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Introduction

Myocardial infarction (MI) leads to the permanent loss of cardiomyocytes, scar formation and the absence of the endogenous repair mechanism which leads to heart failure (1). Bone marrow-derived mesenchymal stem cells (MSCs) are self-renewing, multipotent adult cells (2), which are able to differentiate into osteoblasts, chondrocytes (3), astrocytes (4), neurons (5), endothelial cells (6), skeletal muscle cells (7), vascular smooth muscle cells (6) and cardiomyocytes in vitro (8) and in vivo (9–10). Animal and clinical studies have provided evidence that cell therapy with MSCs is able to improve cardiac function potentially via angiogenesis and myogenesis following MI (11–12). However, the clinical exploitation of MSC transplantation is hampered by their poor viability in the diseased myocardium post-transplantation. For instance, the majority of MSCs injected into the left ventricle of the adult murine heart died within 1 week during the injection period (9). This reflects that the ischemic microenvironment of the infarcted myocardium was not conducive of MSC survival. Furthermore, a large number of studies have demonstrated that increasing the differentiation of implanted MSCs into cardiomyocytes was able to promote heart function during the acute phase of MI (13–15). Therefore, promoting the survival of implanted MSCs and improving the differentiation of implanted MSCs into cardiomyocytes following transplantation is critical for successful cellular therapy.

The extract of Ginkgo biloba (EGb) leaves has been applied as a traditional Chinese medicine for numerous years. At present, the extract of the standardized G. biloba leaf with well-defined components, labeled EGb 761, which contains 24% ginkgo-flavone glycosides (e.g. kaempferol, quercetin and isorhamnetin derivatives) and 6% terpenoid (e.g. ginkgolides A, B, C, J and bilobalide), has been developed and is extensively consumed as a dietary supplement and a herbal remedy (16). Numerous studies have verified that EGb 761 protects the cell from a variety of extrinsic toxic stimuli, which induce damage in different experimental disease models (17–20). Previous studies have reported that EGb 761 mediates its protective effects against doxorubicin-induced cardiac injury through antioxidant, anti-inflammatory and antiapoptotic mechanisms. Furthermore, it may induce neuronal differentiation of cultured PC12 cells and neuroblast differentiation in the mouse hippocampal dentate gyrus, and promote the differentiation of human umbilical cord-derived mesenchymal stem cells (21–24). However, it is unclear whether EGb 761 is able to improve the cardiac performance of infarcted hearts through increasing the viability of implanted MSCs and improving the differentiation of implanted MSCs into cardiomyocytes following cellular therapy. The present study aimed to investigate the roles and the potential mechanisms of EGb 761 on implanted MSCs in the infarcted myocardium, and to provide experimental evidence for the potential application of EGb 761 for cellular therapy during ischemic heart disease.

Materials and methods

Reagents

The reagents and detection kits used were as follows: Low-Dulbecco’s modified Eagle’s medium (L-DMEM), trypsin-EDTA, fetal bovine serum, penicillin and streptomycin were purchased from Gibco-BRL (Carlsbad, CA, USA); malondialdehyde (MDA) and superoxide dismutase (SOD)assay kits were obtained from Nanjing Jiancheng Biochemical Institute (Nanjing, Jiangsu, China); EGb 761 was obtained from Invitrogen Life Technologies (Karlsruhe, Germany); the In situ Cell Death Detection kit was purchased from Nanjing KeyGen Biochemical Institute (Nanjing, China); Fas and cTnI antibodies were obtained from Antibody Design Labs (San Diego, CA, USA); and IRDye 800 CW Donkey anti-rabbit IgG, IRDye 800 CW Donkey anti-mouse IgG and IRDye 680 Donkey anti-goat IgG were purchased from Li-Cor Biosciences, (Lincoln, NE, USA). Unless stated otherwise, all other chemicals were purchased from Sigma (St. Louis, MO, USA).

