Scholarly article on topic 'Antioxidant properties of magnesium lithospermate B contribute to the cardioprotection against myocardial ischemia/reperfusion injury in vivo and in vitro'

Antioxidant properties of magnesium lithospermate B contribute to the cardioprotection against myocardial ischemia/reperfusion injury in vivo and in vitro Academic research paper on "Basic medicine"

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{Ischemia / "Reperfusion injury" / "Myocytes / Cardiac" / "Oxidative stress" / Antioxidants / " In vitro " / "Magnesium lithospermate B"}

Abstract of research paper on Basic medicine, author of scientific article — Wei Quan, Ying Yin, Miaomiao Xi, Dan Zhou, Yanrong Zhu, et al.

Abstract Objective To determine the cardioprotective effect of magnesium lithospermate B (MLB) on myocardial ischemia/reperfusion (MI/R) injury and to investigate the antioxidant potential in vivo and in vitro. Methods MI/R injury was induced by the occlusion of left anterior descending coronary artery for 30 min followed by reperfusion for 3 h in rats. After reperfusion, hearts were harvested to assess infarct size, histopathological damages, the levels of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), reduced glutathione (GSH) and malondialdehyde (MDA). Blood samples were collected to determine serum levels of creatine kinase-MB (CK-MB), cardiac troponin (cTnI) and lactate dehydrogenase (LDH). Furthermore, simulated ischemia/reperfusion (SI/R) injury in vitro was established by oxygen and glucose deprivation (OGD) for 2 h followed by 24-hour recovery period in cardiomyocytes. The activity of LDH in the cultured supernatant and the levels of intracellular reactive oxygen species (ROS), SOD and MDA in cardiomyocytes were also measured. Finally, cardiomyocytes apoptosis was determined with flow cytometry. Results MLB significantly limited infarct size, ameliorated histopathological damages and prevented leakage of CK-MB, cTnI and LDH. Additionally, SOD, CAT, GPx and GSH activities were notably increased by MLB, along with the MDA content decreased as compared with the model group in rats. In vitro study, MLB also decreased LDH activity in the cultured supernatant, increased SOD activity in cardiomyocytes, reduced intracellular ROS and MDA levels, and significantly suppressed cardiomyocytes apoptosis. Conclusion MLB possessed remarkably cardioprotective effects on MI/R injury in vivo and in vitro. The protection of MLB may contribute to its antioxidant properties.

Academic research paper on topic "Antioxidant properties of magnesium lithospermate B contribute to the cardioprotection against myocardial ischemia/reperfusion injury in vivo and in vitro"

JTCM

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JTradit Chin Med 2013 February 15; 33(1): 85-91 ISSN 0255-2922 © 2013 JTCM. All rights reserved.

EXPERIMENTAL STUDY

Antioxidant properties of magnesium lithospermate B contribute to the cardioprotection against myocardial ischemia/reperfusion injury in vivo and in vitro

Wei Quan, Ying Yin, Miaomiao Xi, Dan Zhou, Yanrong Zhu, Yue Guan, Chao Guo, Yanhua Wang, Jialin Duan, Aid-ong Wen

Wei Quan, Ying Yin, Miaomiao Xi, Dan Zhou, Yanrong Zhu, Yue Guan, Chao Guo, Yanhua Wang, Jialin Duan, Aidong Wen, Department of Pharmacy, Xijing Hospital, Fourth Military Medical University, Xi'an 710032, China Supported by the National Natural Science Foundation of China (No. 81173514,No.81001673), the Xijing Research Boosting Program (No. XJZT10D02) and the Excellent Civil Service Training Fund of Fourth Military Medical University (No. 4138C4IDK6).

Correspondence to: Prof. Aidong Wen, Department of Pharmacy, Xijing Hospital, Fourth Military Medical University, Xi'an 710032, China. adwen-7171 @hotmail.com. Telephone: +86-29-84773636 Accepted: September 28,2012

Abstract

OBJECTIVE: To determine the cardioprotective effect of magnesium lithospermate B (MLB) on myocardial ischemia/reperfusion (MI/R) injury and to investigate the antioxidant potential in vivo and in vitro.

