reduced by thioredoxin with NADPH in the presence of excess thioredoxin reductase. Briefly, after tissue homogenization, 40μg protein in a volume of 68μL plus 2μL DTT activation buffer (composed of 50mM HEPES [pH 7.6], 1mM EDTA, 1mg/mL BSA and 2mM DTT) were incubated at 37°C for 15min to reduce thioredoxin, then 40μL of the reaction mixture (containing 250mM HEPES [pH 7.6], 10mM EDTA, 2.4mM NADPH and 6.4mg/mL insulin) were added. The reaction began with the addition of 10μL of rat thioredoxin reductase (90.899 A412U/mL; Sigma-Aldrich Inc., St. Louis, MO, USA) followed by incubation for 20min at 37°C. The reaction was stopped by the addition of 0.5mL of 6M guanidine-HCl and 1mM DTNB (3-carboxy-4-nitrophenyl disulfide). For each sample, the absorbance at 412nm was adjusted by subtracting the absorbance reading without thioredoxin reductase. One unit of activity was calculated using the following formula: A412 ×0.62/(13.6×2).

Western blotting

Tissue homogenates was separated by 12% SDS-PAGE and then transferred to a polyvinylidene-difluoride membrane (Millipore, Billerica, MA, USA). After blocking with 5% skimmed milk, membranes were incubated with a first antibody targeting Bcl-2 and Bax (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), followed by the corresponding HRP-conjugated second antibody. An enhanced chemiluminescence reagent western blotting detection kit (Pierce Biotechnology, Inc., Rockford, IL, USA) was finally used to measure immunoreaction with a light-sensitive film (Kodak, Rochester, NY, USA). The band for each protein was then quantified by densitometry with ImageJ software.

Statistical analysis

All data are presented as means±standard deviations. Intergroup differences were determined by one-way analysis of variance and when statistical significance was found in a group, pairwise comparisons were made using the Student-Newman-Keuls post-hoc test. A value of P <0.05 was considered statistically significant.

Results
Time course changes in haemodynamics: pilot study

In the pilot study, during repeated MCE examination and microsphere injection, no significant changes were detected in heart rate, blood pressure and LV systolic function, or in A, β, (A×β) and MBF (Table 1).

Haemodynamics, cardiac function and infarct size assessment

Five pigs died of ventricular fibrillation after occlusion. During the initial 12h of reperfusion, one pig died in Group I, one died in Group III and two died in Group IV. The data from these animals were excluded. The remaining 33 pigs (9 from Group I; 8 from Group II; 8 from Group III; 8 from Group IV) were evaluated. Heart rate was significantly increased (P <0.001) and blood pressure was significantly lower (P <0.001) after LAD occlusion (Table 2). However, there was no significant difference between the four groups in terms of heart rate and blood pressure.

LAD occlusion caused a significant decrease in regional anterior wall thickening, as well as LV ejection fraction and fractional area change (P <0.001, respectively). At 14 days of reperfusion, these variables were all significantly improved with high-dose treatment (Groups III and IV) compared with Group I (P <0.001, respectively; Table 3).

High-dose salvianolate resulted in a significant decrease in infarct size in Group III (p =0.025) and Group IV (p =0.032) compared with Group I, although there was no significant difference between Groups III and IV (Table 4).

Microvascular reflow by real-time myocardial contrast echocardiography

The risk area measured by MCE at 2h of LAD occlusion was similar in all groups (Table 4). High-dose salvianolate administration (Groups III and IV) resulted in a significant decrease in the defect area after reperfusion (Figure 1; P <0.001, respectively; Table 4).



Figure 1


Figure 1. 

Representative myocardial contrast echocardiography images of the left ventricle at papillary muscle level taken at 2h of reperfusion in different groups. Panels A, B, C and D show images from Groups I, II, III and IV, respectively. The risk area is shown between bold white arrows. The perfusion defect area is shown between slim white arrows. The ratio of perfusion defect area to risk area is significantly smaller in Panels C and D compared with Panel A.

Zoom

Quantitative MCE showed a reduction in A, β and (A×β) in LAD territories after occlusion (P <0.001). The variables recovered at the onset of reperfusion but deteriorated gradually over the following 14 days. Compared with Group I, after reperfusion for 14 days, the variables were significantly improved in Groups III and IV (P <0.001; Table 5). No significant difference was detected between Groups III and IV.