Animals

Sprague-Dawley rats weighing 250±15 g were obtained from the Experimental Animal Center of Shanghai Medical College (Shanghai, China). All animals were housed in a light-controlled room with a 12 h light/dark cycle and were allowed access to food and water ad libitum. Experimental protocols and animal care methods were approved by the Experimental Animal Research Committee of Fudan University (Shanghai, China).

Cell culture

The MSCs were obtained from bone marrow aspirates of femurs and tibias from rats using a modified method originally described by Xie et al (25). The marrow pellet was washed in phosphate-buffered saline (PBS), centrifuged at 2,000 × g for 18 min and then resuspended in L-DMEM. Nucleated cells were isolated with a density gradient centrifugation (Ficoll or Paque, GE Healthcare Life Sciences, Schenectady, NY, USA), then introduced into a 25-cm2 flask and cultured (at a density of 5×107 cell/ml) at 37°C in humidified air with 5% CO2 in L-DMEM containing 15% fetal calf serum (FCS), penicillin (100 U/ml) and streptomycin (100 mg/ml). The medium was changed to remove non-adherent cells 72 h after seeding and every 3 days for ~10 days.

The primary cultured cells were replated into two new flasks when the MSCs had grown to ~80% confluence. For subculture, the cells were resuspended with 0.25% trypsin + 0.02% EDTA and passaged at a ratio of 1:2 plates. For the four passages, homogeneous MSCs devoid of hematopoietic cells were used for the experiments.

The cultured cells were initially identified by fluorescence-activated cell sorting (FACS) analysis prior to the experiments. Briefly, MSCs were trypsinized and washed with 4% FCS buffer in PBS. Then, ~106/100 μl cells were incubated at 4°C for 30 min with 20 μl antibodies, i.e phycoerythrin-conjugated anti-rat CD14, CD90 and CD45 (AbD Serotec, Düsseldorf, Germany), fluorescein isothiocyanate (FITC)-conjugated anti-rat CD105 and CD44 (BioLegend, San Diego, CA, USA) and FITC-conjugated anti-rat CD34 antibodies (Santa Cruz Biotechnology, Inc, Santa Cruz, CA, USA). A normal mouse IgG was considered as the negative isotype control. The cells were washed twice with PBS and were then labeled with rabbit or mouse-FITC conjugated IgG for 20 min in a dark environment at room temperature. Flow cytometric analyses were performed on a FACS Calibur system using Cell Quest TM software (Becton-Dickinson, Franklin Lakes, NJ, USA). In total, 10,000 events were recorded for each sample.

MI animal model and MSC transplantation

The rats underwent myocardial ischemia by occlusion of the left coronary artery. Briefly, the rats were anesthetized by intraperitoneal injection of sodium pentobarbital (45 mg/kg). The animal hearts were exposed by a left thoracotomy. Ligation of the left anterior descending (LAD) artery was performed 1–2 mm distal to the line between the left border of the pulmonary conus and the right border of the left atrial appendage. MI was confirmed by electrocardiogram (ECG; ADI Instruments, Houston, TX, USA). A final 200 μl suspension containing a total of 1×105 MSCs labeled with PKH-26 red fluorescent cell tracker dye (Red Fluorescent Cell Linker Kit; Sigma) was then transplanted into four sites around the ischemic myocardium marginal zone of the left ventricle via intramyocardial injection. The animal chest cavity was closed following transplantation of MSCs. The rats were left to recover in a temperature-controlled chamber until they resumed full alertness and mobility, at which point they were returned to their cages.

The animals were randomly divided into two groups. The control group and the EGb 761-treated groups. In the control group, following MSC transplantation, 3 ml normal sodium (0.9% NaCl) was injected intraperitoneally (n=18) and in the EGb 761-treated groups, following MSC (1×105) transplantation, 100 mg/kg/day EGb 761 (3 ml) was injected intraperitoneally (n=18). For each animal, the total quantity of normal sodium and EGb 761 was divided into two intraperitoneal injections per day, respectively. Following 1, 2 and 7 days of MSC transplantation and EGb 761 treatment, the animal hearts from each group (six animals per group) were harvested under inhalation anesthesia, respectively, and fixed with 4% neutral formaldehyde for further analysis.

