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Acute treatment with Danshen-Gegen decoction protects the myocardium against ischemia/reperfusion injury via the redox-sensitive PKC[epsilon]/m[K.sub.ATP] pathway in rats.

doi: 10.1016/j.phymed.2011.03.006

ABSTRACT

Danshen-Gegen (DG) decoction, an herbal formulation comprising Radix Salvia Miltiorrhiza and Radix Puerariae Lobatae, is prescribed for the treatment of coronary heart disease in Chinese medicine. Experimental and clinical studies have demonstrated that DG decoction can reduce the extent of atherosclerosis. In the present study, using an ex vivo rat model of myocardial ischemia/reperfusion (I/R) injury, we investigated the myocardial preconditioning effect of an aqueous DG extract prepared from an optimized weight-to-weight ratio of Danshen and Gegen. Short-term treatment with DG extract at a daily dose of 1g/kg and 2g/kg for 3 days protected against myocardial I/R injury in rats. The cardioprotection afforded by DG pretreatment was paralleled by enhancements in mitochondrial antioxidant status and membrane structural integrity, as well as a decrease in the sensitivity of mitochondria to [Ca.sup.2+]-stimulated permeability transition in vitro, particularly under I/R conditions. Short-term treatment with the DG extract also enhanced the translocation of PKC[epsilon] from the cytosol to mitochondria in rat myocardium, and this translocation was inhibited by [alpha]-tocopherol co-treatment with DG extract in rats. Short-term DG treatment may precondition the myocardium via a redox-sensitive PKC[epsilon]/m[K.sub.ATP] pathway, with resultant inhibition of the mitochondrial permeability transition through the opening of mitochondrial m[K.sub.ATP] channels. Our results suggest that clinical studies examining the effectiveness of DG extract given prophylactically in affording protection against myocardial I/R injury would be warranted.

[C] 2011 Elsevier GmbH. All rights reserved.

ARTICLE INFO

Keywords: Danshen-Gegen Root of Salvia miltiorrhiza/Pueraria Lobata Myocardium Ischemia Reperfusion Redox Protein kinase C

Introduction

Despite significant improvement in medical and surgical management, coronary heart disease remains one of the major causes of morbidity and mortality in industrialized countries, with a large portion of patients suffering from myocardial infarction caused by ischemia and/or reperfusion injury (Ferdinandy et al. 2007). The exploration for therapeutic agents aimed at reducing the extent of myocardial infarction has become an area of intensive research. Ischemic preconditioning, which involves the procedure of exposure of myocardial tissue to brief episodes of ischemia prior to the induction of ischemia, has been demonstrated to protect the myocardium against ischemia/reperfusion (I/R) injury (Tyagi and Tayal 2002). The cardioprotection afforded by ischemic preconditioning possibly involves the activation of an adenosine-mediated reperfusion-injury salvage kinase (RISK) pathway (Hausenloy and Yellon 2004) and a tumor necrosis factor-[alpha]-mediated survivor activating factor enhancement (SAFE) pathway (Lacerda et al. 2009). Both RISK and SAFE signaling pathways target mitochondria through the activation of protein kinase C[epsilon]1/2 (PKC[epsilon]l/2), which in turn opens a mitochondrial ATP-dependent potassium channel (m[K.sub.ATP), caused inhibition of a mitochondrial permeability transition (MPT) and resulting in cardioprotection (Costa and Garlid 2009; Ovize et al. 2010). Although ischemic preconditioning of the myocardium is clinically impractical except in cases of patients undergoing cardiac surgery, the prophylactic use of pharmacological agents mimicking the effect of ischemic preconditioning may provide an effective means of reducing the extent of myocardial damage caused by I/R challenge, particularly in patients suffering from coronary heart disease.

Danshen and Gegen, which are roots of Salvia miltiorrhiza and Puerariae lobata, respectively, are traditional Chinese medicinal herbs that have long been used in China, Japan, Korea and Taiwan for the treatment of cardiac and cerebral ischemic conditions (Ji et al. 2003; Kim et al. 2009; Lam et al. 2010). The herbal formulation made of Danshen and Gegen (referred to as "DC decoction") has long been prescribed for the treatment of coronary heart disease in the practice of Chinese medicine (Adams et al. 2006). Recently, it has been shown that treatments with DG decoction alleviate key early atherogenic events in human monocyte-derived macrophages and thereby modulate the key early events in atherosclerosis (Sieveking et al. 2005). DG decoction has also been found to be effective in improving vascular structure and function in patients suffering from cardiovascular diseases (Tarn et al. 2009). Given the traditional usage for the treatment of coronary heart disease, DG decoction may also protect the myocardium against I/R injury. However, the cardioprotective action of DG decoction has not been fully investigated. In the present study, an aqueous extract was prepared from a mixture of Danshen and Gegen (DG) in an optimized weight-to-weight ratio in terms of biological activity, and its effectiveness in ameliorating I/R injury was investigated using an ex vivo rat heart model system. The biochemical mechanism underlying the cardioprotection was investigated by examining changes in mitochondrial antioxidant status and membrane structural integrity, as well as the sensitivity of mitochondria to [Ca.sup.2+]-induced permeability transition in control and ischemic/reperfused rat hearts. The involvement of a PKC[epsilon] and m[K.sub.ATP]-mediated signaling pathway in the cardioprotection afforded by DG was also investigated using specific inhibitors of these biochemical entities.