METHODS: MI/R injury was induced by the occlusion of left anterior descending coronary artery for 30 min followed by reperfusion for 3 h in rats. After reperfusion, hearts were harvested to assess infarct size, histopathological damages, the levels of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), reduced glutathione (GSH) and malondialdehyde (MDA). Blood samples were collected to determine serum levels of creatine ki-nase-MB (CK-MB), cardiac troponin (cTnl) and lactate dehydrogenase (LDH). Furthermore, simulated

ischemia/reperfusion (Sl/R) injury in vitro was established by oxygen and glucose deprivation (OGD) for 2 h followed by 24-hour recovery period in car-diomyocytes. The activity of LDH in the cultured supernatant and the levels of intracellular reactive oxygen species (ROS), SOD and MDA in cardiomyo-cytes were also measured. Finally, cardiomyocytes apoptosis was determined with flow cytometry.

RESULTS: MLB significantly limited infarct size, ameliorated histopathological damages and prevented leakage of CK-MB, cTnl and LDH. Additionally, SOD, CAT, GPx and GSH activities were notably increased by MLB, along with the MDA content decreased as compared with the model group in rats. In vitro study, MLB also decreased LDH activity in the cultured supernatant, increased SOD activity in cardiomyocytes, reduced intracellular ROS and MDA levels, and significantly suppressed cardiomy-ocytes apoptosis.

CONCLUSION: MLB possessed remarkably cardio-protective effects on MI/R injury in vivo and in vitro. The protection of MLB may contribute to its antioxidant properties.

© 2013 JTCM. All rights reserved.

Key words: Ischemia; Reperfusion injury; Myocytes, Cardiac; Oxidative stress; Antioxidants; In vitro; Magnesium lithospermate B

INTRODUCTION

Ischemic heart disease (IHD) is the most common cause of death in the world wide.1 After an acute myo-

cardial infarction, myocardial reperfusion with the use of thrombolytic therapy or primary percutaneous coronary intervention (PCI) is the most effective strategy for improving the clinical outcome. The process of restoring blood flow to the ischemic myocardium, however, can induce myocardial injury.1'2 This phenomenon, termed as a myocardial ischemia/reperfusion (MI/ R) injury, draws more attentions of cardiologists.2 It is well known that the pathogenesis of MI/R injury is known to involve interplay of multiple mechanisms.1 Numerous studies have implicated the reactive oxygen species (ROS), including superoxide radical (O2-), hydrogen peroxide (H2O2X hydroxyl radical (• OH), and peroxynitrite (ONOO-), which are generated upon reperfusion.34 They may injure cells by causing membrane lipid peroxidation and consequently lead to apoptosis or death in the process of MI/R injury.15 Scientific studies support that compounds with antioxi-dant properties are more helpful in treatment of MI/R injury.6-8 In recent years, considerable attention has been focused on the protective properties of exogenous antioxidants, and on the mechanisms of their action.8 9 Searching for antioxidants with minimal side effects from natural sources such as herbs or plants probably represent an ideal strategy to develop safe and effective agents for MI/R injury treatment. The dry roots of Danshen (Radix Salviae Miltiorrhizae) are a representative oriental medicine used for the treatment of coronary heart disease.10 Magnesium lithosper-mate B (MLB, Figure 1), the tetramer of caffeic acid with various pharmacological actions, is an effective component extracted from the root of Danshen.1112 A recent study indicate its protective effect against glucose-induced intracellular oxidative damage in vitro.13 In hepatic stellate cells, MLB strongly suppresses H2O2-induced ROS generation.14 Further, MLB is also found as an active component of Danshen in protecting cardiomyocytes from ischemic injury through specific inhibition of TAB1-p38 apoptosis signaling.15 Interestingly, another caffeic acid derivative, salvianolic acid A, possesses protective effect on MI/R injury, which is related to its ability to reduce myocardial cell apoptosis and damage induced by oxidative stress.6 These all evidences above suggest that MLB may have a potential ability to enhance the antioxidant status in cardiomyocytes, which may be contributed to its use in MI/R injury. However, up to now, MLB is rarely reported to ameliorate MI/R injury systematically. Moreover, the cardiac protection of MLB related to antioxi-dant mechanism remains largely unknown. Therefore, the aim of this study is to determine cardioprotective effects of MLB on MI/R injury and to investigate the antioxidant properties in vivo and in vitro.