Measurement of cardiac function

Following 1, 2 and 7 days of normal sodium and EGb 761 treatment, the cardiac function of rats was assessed by a multichannel physiological recorder (Jinjiang Tongyong Industry Co., Ltd., Sichuan, China). Briefly, after obtaining the body weight of each animal, animals were sedated (100 mg/kg ketamine and 1.5 mg/kg xylazine) and a transducer (Jinjiang Tongyong Industry Co., Ltd., Chengdu, China) was applied to the left hemithorax. The heart was visualized using a 2-dimensional mode with the axial view of the left ventricle. Next, through the right carotid artery, a cannula was inserted into the left ventricle. The ejection fraction (EF), left ventricular end-diastolic volumes (LVEDV), left ventricular end-diastolic pressure (LVEDP), left ventricular ± dp/dt (maximum rate of pressure rise), left ventricular end diastolic diameters (LVDd), fractional shortening (FS) and left ventricular end-systolic pressure (LVESP) were calculated by the standard formulas of ECToolbox software (Emory University, Atlanta, GA, USA).

Histopathological examination

Following 1, 2 and 7 days of normal sodium and EGb 761 treatment, each group of animals were sacrificed by high dose anesthesia (45 mg/kg, respectively, and perfused with 0.9% saline followed by 4% paraformaldehyde through a needle inserted into the left ventricle of the rat through the left atrium, for in situ perfusion fixation. Myocardial tissues in the left ventricle of sacrificed rats were collected and fixed in 4% pre-cooled paraformaldehyde for 72 h and embedded in paraffin for histological studies. Paraffin-embedded tissues were sectioned into 5-μm thick slices. The sections were stained for hematoxylin and eosin (H&E) according to the manufacturer’s instructions (Beyotime Biochemical Institute, Shanghai, China). Images were visualized under an optical microscope at ×100 and ×400 magnifications.

Determination of reactive oxygen species production

Following 1, 2 and 7 days of normal sodium and EGb 761 treatment, each group of animals was sacrificed using high-dose anesthesia (sodium pentobarbital, 45 mg/kg), respectively. The hearts of the animals were then removed and opened along the greater curvature and washed with PBS. The homogenate was centrifuged (10,000 × g for 15 min) and then the supernatant was subjected to glutathione peroxidase (GSH-Px), catalase (CAT) and superoxide dismutase (SOD) activity, and malondialdehyde (MDA) content assays, according to the manufacturer’s instructions.

Terminal dUTP nick-end labeling (TUNEL) assay

The cryosections (40-μm thick) from animal myocardial samples were stained with an in situ cell death detection kit (Kaiji Biological Technology development Co., Ltd., Nanjing, China) according to the manufacturer’s instructions, and then the stained cryosections were observed with a confocal microscope (Leica TCS SP8 MP, Leica, Wetzlar, Germany). The apoptotic index (AI) was determined using the following formula: AI = number of TUNEL-positive cells/total transplanted cells pre-labeled with PKH-26 × 100.

Western blot analysis

The infarcted heart tissues were lysed in Tris-HCl buffer (0.05 mol/l) containing 0.15 mol/l NaCl, 0.02% NaN3, 0.1% SDS, 1% nonidet P (NP-40), 11 μg/ml aprotinin and 0.1 mol/l phenylmethyl sulfonylfluoride (pH 8.0). The expression of the Fas protein was determined by immunoblotting. Briefly, the protein content of the supernatant prepared from cultured cells was quantified by Lowry’s method. A 100 μg sample of total protein was separated by 10% SDS-PAGE and then transferred onto a polyvinylidene fluoride membrane (pore size, 0.45 μm; 3.0 mA/cm2 for 40 min). Following inhibition with 5% non-fat milk powder/PBS containing 5% Tween-20 (PBS-T) for 3 h at room temperature, the membranes were incubated with the following antibodies: Mouse anti-fas and rabbit anti-GAPDH. The membranes were probed with the primary antibodies overnight at 4°C. Then, IRDye-conjugated secondary antibodies (IRDye donkey anti-mouse IgG and IRDye donkey anti-rabbit IgG; 1:100) were added for 2 h and subsequently scanned by the Odyssey Infrared Imaging system (Li-Cor Biosciences). Specific densitometry values for the bands were calculated by normalization to GAPDH.