Myocardial blood flow examination

MBF measured by microspheres revealed the severe decrease in blood flow in the LAD regions after occlusion (P <0.001). At the onset of reperfusion, MBF recovered significantly but decreased gradually thereafter. Compared with Group I, MBF was significantly higher in the high-dose groups (Groups III and IV; P <0.001; Table 5). No significant difference was found between Groups III and IV.

The variable (A×β) derived by MCE correlated with MBF assessed by microspheres (r =0.785; P <0.001).

Immunohistochemical staining for capillary density measurement

The density of capillaries that stained positive for vWF in the ischaemic area was significantly higher in Group II (895±311/mm2), Group III (855±92/mm2) and Group IV (588±99/mm2) compared with Group I (350±146/mm2; P <0.001, respectively; Figure 2) with no significant differences between the salvianolate groups.



Figure 2


Figure 2. 

A: representative immunohistochemical staining with antibody against von Willebrand factor for endothelial cells (stained in brown); panels a, b, c and d are from Groups I, II, III and IV, respectively. B: quantification of capillary density is shown for each group. * P <0.05 vs Group I. Data are expressed as means±standard deviations.

Zoom

Effect of salvianolate on myocardial oxidative stress

High-dose salvianolate (Groups III and IV) resulted in a higher level of superoxide dismutase activity and a higher concentration of glutathione in heart tissue, whereas the concentration of malondialdehyde was significantly decreased, compared with Group I (P <0.001, respectively; Table 6). Interestingly, the concentration of nitric oxide in the infarcted area was not significantly different between the four groups.

Effect of salvianolate on myocardial thioredoxin activity

Myocardial thioredoxin activity was significantly increased in Groups III and IV compared with Group I (P <0.001, respectively).

Effect of salvianolate on apoptosis

TUNEL staining showed that the apoptosis ratios for total cells and endothelial cells in the infarct area were significantly decreased in Groups III and IV, compared with controls (P <0.001, respectively, Figure 3).



Figure 3


Figure 3. 

Upper panel: representative terminal deoxynucleotide transferase-mediated dUTP nick end labelling staining from each group; panels A, B, C and D are from Groups I, II, III and IV, respectively. Apoptotic nuclei are stained green, nuclei stained with 4’6-diamidino-2-phenylindole (DAPI) are blue and endothelial cells stained with antibody against von Willebrand factor are red. Lower panel: quantification data, the apoptosis ratios of total cells and endothelial cells. * P <0.05 vs Group I. Data are expressed as means±standard deviations.

Zoom

Consistently, the ratio of Bcl-2/Bax was significantly higher in Groups III and IV than in Group I (P <0.001, respectively; Figure 4).



Figure 4


Figure 4. 

Panel A: representative protein band for Bcl-2 (upper lane) and Bax (lower lane) from different group animals. Panel B: Bcl-2/Bax protein expression ratio. * P <0.05 vs Group I. Data are expressed as means±standard deviations.

Zoom

Discussion

The present study for the first time investigated the protective effect of salvianolate, a highly purified aqueous extract from Danshen, on microcirculation after myocardial ischaemia/reperfusion injury. Impaired microvascular circulation is a pathological process that begins during ischaemia and progresses upon reperfusion, the mechanism of which is still not fully understood [15]. We deliberately delivered the drug during the ischaemic period, allowing a stable blood concentration be established before reperfusion started, although the protective effects might also involve attenuation of ischaemic injury rather than pure reperfusion injury. Nevertheless, in the present study, we clearly showed that salvianolate given intravenously at a dose of 10mg/kg/day for 7 days, with the first dose delivered before reperfusion, significantly limited reperfusion defect size expansion on MCE, and enhanced MBF recovery, as evidenced by an elevated (A×β) value on MCE and MBF measured by coloured microspheres. The protection provided by salvianolate, evidenced by a decrease in infarct size and enhanced LV systolic function, was also present at a high dose of salvianolate. Previous pharmacokinetic studies have verified that blood concentration linearly correlates with the dose of magnesium lithospermate B, the major component of salvianolate, when given intravenously [16]. We also observed a trend of dose-dependent effects with most of the variables we tested, which was not statistically significant. However, our results suggest that a high dose may be necessary to achieve the maximal minimizing effect on ischaemia/reperfusion injury; this has been suggested in previous studies [4, 17]. In addition, we also observed that one extra intracoronary injection of 20mg salvianolate, delivered directly to the ischaemic myocardium, failed to provide further protection, indicating that maximal effects might be reached with the high dose of intravenous salvianolate alone.