MATERIALS AND METHODS

Drug and chemicals

MLB (molecular formula: C36H28 MgO 16, molecular weight: 740.90, batch number: 1109181) was provid-

0 0 II HvJ.....,,«C02-"—;.'Mg2+ \ 0 Ö2C f* V OH ^^OH

Figure 1 Structure of magnesium lithospermate B

ed by Green Valley Company (Shanghai, China). Evans blue, TTC (2, 3, 5-triphenyltetrazolium chlorid), Annexin V-FITC/PI detection kit and DCFH-DA detection kit were purchased from Sigma Chemical (Perth, WA, Australia). Creatine phosphokinase-MB (CK-MB) and cardiac troponin (cTnI) ELISA kits were purchased from Westang Reagent Company (Shanghai, China). Lactate dehydrogenase (LDH), reduced glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and malondialdehyde (MDA) detection kits were purchased from Jiancheng Reagent Company (Nanjing, China). Fetal bovine serum (FBS) was purchased from Sijiqing Biological Engineering Materials Company (Hangzhou, China). Dulbecco's modified Eagle's medium (DMEM) was purchased from Gibco (Grand Island, NY, USA).

Experimental animals and MI/R protocol

Adult male Sprague-Dawley rats [(250 ± 20) g] were supplied by the animal research center at Fourth Military Medical University, Xi'an, China [Animal Certificate of Conformity: SCXK-(Army) 2007-007]. All animal protocols were approved by the Fourth Military Medical University Committee on Animal Care. The MI/R surgical procedure was used as described previ-ously.16 Briefly, Sprague-Dawley rats were intubated and artificially ventilated with a rodent ventilator (HX-100E, Taimeng, China) under anesthesia with 3% pentobarbital sodium (40 mg/kg, i.p.). The normal electrocardiogram was recorded after electrodes were placed subcutaneously and connected to an electrocardiograph (BL-420S, Taimeng, China). Coronary artery ligation was achieved with a gab occluder fixed onto the LAD coronary artery. A 7-0 silk suture was passed under neath the LAD (2-3 mm inferior to the left auricle) and tied. MI/R was induced by 30 min of ischemia followed by 3 h of reperfusion. Rats were divided into five groups: I) sham group, the silk suture crossed without ligation; II) MI/R + vehicle group (model group), rats received saline alone; III) MI/R + MLB (15 mg/kg) group; IV) MI/R+MLB (30 mg/kg) group; V) MI/R+MLB (60 mg/kg) group. MLB was dissolved in saline to give a final concentration. The MLB groups received MLB injection intravenously (i.v.) at the onset of reperfusion.

Myocardial infarct size

Evans blue (EB) was perfused into the aorta and coronary arteries after the ligation of the coronary artery. The entire ventricular tissue was sliced into five sections through the transverse axis from the apex to the atrioventricular groove. Tissues were incubated in 3 mL 2% TTC at 37T! for 15 min. TTC stains viable tissue red while the infarct portion remains white. The area of infarction was determined by computerised planimetry (Image-ProPlus, Media Cybernetics, Bethesda, MD, USA). The size of the risk zone (RZ) was estimated by the area as a percentage of the total left ventricle (TV). Similarly, the size of the infarct zone (IZ) was estimated by the area as a percentage of the RZ.

Histopathological examination in tissues

The heart tissue obtained from all experimental animals was washed with 0.9% saline and then fixed in 10% buffered neutral formalin solution. After fixation, the heart tissue was processed and embedded in paraffin. Then, the tissue was sectioned and stained with he-matoxylin and eosin (HE) dye and examined with a high-power microscope and photomicrographs were captured light microscopy (Olympus, Tokyo, Japan).

Myocardial injury markers in serum

Arterial blood samples were drawn at the end of reperfusion, and serum was separated at 4000 r • min-1 and -20T: for 15 min (TDZ4A-WS, Xiangyi, China). The levels of cTnI, CK-MB and LDH were measured according to manufactures' instructions.