Materials and methods

Preparation of herbal extracts

Radix Salviae miltiorrhizae (Danshen) and Radix Puerariae Lobatae (Gegen) were purchased from mainland China. They were authenticated by an in-house herbalist in the Institute of Chinese Medicine (ICM) at the Chinese University of Hong Kong, Hong Kong. Voucher specimens of Danshen (#2008-3088b) and Gegen (#2008-3167b) were deposited in the ICM. Danshen-Gegen (DG) extract with the optimal ratio of Danshen to Gegen (7:3, w/w), as assessed by cardioprotection against I/R injury (Chiu et al. 2010), was prepared in large-scale for experimental and clinical investigations. Herbs were soaked in water (1:10, w/v) for 75 min, followed by extraction in boiling water for 1 h. The extraction procedure was repeated twice with boiling water (1:8) for 1 h and 30 min, respectively. The pooled aqueous extracts were concentrated under reduced pressure at 60 CC, and the concentrate was spray-dried to obtain the powdered form of DG extract at a yield of 10.1%.

Chemical analysis of the DG extract

Identification and quantification of the major components in the DG extract were performed according to our previous study with minor modifications regarding the instrument and chromatographic conditions (Chang et al. 2008). Briefly, a Waters HPLC system (Waters, Milford, MA, USA) equipped with a 2695 solvent delivery module and a 996 photodiode UV detector was used. The chromatographic separation of the analytes was achieved by an Agilent Eclipse XDB-C18 column (5250mm x 4.6mm i.d.; 5[micro]m particle size) connected to an Agilent CI8 guard column. The mobile phase consisting of 0.5% acetic acid in acetonitrile (solvent A) and 0.5% acetic acid in water (solvent B) was run with gradient elution at a flow rate of 1 ml/min. The linear gradient elution was carried out as follows: solvent A was kept at 5% for the first 5min, and then increased to 10%, 17%, 35% and 90% in the next 13min, 12min, 10min and 30min, respectively, then returned to 5% in 5min and equilibrated for 15min before the next injection. HPLC analysis indicated that the DG extract contained the following marker compounds ([micro]g/100mg; mean[+ or -]SD, n = 3): danshensu (1868.2 [+ or -] 33.7), salvianolic acid B (1345.7 [+ or -] 18.5), protocatechuic aldehyde (78.3[+ or -]3.9), puerarin (1760.1 [+ or -]23.4), daidzein 8-C-apiosyl-glucoside (404.1 [+ or -]8.1), daidzin (159.4[+ or -]3.3) and daidzein( 162.9 [+ or -]1.4).

Animal care

Female Sprague-Dawley rats (8-10 weeks old, weighing 200-250g) were maintained under a 12h dark/light cycling at 22 [degrees]C and allowed food and water ad libitum in the Animal & Pant Care Facility at the Hong Kong University of Science and Technology (HKUST, Hong Kong SAR, China). All experimental protocols were approved by the University Committee on Research Practice at HKUST. Male or female rats could be used for the model of myocardial I/R injury, but female rats were chosen because of their availability.

Drug treatment

Animals were randomly divided into groups of 4-6 animals in each. In preliminary screening experiments, rats were treated intra-gastrically by gavage with DG extracts of varying composition at a dose of 1g/kg for 3 days. In short-term DC treatment, rats were treated intragastrically with DG extract (7:3, dissolved in water) at a daily dose of 1 and 2g/kg for 3 days. Control rats received water only by gavage. Twenty-four hours after the last dosing with the DG extract, hearts were isolated from phenobarbital-anesthetized animals and subjected to I/R challenge, cls described below. In order to determine the role of PKC[epsilon] and m[K.sub.ATP] in the effects of short-term DG treatment on I/R injury, isolated hearts were perfused with 2 [micro]M PKC[epsilon] translocation inhibitor (Peptide EAVSLKPT; Calbiochem, Darmstadt, Germany) or 2 [micro]M 5-hydroxydecanoate, 5-HD (Sigma Chemical Co., St. Louis, MO) in Krebs-Henseleit buffer for 15min prior to I/R challenge.