Microsphere injection has been shown to quantify microvascular blood flow precisely without affecting systemic haemodynamic variables [10, 18]; this was confirmed in our pilot study, with dose carefully adjusted by weight [11]. Thus, our data suggest that repeated injections of microspheres do not significantly affect microcirculation.

In our study, triphenyltetrazolium chloride staining was performed 14 days after reperfusion. However, when we injected Evans Blue dye to delineate the risk area, we noticed that the dye can perfuse into the risk zone, making this methodology impractical for quantification of infarct size at the late phase of reperfusion. This phenomenon could be due to neovascularization in the risk zone after 14 days of reperfusion [19]. Fortunately, however, the risk zone can still be clearly defined by MCE examination. Thus, the infarct size was expressed as a percentage of the total left ventricle rather than the risk area.

Overproduction of oxygen-derived free radicals plays a key role in ischaemia/reperfusion injury [20], leading to increased apoptosis activation during reperfusion [21, 22]. Our data show that a high dose of salvianolate resulted in a decrease in malondialdehyde concentration and an increase in superoxide dismutase activity, glutathione concentration and thioredoxin activity in the ischaemic myocardium. Thioredoxin has been recently proven to be crucial for maintaining activity of antioxidative enzymes [23] and plays a key role in the antiapoptotic signalling pathway [24]. In our study, this protection was evidenced by an increase in the Bcl-2/Bax protein expression ratio and suppressed apoptotic cells, especially apoptotic endothelial cells. Our observations were consistent with previous reports [25, 26, 27]. Thus, we have confirmed that salvianolate improves microvascular reflow via suppression of oxidative stress and apoptosis.

The recommended dose of 200mg/day has been proven to be safe by several experimental and clinical studies [7]. In our present study, no significant changes in arterial blood pressure and heart rate were observed during salvianolate infusion. Thus, an intravenous dose of 10mg/kg/day (equivalent to a dose of 400mg/day in humans) is safe for pigs. A large-scale clinical study is certainly warranted to further evaluate its beneficial effects and drug-related side effects.

In conclusion, using a closed-chest porcine model of myocardial ischaemia/reperfusion injury, we demonstrated that 7-day treatment with high-dose salvianolate (10mg/kg per day, intravenously) significantly improved microvascular reflow, resulting in smaller infarct size and enhanced LV systolic function. The protective effect of salvianolate was closely associated with its strong antioxidative and antiapoptotic effects.

Disclosure of interest

The authors declare that they have no conflicts of interest concerning this article.


Acknowledgement

This research project was supported by the Science and Technology Commission of Shanghai Municipality (Programme 064119507 to M. W.), and partly supported by China Natural Science Foundation (81070110, to WEI M).