Antioxidant status and lipid peroxidation product in tissues

Tissue samples from the IZ were saved. The tissue was homogenized in 5.0 mL of 0.1 M Tris-HCl buffer (pH=7.4) in ice-cold condition. The homogenate was centrifuged and the supernatant was used to assay CAT, GSH, GPx, SOD levels and MDA content, according to the manufacturer's instructions. GSH level was assayed at 420 nm and calculated on the basis of GSH calibration curve. SOD level was assayed at 560 nm on the basis of its ability to inhibit the oxidation of hy-droxylamine by superoxide anion from xanthine oxidase system. GPx level was measured at 412 nm on the basis of the rate of oxidation of the reduced glutathi-one to the oxidized glutathione by H2O2 under the catalysis of GPx. The principles of the assay for CAT are based on the determination of the H2O2 decomposition rate at 240 nm. Thiobarbituric acid reactive substances were assessed by measuring the MDA concentration at 532 nm with the thiobarbituric acid method, which is based on the reaction of MDA with thiobarbi-turic acid to form a stable chromophoric product.

Cell culture and MI/R protocol

Neonatal rat ventricular myocytes (NRVM) were isolated from 1- or 2-day old Spragur-Dawley rats and cultured as previously described.17 The oxygen and glucose deprivation (OGD) technique was based on a previous-

ly described protocol.18 In our study, the OGD injury was produced by incubating with blank solution and exposured to a hypoxic environment of 95% nitrogen and 5% CO2 in airtight gas chambers (Billups-Rothen-berg, San Diego, CA, USA) at 37^ for 2 h. After OGD treatment, cells were removed from the gas chamber, and the OGD solution was replaced with warmed culture medium for 24 h (recovery period) in a CO2 incubator at 37^. After 24 h cultured, cardiomyocytes were used in subsequent experiments. Cardiomyocytes were divided into five groups: (I) control group, cardiomyocytes without any treatment; (II) SI/ R group (model group), cardiomyocytes were cultured under OGD conditions for 2 h and then under recovery conditions for 24 h; (III) SI/R+MLB (20 ^g/mL) group; (IV) SI/R+MLB (40 ^g/mL) group; (V) SI/R+ MLB (60 |jig/mL) group. All MLB groups were pretreat-ed with MLB at concentrations of 20, 40 and 60 |jig/mL for 24 h before SI/R injury.

Intracellular biochemical index in cultured cardiomyocytes

The levels of SOD, MDA and LDH in cardiomyocytes were measured according to manufactures' instructions. The intracellular ROS level was measured using 2', 7'-dichloro fluorescin diacetate (DCFH-DA) detection kit. Briefly, cardiomyocytes were first washed with phosphate buffered saline (PBS) three times and then incubated for 30 min at 37T! with the same solution containing 2.5 |Jig/mL of DCFH-DA.

Apoptosis in cultured cardiomyocytes

The apoptotic cardiomyocytes were measured by flow cytometer using the Annexin V-FITC/PI kit as manufacturer's instructions. In brief, cardiomyocytes were collected, washed with PBS, resuspended with binding buffer and incubated with Annexin V at room temperature in dark for 10 min. Then the cardiomyocytes were centrifuged and resuspended with binding buffer. PI was added to the resuspended cardiomyocytes before they were analyzed with flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA).

Statistical analyses

Data were presented as mean ± SD and analyzed by Graphpad Prism software version 5.01 (GraphPad Software, La Jolla, CA, USA). One-way ANOVA followed by Tukey's test was applied for the statistical analysis of the parameters. Value of at P<0.05 was considered statistically significant.

RESULTS

Effect of MLB on myocardial infarct size

In Figure 2, no difference was observed in the RZ between the model group and MLB groups. The infarct size among MLB groups was smaller than that in the model group. The size of IZ was 46% ± 10% in the model group. Treatment with MLB was followed by lower IZ values (24% ± 5%, P<0.05).

Effect of MLB on histopathological damage in tissues

Histopathological changes of the heart in each group are shown in Figure 3. Heart tissues from the model group showed anomaly wave form, staining uneven, interstitial region enlarged, a small amount of inflammatory cells infiltration around the interstitial region and small hemorrhage. After treated with MLB (60 mg/ kg), pathological extent was generally pathologically better improved than the model group. Some cardio-myocytes denaturation could be seen, whereas necrosis was not obvious, with slight amount of inflammatory cells infiltration being seen around the interstitial region.