Isolated-perfused rat hearts

Hearts were quickly excised and immediately immersed in ice-cold and heparinized (50 unit/ml) saline.The aorta was cannulated and transferred to a warm, moist chamber (double-wall jacket with temperature being maintained at 37 [degrees]C by circulating warm water) of the Langendorff perfusion apparatus (Radnoti, Monrovia, CA, USA). The hearts was retrogradely perfused at constant pressure of 70 mm Hg with Kreb's Henseleit solution of pH 7.4 which was gassed with 95% [O.sub.2] and 5% C[O.sub.2], as previously described (Chiu and Ko 2004).

Myocardial I/R injury

After an initial 30-min of perfusion for equilibration, the isolated heart was subjected to a 15-(screening experiment) or 40-min (long-term study) period of'no-flow global ischemia by stoppage of perfusate, followed by 10- or 20-min reperfusion, respectively. For convenience, brief durations of ischemia and reperfusion were adopted for screening experiments. Coronary effluent was collected in 1 -min fractions at various time intervals during the course of equilibration and reperfusion. The fractions were immediately placed on ice until assayed for lactate dehydrogenase (LDH) activity. The extent of LDH leakage during the reperfusion period, an indirect index of myocardial injury, was estimated by computing the area under the curve (AUC) of the graph plotting the percent LDH activity (with respect to the mean pre-ischemic value measured during the equilibration period) against the reperfusion time (1-20 min), as previously described (Chiu and Ko 2004), and the value was expressed in arbitrary units. Non I/R hearts were perfused for 90min. After the non I/R or I/R procedure, ventricular tissue samples were obtained and subjected to biochemical analysis.

[FIGURE 1 OMITTED]

Preparation of cytosolic and mitochondrial fractions

Cytosolic and mitochondrial fractions were prepared by differential centrifugation as described by Starnes et al. (2007). Myocardial ventricular tissue samples were rinsed with ice-cold isotonic buffer (210 mM mannitol, 70 mM sucrose, 5mM HEPES, 1 mM EGTA, pH 7.4,0.2 mg/ml soybean trypsin inhibitor, 0.2 mg/ml bacitracine, 0.16 mg/ml benzamidine). Tissue homogenates were prepared by homogenizing 0.6 g of minced tissue in 6 ml ice-cold isotonic buffer using a Teflon-in glass homogenizer (Glas-Col, Terre Haute, IN) at a speed of 1600 rpm for 20 strokes on ice. The homogenates were centrifuged at 600 xg for 20min at 4CC. The supernatants were centrifuged at 9200 x g for 30 min, and the mitochondrial pellets were collected. The supernatants were saved for the preparation of cytosolic fractions. The mitochondrial pellets were then washed with the same volume of ice-cold sucrose buffer containing 210 mM mannitol, 70 mM sucrose, 5mM HEPES-KOH, pH 7.4, and the mixtures were centrifuged at 9200 x g for 30 min. The washing procedure was repeated. The mitochondrial pellets were resuspended in 1.0 ml of ice-cold sucrose buffer and constituted the mitochondrial fractions. Cytosolic fraction was prepared by centrifuging the above supernatant at 100,000 x g for 60 min at 4 C The protein concentrations of the mitochondrial and cytosolic fractions were determined using a protein assay kit (Bio-Rad, Hercules, USA).

Mitochondrial ROS generation

The extent of mitochondrial reactive oxygen species (ROS) generation in vitro was measured as previously described (Degli Esposti 2002). An aliquot (50 [micro]g) of mitochondrial fraction (50[micro]g protein/ml) and 60 ([micro]1 of 2',7'-dichlorofluorescin diacetate (DCFDA) (Fluka, Switzerland) solution (5[micro]M in the incubation buffer) were added to wells of a black micro-titer plate. The mixture was incubated at 37 C for 10 min in the dark in a Victor [3.sup.TM] Multi-Label Counter (Perkin-Elmer, Wellesly, MA). After the incubation, 50[micro]1 of incubation buffer (0.1 mM EGTA, 5mM K[H.sub.2]P[O.sub.4], 3mM Mg[Cl.sub.2]. 145 mM KC1, 30 mM Hepes, pH 7.4) and 50 [micro]1 of substrate solution (20 mM pyruvate and 10 mM malate) were added. Fluorescence intensity (excitation: 485 nm and emission: 535 nm) of the reaction mixture was taken every 5min for 30min. Mitochondrial ROS generation was determined using the fluorescence intensity of the sample after subtracting the value of a blank sample containing incubation buffer, substrate solution and DCFDA. The extent of ROS generation over the 30-min period of incubation was estimated by computing the AUC of the graph plotting fluorescent intensity against time (0-30 min) and expressed in arbitrary unit(AU).