Differentiation of implanted cells

Animals were sacrificed using high-dose anesthesia (sodium pentobarbital, 45 mg/kg). The hearts were quickly harvested and tissues from the free wall of the left ventricle, including the infarct and peri-infarct regions were then embedded in optimal cutting temperature tissue-freezing medium. The frozen sections (40-μm thick) of the left ventricular samples were prepared for identification of implanted cells. Briefly, the mounted frozen sections on glass slides were fixed with methanol at −20°C for 20 min. Following washing three times with PBS, the sections were incubated with blocking buffer (1% donkey serum, 1% bovine serum albumin and 0.2% Tween-20 in PBS) for 30 min at room temperature and then with the mouse anti-rat cTnI antibodies in blocking buffer for 1 h at room temperature. The sections were then washed three times in PBS, followed by incubation with FITC-conjugated goat anti-mouse IgG second antibody for 1 h at room temperature. Following washing three times with PBS, the stained cryosections were observed under a confocal microscope (Leica).

Statistical analysis

All data are expressed as the mean ± standard error of the mean and analyzed by analysis of variance and a Student-Newman-Keuls test using SPSS 18.0 software (SPSS Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

Results

Expression of surface markers of MSCs

Consistent with previous studies (25,26), the cultured MSCs expressed typical mesenchymal markers, including CD44, CD90 and CD105. However, the surface markers of hematopoietic stem cells (CD45), macrophages (CD14) and lymphocytes (CD34) were not expressed (data not shown).

Establishment of the MI model

Compared with the ECG trace from a normal rat that had not undergone ligation of the LAD coronary artery (Fig. 1A), a deep S wave, R-wave elimination ST segment elevation was observed on the ECG from a rat that had undergone ligation of LAD coronary artery (Fig. 1B).

Figure 1 ECG recordings in rats. (A) surface ECG from a normal rat that had not undergone ligation of athe LAD coronary artery; (B) typical ECG pattern produced during the ligation procedure, indicating MI-induced deep S wave, diminished R-wave amplitude and marked ST segment elevation. ECG, electrocardiogram; LAD, left anterior descending; MI, myocardial infarction.

Amelioration of cardiac function by EGb 761 treatment

The parameters of echocardiography and hemodynamics were analyzed. Compared with the respective control group, FS, EF, LVESP and dp/dtmax significantly increased, and LVDd, LVEDV and LVEDP significantly decreased following EGb 761 treatment (P<0.05 vs. the control group; Table I).

Table I EGb 761 ameliorates cardiac function.

Table I

EGb 761 ameliorates cardiac function.

	1 day		2 days		7 days
Group	Control	EGb 761	Control	EGb 761	Control	EGb 761
EF (%)	72.32±1.46	75.29±2.43a	70.34±0.51b	78.53±0.95a,b	68.57±0.43c	83.66±1.57a,c
FS (%)	40.20±0.56	43.32±1.82a	35.43±1.43b	47.64±1.55a,b	30.12±2.00c	53.00± 2.51a,c
LVDd (cm)	0.62±0.04	0.57±0.05a	0.67±0.02b	0.53±0.08a,b	0.73±0.06c	0.48±0.03a,c
LVEDV (ml)	0.310±0.021	0.300±0.034a	0.315±0.038b	0.290±0.033a,b	0.320±0.030c	0.280±0.040a,c
LVESP (mmHg)	105.40±3.28	115.40±3.38a	98.40±3.16b	125.40±3.68a,b	92.10±2.32c	135.400±3.41a,c
LVEDP (mmHg)	9.300±0.31	7.300±0.22a	11.300±0.28b	6.306±0.21a,b	12.320±0.31c	5.300±0.18a,c
dp/dtmax (mmHg/sec)	6560.5±133.5	7260.5±135.9a	5860.5±115.5b	7950.4±136.6a,b	4860.3±210.3c	8500.0±234.3a,c