Effect of MLB on myocardial injury markers in serum

The levels of acute serum myocardial injury marker enzymes cTnl, CK-MB and LDH are given in Table 1. After MI/R, there was a significant increase in the levels of cTnl, CK-MB and LDH compared with the sham group. Treatment with MLB restored all of them to low levels.

Effect of MLB on antioxidant status and lipid peroxidation product in tissues

Table 2 shows the effect of MLB on antioxidant status and lipid peroxidation product in heart homogenate. In the model group, the homogenate levels of SOD, CAT, GPx and GSH were decreased significantly, while the MDA content was increased significantly compared with the sham group. Treatment with MLB

increased the homogenate levels of SOD, CAT, GPx ■-

able 1 Effect of MLB on myocardial injury markers in serum (x±s)

and GSH, and decreased the homogenate MDA content.

Effect of MLB against OGD induced injury in cultured cardiomyocytes

The levels of intracellular ROS, LDH, SOD and MDA are summarized in Table 3. Intracellular ROS level was measured by DCFH-DA. A significant increase in DCF fluorescence was observed when cardio-myocytes were induced by OGD, which was attenuated by MLB. In the model group, the level of SOD in cardiomyocytes was decreased, while the content of MDA in cardiomyocytes and the level of LDH in the cultured supernatant were increased compared with the control group respectively. Treatment with MLB significantly increased the level of SOD, but reduced the content of MDA and the level of LDH.

Effect ofMLB on cardiomyocytes apoptosis

In the control group, most cardiomyocytes were viable. SI/R stimulation induced apoptotic damage in cardio-myocytes compared with the control group (15.73% ± 0.77%, P<0.05). While treated with MLB, the apoptot-ic cardiomyocytes were decreased markedly compared with the model group (6.95% ± 1.05%, P<0.05).

DISCUSSION

It is generally accepted that the intense and sustained changes of myocardial injury occur in myocardial ischemia period laid the foundation of reperfusion injury following. This reperfusion injury testified as continua-

Group n Dose (mg/kg) cTnl (ng/mL) CK-MB (ng/mL) LDH (U/mL)

Sham 6 - 25.7 ±7.3 3.9 ± 2.5 29.5 ± 0.7

MI/R+vehicle 6 - 94.4 ± 11.6a 13.0 ± 1.9a 63.0 ± 3.5a

MI/R+MLB 6 15 92.0 ± 9.7 10.1 ± 0.9b 41.4 ± 5.8b

6 30 74.4 ± 8.3b 9.2 ± 1.3b 31.5 ± 5.1b

6 60 44.8 ± 13.5b 7.3 ± 1.2b 29.8 ± 1.7b

Notes: MI/R: myocardial ischemia/reperfusion; MLB: magnesium lithospermate B; cTnl: cardiac troponin; CK-MB: creatine kinase-MB; LDH: lactate dehydrogenase. aP<0.05 vs Sham group; kP<0.05 vs MI/R+vehicle group.

able 2 Effect of MLB on myocardial antioxidants and lipid peroxidation in tissues (x ±s)

Dose SOD CAT GPx GSH MDA

(mg/kg) (U/mg protein) (U/mg protein) (U/mg protein) (nmol/g protein) (nmol/mg protein)

Sham 6 - 9.56 ± 1.14 22.82 ± 2.89 1.66 ± 0.48 2.60 ± 1.04 6.22 ± 0.91

MI/R+vehicle 6 - 4.51 ± 1.52a 13.12 ± 1.35a 0.50 ± 0.27a 0.68 ± 0.32a 11.33 ± 1.59a

MI/R+MLB 6 15 7.24 ± 1.40b 19.69 ± 1.20b 1.03 ± 0.34 2.02 ± 0.43b 9.79 ± 1.13

6 30 7.79 ± 2.19b 23.09 ± 1.79b 1.45 ± 0.45b 2.45 ± 1.04b 9.46 ± 0.93b

6 60 9.08 ± 2.00b 21.27 ± 2.96b 1.49 ± 0.57b 2.46 ± 1.07b 8.92 ± 1.22b

Notes: MI/R: myocardial ischemia/reperfusion; MLB: magnesium lithospermate B; SOD: superoxide dismutase; CAT: catalase; GPx: glutathione peroxidase; GSH: reduced glutathione; MDA: malondialdehyde. 'P<0.05 vs Sham group; bP<0.05 vs MI/R+vehicle group.