[FIGURE 2 OMITTED]

Mitochondrial [Ca.sup.2+] content

Mitochondrial [Ca.sup.2+] content was measured using a [Ca.sup.2+]-sensitive fluorescence probe Fluo-5N AM ester (Molecular Probe, OR), as previously described (Menze et al. 2005). The [Ca.sup.2+] dissociation constant ([K.sub.d]) was determined using a [Ca.sup.2+] calibration kit in a concentration range of 1-1000 [micro]M, with the Kd value being estimated to be ~98 [micro]M, which is in good agreement with the data provided by the manufacturer. An aliquot (25[micro]l) of mitochondrial fraction (0.5 mg/ml final concentration) was mixed with 25 [micro]l of incubation buffer (100 mM KG, 30 mM MOPS, pH 7.2) in 96-well black micro-titer plate. The mixture was incubated at 25 "C for 15 min and then added with 25[micro]l digitonin (50[micro]g/ml) and 25[micro]l Fluo-5N AM ester (1[micro]M in 0.005% Pluronic F-127). The reaction mixture was incubated at 25 CC for 30 min, and the fluorescence reading was measured at excitation wavelength of 488 nm and emission wavelengths at 532 nm. The mitochondrial [Ca.sup.2+] content was estimated from a standard calibration curve and expressed in [micro]mol/mg protein.

[FIGURE 3 OMITTED]

Mitochondrial cytochrome c release

Mitochondrial cytochrome c release was indirectly assessed by the measurement of cytosolic cytochrome c levels using Western blot analysis, as previously described (Kavazis et al. 2008). Total cytosolic fractions with equal amounts of protein (40 [micro]g protein) were subjected to 15% SDS-PAGE, followed by immunoblotting using specific antibodies of cytochrome c (clone 7H8.2C12, BD PharMingen, San Diego, CA, USA). The extent of mitochondrial contamination in the cytosolic fractions was determined using specific antibodies against complex IV. The protein-blot analysis was carried out using ECL Western Blotting System (Cell Signaling Technology, Beverly, MA) according to manufacturer's recommendation and the protein bands were quantified by densitometry. The cytochrome c release was estimated from the amount (arbitrary units) of cytochrome c normalized with reference to actin (1:5000, Sigma Chemical Co., St. Louis, MO, USA) levels (arbitrary units) in the sample.

Mitochondrial permeability transition

Mitochondrial swelling was taken as an indirect measure of MPT in vitro, as previously described (Fauvel et al. 2002). An aliquot (1.6 ml) of mitochondrial sample (0.5 mg protein/ml) was prepared by mixing the mitochondrial fraction with incubation buffer containing 125 mM sucrose, 65 mM KC1,10 mM Hepes (pH 7.2), 5 mM succinate (freshly prepared)and 5[micro]M rotenone (freshly prepared). Aliquots (200[micro]l) of mitochondrial homogenate were mixed with 10[micro]1 of cyclosporine A (CsA) (5[micro]M) in 0.5% (w/w) ethanol (final concentration in incubation buffer). The mixtures were incubated at 30[degrees]C for 5min. An aliquot (10[micro]l) of calcium chloride ([Ca.sup.2+]) solution (1[micro]M final concentration) was then added, and the mixtures were incubated at 30[degrees]C for 5min. Aliquots (180[micro]l) of the mixtures were added into 96-well micro-titer plate, and the initial absorbance at 520 nm was monitored for 5min at 30 C The swelling reaction was then initiated by adding 20[micro]l of potassium phosphate (0.5 mM, pH 7.2), and the absorbance at 520 nm of the reaction mixtures was read every 2 min for 30 min at 30 [degrees]C, using Victor [3.sup.TM] Multi-Label Counter. The extent of mitochondrial swelling was estimated by computing the AUC of the declining graph plotting percent initial absorbance (100% as baseline) against time (min) to obtain AU[C.sub.1]. The extent of M PT([DELTA]AU[C.sub.1]) was estimated by subtracting the AU[C.sub.1] with CsA from the AU[C.sub.1] without CsA. The [Ca.sup.2+]-induced MPT was expressed as the ratio of AAUCi induced by both [Ca.sup.2+] and [P[O.sub.4].sup.3-] to that induced by [P[O.sub.4].sup.3-]only.