{ label (or @symbol) needed for fn[@id='tfn1-mmr-09-04-1112'] } Data are shown as the mean ± standard error of the mean (n=8). EF, ejection fraction; FS, fractional shortening; LVDd, left ventricular end diastolic diameters; LVEDV, left ventricular end-diastolic volumes; LVESP, left ventricular end-systolic pressure; LVEDP, left ventricular end-diastolic pressure; dp/dtmax, left ventricular maximum rate of pressure rise.

a P<0.05, vs. the respective control group,

b P<0.05, vs. the 1 day group;

c P<0.05, vs. the 2 days group.

{ label (or @symbol) needed for fn[@id='tfn5-mmr-09-04-1112'] } EGb, extract of Ginkgo biloba.

EGb 761 reduces MI-induced heart inflammation

H&E staining images demonstrated that MI elicited typical cardiac histopathological changes in the myocardium, which were manifested as the presence of mononuclear cells, fragments of necrotic myocardial fibers, eosinophils, polymorphonuclear neutrophils and macrophagocytes. Compared with the respective control group, EGb 761 significantly reduced the number of infiltrated inflammatory cells in the infarcted myocardium following 1, 2 and 7 days of MSC transplantation (Fig. 2).

Figure 2 Pathological structures of the heart by hematoxylin and eosin staining (magnification, ×100 and magnification, ×400). (A1–C1) Control group; (A2–C2) EGb 761-treated groups; (A1 and A2) Pathological images of the heart following 1 day of MI; (B1 and B2) Pathological images of the heart following 2 days of MI; (C1 and C2) Pathological images of the heart following 7 days of MI. MI, myocardial infarction; EGb, extract of Ginkgo biloba.

Anti-oxidative effects of EGb 761 on the heart

The effects of EGb 761 on the activity of the antioxidant enzymes SOD, CAT and GSH-Px, and the level of MDA in the heart tissues of rats are shown in Fig. 3. The results indicated that MDA content (a product of lipid peroxidation) significantly decreased and the SOD, CAT and GSH-Px activity increased following EGb 761 treatment. With the extension of time of MI, MDA content gradually increased and the activity of SOD, CAT and GSH-Px gradually decreased in the control group. Compared with the respective control group, the activity of SOD, CAT and GSH-Px gradually increased and MDA content gradually decreased in the EGb 761 treatment group (P<0.05).

Figure 3 Effects of EGb 761 on MI-induced oxidative stress. Activity of (A) SOD, (B) CAT, (C) GSH-Px and (D) MDA are presented as histograms. *P<0.05, vs. the respective control group, #P<0.05, vs. the 1 day group. ▲P<0.05, vs. the 2 days group.(n=6). MI, myocardial infarction; SOD, superoxide dismutase; CAT, catalase; GSH-Px, glutathione peroxidase; MDA, malonyldialdehyde; EGb, extract of Ginkgo biloba.

Antagonism of EGb 761 on MI-induced MSC apoptosis

Compared with the respective control group, the AI in the EGb 761 treatment group was significantly decreased (P<0.05). With the extension of MI time, the AI gradually increased in the control groups and the EGb761 treatment groups (P<0.05; Fig. 4A and B).