MI/R+MLB (mg/kg)

30.0 25.0 20.0 15.0 10.0 5.0 0.0

50.0 40.0 30.0 20.0

MI/R+MLB (mg/kg)

MI/R+MLB (mg/kg)

B ^ .......C

Figure 2 Effect of MLB on myocardial risk and infarct area

TV: total left ventricle; RZ: risk zone; IZ: infarct zone; MI/R: myocardial ischemia/reperfusion (30 min/3 h); MLB: magnesium litho-spermate B. A: The myocardial risk and infarct area was determined by Evans blue and TTC method. B:The effect of MLB administration on RZ (%TV) in rat after MI/R. C:The effect of MLB administration on IZ (% RZ) in rat after MI/R. Values are expressed as the mean ± SD (n=8). aP<0.05 vs sham group; bP<0.05 vs MI/R+vehicle group.

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Figure 3 Histopathological changes of cardiac muscle (HEx400) HE: hematoxylin and eosin; MI/R: myocardial ischemia/reperfusion (30 min/3 h); MLB, magnesium lithospermate B. A: sham group; B: MI/R+vehicle group; C: MI/ R + MLB (15 mg/kg) group; D: MI/R+MLB (30 mg/kg) group; E: MI/R + MLB (60 mg/ kg) group.

Table 3 Effect of MLB in cultured cardiomyocytes (x±s)

Group n Dose (^g/mL) Intracellular ROS level (fluorescence density) LDH (U/L) SOD (U/mg protein) MDA (nmol/g protein)

Control 6 - 18.46 ± 2.12 733.19 ± 33.18 73.72 ± 0.48 1.39 ± 0.13

SI/R 6 - 62.76 ± 3.59a 1714.74 ± 57.99* 25.18 ± 1.21* 2.89 ± 0.14*

SI/R+MLB 6 20 49.92 ± 3.39b 1096.65 ± 65.94b 41.92 ± 4.64b 2.56 ± 0.34

6 40 45.39 ± 2.90b 1074.50 ± 64.46b 53.78 ± 2.20b 2.31 ± 0.22b

6 60 33.92 ± 7.00b 987.59 ± 50.64b 60.11 ± 5.63b 1.78 ± 0.40b

Notes: SI/R: simulated ischemia/reperfusion; MLB: magnesium lithospermate B; LDH: lactate dehydrogenase; SOD: superoxide dismutase; MDA: malondialdehyde. "P<0.05 vs Control group; bP<0.05 vs SI/R group.