Western blot analysis for PKC[epsilon]

Mitochondrial and cytosolic fractions were prepared from myocardial ventricular tissue samples by differential centrifugation as described above, except using a different homogenizing buffer which contained 300 mM sucrose, 4 mM HEPES, 2 mM EGTA, 1 mM PMSF and 20[micro]M leupeptin, pH 7.4. After centrifugation, the resultant pellets were resuspended in ice-cold mitochondrial buffer (20 mM Tris-HCl, 1 mM EGTA, 1 mM PMSF, 20[micro]M leupeptin, 0.5% Triton X-l00, pH 7.4) and sonicated on ice for 3 min, and this constituted the mitochondrial fraction. The cytosolic and mitochondrial fractions were mixed with 6 volumes of SDS sample loading buffer (1 M Tris-HCl, pH 6.8,10% SDS, 30% glycerol, 6 mM DTT and 0.2 mM bromophenol blue). Mitochondrial and cytosolic proteins (50[micro]g) were resolved using 10% SDS-PAGE gels and transferred onto nitrocellulose membranes. An equal loading of proteins was confirmed by staining with Ponceau-S solution [0.5% (w/v) Ponceau S in 1% (v/v) acetic acid] (Bannur et al. 1999). After blocking with 5% skim milk, the membranes were incubated with anti-PKC-[epsilon] antibody (Upstate, Temecula, CA) at 4C overnight. The membrane was subsequently washed and incubated with peroxidase-conjugated secondary antibody (1:2000, Cell Signaling Technology, Beverly, MA) for 2 h at room temperature. Immuno-stained bands were quantitatively analyzed by densitometry using an ECL Western BlotSystem (Cell Signaling Technology, Beverly, MA). The intensity of immune-stained PKC-[epsilon] band was normalized to that of the sum of Ponceau S-stained bands in the sample. The ratio of mitochondrial(m)PKC-[epsilon] level to cytosolic(c)PKC-[epsilon] level indicated the extent of PKC-[epsilon] translocation.

Role ofROS in PKC[epsilon] translocation

To investigate the role of ROS in DG-induced PKC-[epsilon] translocation, rats were treated intragastrically with a-tocopherol (dissolved in oil) at 400 [micro]g/kg once daily 2 h prior to DG treatment (a daily dose of 2 g/kg for 3 days). Mitochondrial and cytosolic fractions were prepared from heart tissues and measured for PKC-[epsilon] level as described above.

Biochemical analyses

LDH activity of coronary effluent was measured as described (Ko and Yiu 2001). Aliquots (210[micro]l) of mitochondrial fractions were taken for measuring reduced glutathione (GSH) level by an enzymatic method described by Griffith (1980). Aliquots (250[micro]l) of mitochondrial fractions were taken for measuring malon-dialdehyde (MDA) level using an HPLC method (Ko and Yiu 2001).

Statistical analysis

Data were analyzed by one-way Analysis of Variance (ANOVA). Post hoc multiple comparisons were done with least significant difference test. P-values <0.05 were regarded as statistically significant.

Results

DG treatment protected against myocardial l/R injury in rats

Short-term treatment with the DG extract (a daily dose of 1 or 2 g/kg for 3 days) did not affect the LDH leakage in isolated-perfused (i.e. non-I/R) rat hearts (Fig. 1). However, I/R challenge caused a 9.6-fold increase in the extent of LDH leakage, when compared with the non-I/R hearts. DG pretreatment dose-dependently reduced the extent of I/R-induced LDH leakage, with the degree of protection being 27% and 51% for 1 and 2 g/kg of DG treatment, respectively, when compared with animals subjected to I/R in the absence of DG pretreatment (i.e. DG untreated I/R hearts).

I/R challenge caused a significant decrease in mitochondrial GSH levels (17%) and an increase in ROS production (40%) in rat hearts, when compared with the non-I/R hearts (Fig. 2). While DG treatment dose-dependently increased mitochondrial GSH levels (27-38%) and ROS production (11-22%) in non-I/R hearts, the cardioprotection afforded by DG pretreatment was associated with an increase in mitochondrial GSH levels (19-29%) and a decrease in ROS production (11-24%), when compared with DG untreated I/R hearts.

[FIGURE 4 OMITTED]

I/R challenge significant increased in MDA production (31%), [Ca.sup.2+] content (25%) and cytochrome c release (36%) in rat hearts, when compared with the non-I/R hearts (Fig. 3). In non-I/R hearts DG treatment dose-dependently decreased mitochondrial MDA production (10-14%), [Ca.sup.2+] content (17-30%) and cytochrome c release (6-30%); the cardioprotection against I/R injury afforded by DG pretreatment was accompanied by decreases in the MDA production (12-25%), [Ca.sup.2+] content (13-21%) and cytochrome c release (10-23%), when compared with the DG untreated I/R hearts.