Figure 4 Effect of EGb 761 on the apoptosis of transplanted MSCs. (A) MSCs labeled with PKH-26 (red) were transplanted into the rat ischemic myocardium. Cellular apoptosis was determined using TUNEL-positive staining (green). The co-labeling of PKH-26 (red) and TUNEL (green) indicated cell apoptosis following MSC transplantation (n=6). (Aa-f) The transplanted cells identified by PKH-26-positive staining (red). (Ag-i) The apoptotic cells identified with TUNEL-positive staining (green). (Am-r) Merged figures show the apoptosis of the grafted MSCs co-labeled with PKH-26 (red) and TUNEL (green; scale bar, 10 μm). (Aa, c, e, g, i, k, m, o and q) Control group; (Ab, d, f, h, j, l, n, p and r) EGb 761-treated groups; (Aa, b, g, h, m and n) Apoptotic images of transplanted MSCs following 1 day of MI; (Ac, d, i, j, o and p) Apoptotic images of transplanted MSCs following 2 days of MI; (Ae, f, k, l, q and r) Apoptotic images of transplanted MSCs following 7 days of MI. (B) Apoptotic indexes are presented as histograms. *P<0.05, vs. the respective control group. There were clear differences between the 1 day group and the 2 day group (#P<0.05), and between the 2 day group and the 7 day group (▲P<0.05). n=6. MSCs, bone marrow-derived mesenchymal stem cells; TUNEL, terminal dUTP nick-end labeling; MI, myocardial infarction; EGb, extract of Ginkgo biloba.

Expression of Fas protein

As shown in Fig. 5, western blotting indicated that Fas protein was upregulated gradually and reached a high level following 7 days of normal sodium treatment in the control group, whereas the Fas protein was downregulated gradually in the EGb 761 treatment group. Compared with the respective control group, the level of Fas expression was significantly decreased (P<0.05).

Figure 5 Western blotting of Fas protein. Expression of Fas protein in the EGb 761 treatment groups was significantly lower than that in the control group. The level of Fas expression gradually increased in the control group, whereas it decreased gradually in the EGb 761 treatment group. *P<0.05, vs. the respective control group, #P<0.05, vs. the 1 day group. ▲P<0.05, vs. the 2 days group. (n=6). EGb, extract of Ginkgo biloba.

Differentiation of MSCs following transplantation

To detect whether implanted MSCs differentiated into cardiomyocytes, the cTnI-expression in the PKH26-labeled MSCs of the transplanted myocardium was assessed using immunohistological staining following 7 days of MSC transplantation. The results demonstrated that, in the control group, the muscle-specific markers, including cTnI staining were not expressed on the PKH26-positive cells as observed by confocal microscopy (Fig. 6A–D). Conversely, co-localization expression of cTnI and PKH26 was observed in the MSCs of the EGb 761 treatment group (Fig. 6E–H).

Figure 6 Cardiac differentiation of PKH 26-labeled MSCs in the transplanted myocardium following 7 days of MSC-transplantation. (A and E) MSCs labeled with PKH-26 (red) were transplanted into rat ischemic myocardium; (B and F) cell differentiation was determined with cTnI-positive staining (green); (C–D and G–H) co-labeling of PKH-26 (red) and cTnI (green) indicated cell differentiation following MSC-transplantation. D and H (Scale bar=10 μm) are an amplification of C and G, respectively. The white arrow indicates the stained positive cells. Scale bar, 100 μm in figure A, B, C, E, F and G. MSCs, bone marrow-derived mesenchymal stem cells; EGb, extract of Ginkgo biloba.

Discussion

MSC therapy prevents disadvantaged remodeling of heart function after MI and improves recovery when introduced into the infarcted heart; however, further promotion of the differentiation of MSCs in the cardiac scar tissue is required. The present study examined a novel strategy to promote the therapeutic effects of MSCs within the ischemic heart and improve cardiac function under pathological conditions. The present study indicated that EGb 761 is able to improve deteriorated cardiac function, which partially contributes to the protective effect of EGb 761 on the grafted MSCs against MI-induced apoptosis and its promotion for the differentiation of MSCs into myocardial cells. Several previous studies have demonstrated that EGb 761 reduces extrinsic damage and anti-oxidative stress, which induced the apoptosis of cardiomyocytes (26,27). The present study demonstrated new evidence that EGb 761 promotes heart function recovery through enhancing implanted cell survival and differentiation in the ischemic myocardium.