References

Ragosta M., Camarano G., Kaul S., and al. Microvascular integrity indicates myocellular viability in patients with recent myocardial infarction. New insights using myocardial contrast echocardiography Circulation 1994 ;  89 : 2562-2569
Ito H., Maruyama A., Iwakura K., and al. Clinical implications of the ‘no reflow’ phenomenon. A predictor of complications and left ventricular remodeling in reperfused anterior wall myocardial infarction Circulation 1996 ;  93 : 223-228
Jin S., Zhao G., Fan Y. Effect of salvianolic acid B on endothelin release and TXA2/PGI2 system in myocardial ischemia/reperfusion injury in rats Chin J Gerontol 2004 ;  24 : 127-128
Xu J.P., Sun L.S., Wu H.Y., and al. Protective effect of salvianolic acid B on myocardial ischemia/reperfusion in rats Chin Pharm J 2003 ;  38 : 595-597
Li X., Yu C., Sun W., and al. Simultaneous determination of magnesium lithospermate B, rosmarinic acid, and lithospermic acid in beagle dog serum by liquid chromatography/tandem mass spectrometry Rapid Commun Mass Spectrom 2004 ;  18 : 2878-2882 [cross-ref]
Xu YM, J. XL, Li T, et al. Methods for identification, purification, and manufacturing of the active constituents in Salvia miltiorrhiza (Dansheng) and the application of this product in enhancing cardiovascular functions. description.html 2001; United States Patent 6299910.
Yan P., Luo X., Shi H., and al. Effect of depside salt from salvia miltiorrhiza on angina pectoris and platelet function Jie Ru Fang She Xue Za Zhi 2004 ;  12 (Suppl. 2) : 55-59
Wei K., Jayaweera A.R., Firoozan S., and al. Quantification of myocardial blood flow with ultrasound-induced destruction of microbubbles administered as a constant venous infusion Circulation 1998 ;  97 : 473-483
Pacella J.J., Villanueva F.S. Effect of coronary stenosis on adjacent bed flow reserve: assessment of microvascular mechanisms using myocardial contrast echocardiography Circulation 2006 ;  114 : 1940-1947 [cross-ref]
Kowallik P., Schulz R., Guth B.D., and al. Measurement of regional myocardial blood flow with multiple colored microspheres Circulation 1991 ;  83 : 974-982
Hoffmann U., Millea R., Enzweiler C., and al. Acute myocardial infarction: contrast-enhanced multi-detector row CT in a porcine model Radiology 2004 ;  231 : 697-701 [cross-ref]
Hasegawa H., Takano H., Iwanaga K., and al. Cardioprotective effects of granulocyte colony-stimulating factor in swine with chronic myocardial ischemia J Am Coll Cardiol 2006 ;  47 : 842-849 [cross-ref]
Holmgren A., Bjornstedt M. Thioredoxin and thioredoxin reductase Methods Enzymol 1995 ;  252 : 199-208 [cross-ref]
Yegorova S., Liu A., Lou M.F. Human lens thioredoxin: molecular cloning and functional characterization Invest Ophthalmol Vis Sci 2003 ;  44 : 3263-3271 [cross-ref]
Rezkalla S.H., Kloner R.A. No-reflow phenomenon Circulation 2002 ;  105 : 656-662 [cross-ref]
Li X.C., Yu C., Sun W.K., and al. Pharmacokinetics of magnesium lithospermate B after intravenous administration in beagle dogs Acta Pharmacol Sin 2004 ;  25 : 1402-1407
Chen Y.H., Du G.H., Zhang J.T. Salvianolic acid B protects brain against injuries caused by ischemia-reperfusion in rats Acta Pharmacol Sin 2000 ;  21 : 463-466
Theilmeier G., Verhamme P., Dymarkowski S., and al. Hypercholesterolemia in minipigs impairs left ventricular response to stress: association with decreased coronary flow reserve and reduced capillary density Circulation 2002 ;  106 : 1140-1146 [cross-ref]
Vandervelde S., van Amerongen M.J., Tio R.A., and al. Increased inflammatory response and neovascularization in reperfused vs. non-reperfused murine myocardial infarction Cardiovasc Pathol 2006 ;  15 : 83-90 [cross-ref]
Kaul S., Ito H. Microvasculature in acute myocardial ischemia: part II: evolving concepts in pathophysiology, diagnosis, and treatment Circulation 2004 ;  109 : 310-315 [cross-ref]
Zhao Z.Q., Velez D.A., Wang N.P., and al. Progressively developed myocardial apoptotic cell death during late phase of reperfusion Apoptosis 2001 ;  6 : 279-290 [cross-ref]
Zhao Z.Q., Vinten-Johansen J. Myocardial apoptosis and ischemic preconditioning Cardiovasc Res 2002 ;  55 : 438-455 [cross-ref]
World C.J., Yamawaki H., Berk B.C. Thioredoxin in the cardiovascular system J Mol Med 2006 ;  84 : 997-1003 [cross-ref]
Liu Y., Min W. Thioredoxin promotes ASK1 ubiquitination and degradation to inhibit ASK1-mediated apoptosis in a redox activity-independent manner Circ Res 2002 ;  90 : 1259-1266 [cross-ref]
Kim S.H., Choi M., Lee Y., and al. Natural therapeutic magnesium lithospermate B potently protects the endothelium from hyperglycaemia-induced dysfunction Cardiovasc Res 2010 ;  87 : 713-722 [cross-ref]
Liu X., Chen R., Shang Y., and al. Superoxide radicals scavenging and xanthine oxidase inhibitory activity of magnesium lithospermate B from Salvia miltiorrhiza J Enzyme Inhib Med Chem 2009 ;  24 : 663-668 [cross-ref]
Wang M.W., Zhang D.F., Tang J.J., and al. Effects of salvianolate on cardiomyocytic apoptosis and heart function in a swine model of acute myocardial infarction Zhong Xi Yi Jie He Xue Bao 2009 ;  7 : 140-144 [cross-ref]



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