DG treatment decreased the sensitivity to [Ca.sup.2+]-induced MPT in control and ischemic/reperfused rat hearts

[Ca.sup.2+]-induced MPT was indirectly assessed by the in vitro measurement of mitochondrial swelling. I/R challenge increased the sensitivity of mitochondria to [Ca.sup.2+]-induced FT (20%) in rat hearts, when compared with the non-I/R hearts (Fig. 4). While DG treatment decreased the sensitivity of mitochondria to [Ca.sup.2+]-induced PT in non-I/R hearts (5-17%), the DG afforded cardioprotection against I/R injury was associated with protection against the alteration in mitochondrial sensitivity to [Ca.sup.2+]-induced PT (10-14%), when compared with DG untreated I/R hearts.

PKC[epsilon] and m[K.sub.ATP] inhibition suppressed the DG-induced cardioprotection against I/R injury

The involvement of a PKC[epsilon]-mediated signaling pathway in the cardioprotective action of DG was investigated using agents known to act as inhibitors of PKC[epsilon] translocation and of m[K.sub.ATP]. While the susceptibility of rat hearts to I/R injury, as assessed by LDH leakage, was increased by inhibition of PKC[epsilon]translocation or m[K.sub.ATP]. DG pretreatment (a daily dose of 2g/kg for 3 days) did not protect against myocardial I/R injury in the presence of PKC[epsilon] or m[K.sub.ATP] inhibition (Fig. 5a). The abolition of DG-induced cardioprotection was paralleled by the lack of improvement in the I/R-induced impairment in mitochondrial GSH level and ROS production (Fig. 5b), MDA production, [Ca.sup.2+] content and cytochrome c release (Fig. 5c), or the sensitivity to [Ca.sup.2+]-induced PT (Fig. 5d).

[FIGURE OMITTED]

[FIGURE OMITTED]

DG treatment activated the PKC[epsilon] translocation in rat hearts

The ratio of mitochondrial PKC[epsilon] to cytosolic PKC[epsilon] levels was taken as an indirect measure of PKC[epsilon] translocation from the cytosol to mitochondria. DG pretreatment (a daily dose 2g/kg for 3 days) enhanced PKC[epsilon] translocation in rat hearts, as evidenced by a significant increase (49%) in the ratio of mPKC[epsilon] to cPKC[epsilon], when compared with DG untreated hearts (Fig. 6).

While co-treatment with [alpha]-tocopherol (400[micro]g/kg) and the DG extract did not produce any detectable change in PKC[epsilon] translocation in the myocardium of control rats, the combined treatment completely suppressed PKC[epsilon] translocation in DG-treated rats (Fig. 7).

Discussion

I/R challenge caused tissue damage in isolated-perfused rat hearts as assessed by LDH leakage. It has been shown that the I/R-induced increase in the extent of LDH leakage positively correlates with histopathological changes in ventricular tissue in isolated-perfused rat hearts (Pagliaro etal. 2003). The measurement of LDH leakage can therefore be used as a reliable assessment of myocardial injury caused by I/R challenge. Under the present experimental conditions, the myocardial injury caused by ischemic and/or reper-fusion insult was assessed by LDH leakage during the 20-min period of post-ischemic reperfusion. It is now well established that ROS-mediated processes are involved in the pathogenesis of myocardial I/R injury (Baudry et al. 2008), and mitochondria are an important source of ROS production during post-ischemic reperfusion (Giordano 2005). In the present study, myocardial injury induced by I/R was associated with impairment in mitochondrial antioxidant status and MDA production, indicating an increase in oxidative stress.

[FIGURE 6 OMITTED]

Mitochondria, which are important in maintaining cellular structural and functional integrity, serve as coordinators of ceil survival and death (Kroemer and Reed 2000). In this regard, the opening of MPT pores is critically involved in cellular dysfunction and cell death (Halestrap et al. 2002). In the present study, myocardial injury induced by I/R was associated with an increased sensitivity of mitochondria to [Ca.sup.2+]-induced FT. The opening of MPT pores, either under in vivo or in vitro conditions, is triggered by stimuli such as oxidants, high mitochondrial [Ca.sup.2+] content and/or depletion of adenine nucleotides (Brookes et al. 2004). The disruption of mitochondrial membrane structural integrity associated with I/R, as indicated by the increased extent of cytochrome c release, is paralleled by the increase in mitochondrial sensitivity to [Ca.sup.2+]-induced PT. DG pretreatment protected against myocardial I/R injury. The cardioprotection was paralleled by a partial protection against I/R-induced impairment in mitochondrial antioxidant and membrane structural status, particularly the GSH level and sensitivity to [Ca.sup.2+]-induced PT.