Cardiac inflammation is associated with left ventricular remodeling in the MI model. Previous studies have demonstrated that autologous or allogeneic MSCs markedly suppress T-lymphocyte proliferation in the infarcted myocardium (28,29). Furthermore, previous evidence suggests that the transplantation of MSCs attenuated histiocytic infiltration in a rat model of acute myocarditis (30). However, inflammation remains one of the main reasons for the low survival rate of transplanted cells in the infarcted myocardium. The present study revealed that EGb 761 significantly reduced the number of infiltrated inflammatory cells in the infarcted myocardium, which was consistent with other studies demonstrating the anti-inflammatory property of EGb 761 in various animal models (31,32).

Consistent with other studies of the anti-oxidative effect of EGb 761 in vivo and in vitro (33,34), the present study also demonstrated that EGb 761 had an anti-oxidative effect on the infarcted myocardium. In the present study, its effects on the reduction of MDA content and the enhancement of the activity of antioxidant enzymes SOD, CAT and GSH-Px, were demonstrated. In addition, the anti-oxidative effect of EGb 761 was positively correlated with the treatment time of EGb 761.

The present study also demonstrated that EGb 761 has a protective effect on MI-induced MSC apoptosis during MSC transplantation. The TUNEL-positive rate in the transplanted MSCs was significantly decreased following EGb 761 treatment, and the TUNEL-positive rate in the transplanted MSCs was negatively correlated with the treatment time of EGb 761.

Apoptosis, or programmed cell death, can be triggered via the extrinsic (death receptor-mediated) signaling pathways. FasL interacts with its receptor, Fas (CD95/APO-1), and then triggers a cascade of subcellular events involved in the extrinsic signaling pathways, which exhibit a role in cell apoptosis (17). In the present study, the level of Fas expression was positively correlated with the time of MI in the control groups. EGb 761 significantly downregulated the expression of the Fas protein and it was negatively correlated with the treatment time of EGb 761. Therefore, the present study hypothesized that the attenuating effect of EGb 761 on cell apoptosis may be mediated by the Fas death receptor signaling pathways. However, the detailed mechanism of the extrinsic apoptotic pathway requires further investigation.

Previously, it has been demonstrated by Daigneault et al (35) that the transplanted MSCs did not express myocyte-specific markers after 2 weeks of transplantation. Our data also demonstrated that MSCs did not express cTnI, a myocyte-specific marker, 7 days after exogenous MSCs were engrafted into infarcted hearts. Therefore, enhancing the differentiation of MSCs into myocytes is a crucial problem in the regeneration of myocardium via stem cell transplantation.

To identify the enhancement of the differentiation of MSCs into cardiac cells, the rats were treated with 100 mg/kg/day EGb 761 following MSC transplantation. Notably, the present study demonstrated the expression of cTnI in implanted MSCs following 7 days of EGb 761 treatment. However, the detailed mechanisms of the differentiation of MSCs into cardiomyocyte-like cells requires further investigation.

The present study demonstrated the protective effect of EGb 761 on MI following the transplantation of MSCs. These data markedly support the theory that EGb 761 protects implanted-MSCs against MI-induced apoptosis through the activation of the Fas-mediated death receptor signaling pathways. Furthermore, prevention of inflammation, apoptosis and oxidative stress enhanced the differentiation of the transplanted MSCs. The present study provides a promising approach for promoting cardiac repair with a widely available and acceptable therapeutic agent. Therefore, EGb 761 may have considerable significance in improving the efficiency of stem cell therapy for MI. However, due to the complexity of G. biloba composition, further investigation of the detailed mechanisms of specific components involved, particularly in animal ischemic hearts, need to be conducted in order to improve our understanding of the effects of G. biloba products.

In conclusion, the present study presents unique evidence that EGb 761 is able to ameliorate the decrease in heart function in a rat model with transplanted MSCs via enhancing the survival and differentiation of implanted MSCs, and attenuating inflammation and oxidative stress in the infarcted myocardial microenvironment.

Acknowledgements

This study was supported by grants from the National Nature Science Foundation of China (grant no. 31100838; http://www.nsfc.gov.cn).

References