tion of ischemic injury, and further aggravated irreversibly myocardial injury.1,2 Therefore, therapeutic strategies aim at preventing reperfusion injury may be a reasonable choice for the treatment of related heart disease. In the present study, we demonstrated that administration of MLB could attenuate the severity of heart injury induced by MI/R injury in vivo and in vitro, and these protective effects of MLB were mediated through the reduction of oxidative stress. Infarction size is considered as a key criterion in evaluating MI/R injury, and directly related to the prognosis of patients in clinic.19 It was worthy of noting that MLB could significantly reduce myocardial infarct size. On histopathological examination, obvious myocardial damage was observed in the model group compared with the sham group. Extensive cloudy swelling, hyper-emia, sinusoidal distension and focal vacuolar degeneration were also observed in the model group. Our results indicated that MLB restored most of these heart pathological lesions, showing the cardioprotective action of it. In addition, myocytes release a variety of in-tracellular enzymes into blood during MI/R period.20 Cytosolic enzymes of CK-MB, cTnI and LDH, which serve as the diagnostic markers of myocardial tissue damage, leak out from the damaged tissues to the blood stream when the cell membrane becomes permeable or rupture.21 Our results showed a significant elevation in the levels of cTnI, CK-MB and LDH in serum of the model group. These MI/R-associated serum cTnI, CK-MB and LDH elevations were markedly blunted when rats were treated with MLB. This could be due to its action on maintaining membrane integrity and thereby restricting the leakage of these enzymes. Finally, taking together with the results above, we could concluded that MLB possessed marked cardioprotec-tive activities on MI/R injury in vivo. Accumulating evidence indicates that the pathogenesis of MI/R injury can be divided into several phases.1 The initial injury is triggered by ROS with inflammation involving chemokines and cytokines, followed by neutro-phil-mediated myocardial injury occurring in the late period of reperfusion.1,22 A large body of literature suggests that ROS is a main dominative factor of MI/R injury, and they are natural byproducts of cellular metabolism.1 ROS may injure cells by causing membrane lipid peroxidation and consequently lead to apoptosis or death in the process of MI/R injury.3,4 Therefore, further investigation was performed with focusing on anti-oxidant properties involved in the cardioprotective effects of MLB. Among endogenous antioxidant enzymes, SOD plays an important role in protecting the cells from oxidative damage by converting O2- into H2O2, which is further metabolized by CAT to molecular oxygen and H2O. The decrease in the levels of these antioxidant enzymes might be due to cardiomyocytes damage.23 In myocardial tissues, treatment with MLB significantly increased SOD level. The change of SOD level may be due to the scavenged free radicals of •HO formed by MI/R-induced lipid peroxidation and maintained cellular integrity.24 GSH is an important non-en-

zymatic antioxidant defense required to maintain the normal redox state of cells and to counteract deleterious effects of oxidative stress. GSH depletion ultimately promotes oxidative stress, with a cascade of effects affecting functional and structural integrity of cell and organelle membrane.25 Our results showed that the significant decrease in GSH level in the model group was enhanced by MLB, which indicated the potentiality of MLB to counteract the oxidative damage and to reinforce the antioxidant defense. CAT converts H2O2 to H2O and GPx catalyses the transformation of H2O2 to harmless byproducts. The decrease in GPx and CAT levels in myocardium indicates the severity of the oxida-tive stress-induced by MI/R injury.5 Our results clearly demonstrated that MLB indeed enhanced the levels of CAT and GPx respectively. In addition, MDA is an important product of lipid peroxidation, and the changes of the concentrations of MDA indicate the degree of lipid peroxidation.26 Treatment with MLB significantly decreased MDA content in our present study. In summary, all these findings above suggested that MLB might affect the levels of endogenous antioxidants or oxidative stress or both.

To further confirm the antioxidative activities of MLB, we observed cultured NRVM induced by OGD. Our researches revealed that the level of SOD in cultured cardiomyocytes was decreased, while LDH and MDA were increased in the model group. Treatment with MLB significantly altered these biochemical indexs. In addition, it is widely accepted that oxidative stress, which is associated with increased formation of ROS, plays a major role in the pathogenesis of MI/R injury.27 In our study, intracellular ROS levels were measured by DCFH-DA. A significant increase in DCF fluorescence was observed in the model group, which was attenuated by treatment with MLB. We also found that the results in the cardiomyocytes were consistent with that in the rat model. Finally, taken together the results in vivo and in vitro, we speculated reasonably that car-dioprotective effects of MLB were closely related to its antioxidant properties.

Oxidative stress is a well-known factor promoting programmed cell death (apoptosis).28,29 Apoptosis is a significant cellular mechanism responsible for MI/R inju-ry.6,28 Thus, inhibition of apoptosis caused by oxidative stress could be an available therapy for attenuation of MI/R injury. In the present study, cardiomyocytes apoptosis was quantitatively analyzed by Annexin V-FITC staining. The results strongly suggested that MLB exerted protective effects against cardiomyocytes apoptosis. We speculated that antiapoptotic activity of MLB may be linked to its antioxidant properties. In summary, MLB could provide cardioprotective effect against MI/R injury in vivo and in vitro. The mechanisms might be attributed to scavenging lipid peroxi-dation products and ROS and increasing endogenous antioxidant defense enzymes. These findings might be helpful to understand the beneficial effects of MLB

against MI/R injury, although further study is needed to confirm its mechanism.

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