[FIGURE 7 OMITTED]

Glutathione plays an important role in numerous cellular functions, including the regulation of [Ca.sup.2+] homeostasis and the detoxification of ROS (Hidalgo et al. 2002). The decrease in mitochondrial sensitivity to [Ca.sup.2+] -stimulated FT by DG, as observed in the present study, may be related to the enhancement of mitochondrial glutathione redox status, which is characterized by the increase in the GSH level. In this regard, one of the two voltage-sensitive sites of the MPT pore was found to be gated by a critical dithiol that is sensitive to glutathione redox status, and the oxidation of GSH could open this pore (Armstrong and Jones 2002; Haouzi et al. 2002). Furthermore, the modification of a specific thiol group on the adenine nucleotide translocase either by oxidative stress or by thiol reagents also has been shown to decrease adenine nucleotide binding and activate the MPT pore (McStayet al. 2002). Increased oxidative stress enhances cyclophilin binding and hence increases the sensitivity of MPT (Lu and Armstrong 2007). The observation that a smaller degree of inhibition of [Ca.sup.2+] -induced MPT than that of myocardial I/R injury afforded by DG treatment supports the notion that the decrease in the sensitivity of mitochondria to permeability transition is an effect secondary to the enhancement of mitochondrial glutathione redox status. The direct effect on increasing mitochondrial GSH level and/or the indirect effect on removing mitochondrial-derived ROS produced by DG pretreatment might well increase the threshold for MPT pore opening in the presence of [Ca.sup.2+] (Brookes et al. 2004).

I/R challenge caused an increase in mitochondrial [Ca.sup.2+] content. It has been shown that cytosolic [Ca.sup.2+] content increases during myocardial I/R (Xie et al. 2003), leading to the accumulation of [Ca.sup.2+] in mitochondria via the uptake by the inner membrane [Ca.sup.2+] uniporter (Gunter et al. 2000). The resulting mitochondrial Ca2+ overload not only generates energy-consuming futile cycles that divert the use of the inner membrane proton gradient to cation transport rather than ATP production (Moens et al 2005), but also predisposes the mitochondria to undergo a permeability transition (Kushnareva and Sokolove 2000). The MPT further jeopardizes the cellular energy status and the consequent loss of ion homeostasis can lead to necrotic cell death (Kim et al. 2003). The release of cytochrome c from the mitochondrial inner membrane, an event which is believed to be secondary to the onset of MPT (Zhang et al. 2008), is a key step leading to apoptosis (Jiang and Wang 2004). While, under the present experimental conditions, the relative contribution of necrotic and apoptotic cell death to I/R-induced tissue injury was not determined, the finding of decreased sensitivity of mitochondria isolated from DG-pretreated hearts to [Ca.sup.2+] -stimulated PT suggests that the increase in mitochondrial resistance to this transition may play an important role in protecting against myocardial I/R injury. It has been reported that myocardial preconditioning by tanshinone II A or puerarin from Danshen and Gegen, respectively, confers cardioprotection through the inhibition of MPT or opening of m[K.sub.ATP] channels (Gao et al. 2006; Zhang et al. 2005). The observation that DG treatment decreased the mitochondrial [Ca.sup.2+] level in both non-I/R and I/R hearts may be related to the enhancement of mitochondrial GSH content by DC (Meister and Anderson 1983).

The cardioprotection against I/R injury afforded by DG pretreatment was abrogated by PKC[epsilon] or m[K.sub.ATP] inhibition, suggesting the involvement of PKC[epsilon] activation and m[K.sub.ATP] channel opening in the process. PKC[epsilon] is a member of the novel group of the PKC family of serine and threonine kinases that are involved in a wide range of physiological processes including mitogenesis, cell survival, metastasis and transcriptional regulation (Dempsey et al. 2000). In this connection, activation of PKC[epsilon] prior to ischemia has been demonstrated to afford cardioprotection by mimicking

ischemic preconditioning (Ohnuma et al. 2002). The prolongation of onset time for MPT by ischemic preconditioning was found to be associated with the translocation of PKC[epsilon] from the cytoplasm to mitochondria in isolated adult rat cardiomyocytes (Garlid et al. 2009). Recently, it has been shown that phosphorylation by PKC[[epsilon].sub.1] can open m[K.sub.ATP] channels, which in turn increases intramito-chondrial ROS production and the subsequent activation of PKC[[epsilon].sub.2]-PKC[[epsilon].sub.2] can inhibit MPT directly by phosphorylation of its component proteins, thereby reducing the extent of necrotic and/or apoptotic cell death in the myocardium during reperfusion (Costa and Garlid 2008). In addition, our findings showed that DG treatment enhanced PKC[epsilon] translocation and this DG-induced PKC[epsilon] translocation was abrogated by [alpha]-tocopherol co-treatment. In this connection, the translocation of PKC[epsilon] was found to be triggered by ROS-mediated processes (Jiang et al. 2007). Conceivably, ROS generated from the biotransformation of DG-derived ingredients in cardiomyocytes, as observed in cultured cardiomyocytes (data not shown), may play a role in the cardioprotection afforded by DG pretreatment. In addition to the activation of PKC[epsilon], the enhancement in mitochondrial GSH levels in DG-pretreated hearts may be related to a redox-sensitiveand NF-E2-related factor 2-mediated antioxidant response (Numazawa et al. 2003). Both cellular responses elicited by DG pretreatment precondition the myocardium and render it more resistant to I/R injury. In addition to the short-term and high doses treatment regimen, a paralleled study showed that long-term and low doses of DG treatment produced a similar preconditioning effect on the rat myocardium (Chiu et al. 2010).

Short-term with the DG extract likely pharmacologically preconditions the myocardium rather than exert a direct action in attenuating the I/R injury. This postulation is supported by the observation that an oral administration of the DG extract at a single dose did not produce any cardioprotective effect 24 h after the dosing in rats (data not shown). Consistent with this, long-term oral treatment with salvianolic acid B (an active ingredient present in the DG extract, 400 mg/kg x 4 weeks) protected against myocardial infarction in rats, partly by augmenting the expression of vascular endothelial growth factor and promoting angiogenesis (He et al. 2008). However, acute treatment with puerarin (0.24mmol/l in perfusate for 5min) or daidzein (10 mg/kg, i.p.) prior to ischemia also conferred cardioprotection against I/R injury in rats both in vitro and in vivo, by opening calcium-activated potassium channel and activating PKC or inhibiting nuclear factor-Kappa B activation (Gao et al. 2007; Kim et al. 2009). Interestingly, intravenous administration of a mixture of puerarin and danshensu prior to an ischemic insult was also found to protect against myocardial I/R injury in rats (Wu et al. 2007). While short-term treatment with the DG extract at l-2g/kg results in myocardial preconditioning, acute treatment with higher doses may exert direct cardioprotective effects, as were the cases for active ingredients present in the DG extract. A recent study from our laboratory indicated that an oral administration of the DG extract at 4g/kg immediately after the intraperitoneal injection of isoproterenol protected against the isoproterenol-induced myocardial injury in rats (unpublished data). Conceivably, synergistic interactions among multiple active ingredients present in the DG extract may produce cardioprotection against oxidative challenge through an indirect myocardial preconditioning action or a direct cardiac action on specific targets). Taken together, active ingredients from the DG extract may act directly (via opening calcium-activated potassium channel and inhibiting nuclear kappa B activation) and/or indirectly (via enhancing mitochondrial glutathione antioxidant status, activating PKC[epsilon], opening m[K.sub.ATP] and inhibiting MPT) in protecting the myocardium against I/R injury.

In conclusion, short-term DG treatment at high doses caused a dose-dependent protection against myocardial I/R injury in rats. The cardioprotection afforded by DG pretreatrnent was associated with enhancements in mitochondrial antioxidant status and membrane structural integrity, as well as a decrease in the sensitivity of mitochondria to [Ca.sup.2+]-stimulated PT in vitro, particularly under I/R conditions. DG treatment may precondition the myocardium via a redox-sensitive PKC[epsilon]/m[K.sub.ATP] pathway, with resultant inhibition of MPT. Our results suggest that clinical studies examining the effectiveness of DG extract given prophylactically in affording protection against myocardial I/R injury would be warranted.

Acknowledgement

The work described in this paper (or equipment/facility) was fully/substantially/partially supported by a Grant from the University Grants Committee of the Hong Kong Special Administrative Region, China (Project No. AoE/B-10/01).

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Po Yee Chiu (a), Sze Man Wong (a), Hoi Yan Leung (a), Pou Kuan Leong (a), Na Chen (a), Limin Zhou (b), Zhong Zuo (b), Philip Y. Lam (a), Kam Ming Ko (a), *

(a.) Section of Biochemistry and Cell Biology, Division of Life Science, The Hong Kong University of Science & Technology, Clear Water Bay, Hong Kong, China

(b.) School of Pharmacy, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong, China

* Corresponding author. E-mail address: bcrko@ust.hk (K.M. Ko).

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Title Annotation:ELSEVIER
Author:Chiu, Po Yee; Wong, Sze Man; Leung, Hoi Yan; Leong, Pou Kuan; Chen, Na; Zhou, Limin; Zuo, Zhong; Lam
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:9CHIN
Date:Aug 15, 2011
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