The aim of the present study was to examine the protective effect of proanthocyanidins anoxia-reoxygenation injury of myocardial cells and its association with phosphatidylinositol-3-kinase/Akt and glycogen synthase kinase (PI3K/Akt/GSK)-3β and ATP-sensitive potassium channels. Neonatal rat myocardial cells were cultured and an anoxia-reoxygenation model was established following pretreatment with various drugs. The experiment was divided into five groups according to an experimental scheme. An MTT assay was used to examine the cell survival, and reactive oxygen species (ROS) levels and apoptosis were detected by flow cytometry. Myocardial apoptosis was also examined by terminal deoxynucleotidyl transferase dUTP nick end labeling staining and western blot analysis was employed to detect the expression levels of caspase-3, p-Akt and p-glycogen synthase kinase (GSK)-3β. The results revealed that myocardial cells in the anoxia-reoxygenation group (A/R) exhibited reduced survival rates, increased ROS levels and enhanced caspase-3 expression, as compared with the control group (CN; P<0.05). However, the increase in p-Akt and p-GSK-3β expression was not significantly different. In the proanthocyanidin pretreatment group (PC) the myocardial cell survival rate was increased, ROS levels were reduced, caspase-3 expression was decreased and p-Akt and p-GSK-3β expression levels were significantly increased as compared with the A/R group (P<0.05). Blockade of the PIK3/Akt channel by LY294002 eliminated the protective effects of proanthocyanidins and induced a significant decrease in p-Akt protein and p-GSK-3β expression levels as compared with the PC group. The inhibitor of mitochondrial ATP-sensitive potassium (mitoKATP) channels, 5-HD, also significantly suppressed the protective effects of proanthocyanidins, but had no evident impact on p-Akt and p-GSK-3β expression as compared with the PC group. In conclusion, pretreatment with proanthocyanidins had a protective effect on rat myocardial cell anoxia/reoxygenation injury. This effect is associated with the activation of the PI3K/Akt/GSK-3β signaling pathway and the opening of mitoKATP channels, which may have important roles downstream of PI3K.
Coronary atherosclerosis heart disease (coronary heart disease) is a leading cause of mortality in the 21st century, among which acute myocardial infarction (AMI) is one of the most serious clinical manifestations associated with high occurrence rate, high mortality and poor long-term prognosis (
Myocardial ischemia reperfusion injury is a highly complex process, and currently there are several key pathogenic mechanisms that are considered to be involved, including oxidative stress injury, intracellular calcium overload, cell apoptosis, cellular energy loss and activation of neutrophil inflammatory reaction (
Proanthocyanidins are highly efficient free radical scavengers, which are widely used in the clinic to delay senility, regulate blood fat and to reduce the development of atherosclerosis and tumor growth (
Sprague Dawley rats, aged 1–3 days, were provided by the Animal Center of Shandong University (Jinan, Shandong, China). This study was approved by the Ethics Committee of Shandong University (Jinan, China). Several regents were used, including Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen Life Technologies, Carlsbad, CA, USA), trypsin (Tiangen Biotech (Beijing) Co., Ltd., Beijing, China), dichlorofluorescein probe (Sigma, St. Louis, MO, USA), MTT proliferation detection kit (Sigma, San Jose, CA, USA), a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) kit (Tiangen Biotech (Beijing) Co., Ltd.), Annexin V-fluorescein isothiocyanate (FITC) cell apoptosis detection kit (Invitrogen Life Technologies), LY294002 and 5-hydroxy decanoic acid (5-HD; Sigma, CA, USA), mouse anti-rat Akt antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). Furthermore, mouse anti-rat glycogen synthase kinase (GSK)-3β and caspase-3 antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).
Isolation of neonatal cardiomyocytes is a technically more simple procedure than cell isolation from adult hearts, as it does not require aorta cannulation and perfusion. A two-step procedure was employed, consisting of enzyme digestion and mechanical agitation of the ventricular tissue followed by purification of the cardiomyocyte population. The enzymatic digestion and purification of cardiomyocytes was performed as previously described (
For the culture of neonatal myocardial cells, two methods were employed, including a re-differentiation and a rapid attachment method as previously described (
The experiment was started when the myocardial cells grew close to confluence, exhibiting synchronous growth cycles. Ischemia simulation solution components included NaH2PO4 0.9 mmol/l, NaHCO3 6.0 mmol/l, CaCl2 1.8 mmol/l, MgSO4 1.2 mmol/l, sodium lactate 40 mmol/l, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) 20 mmol/l, NaCl 98.5 mmol/l, KCl 10.0 mmol/l at pH 6.8. Furthermore, cells were presaturated in an atmosphere of 95% N2 and 5% CO2 for 1 h to create an oxygen pressure of P(O2)≤4.0 Kpa, to allow the cells to contain high concentrations of K+, lactic acid and H+, a low oxygen concentration, and no glucose or other energy substrates. Cultured cells were kept in this anoxia solution under anoxic conditions for 3 h. The solution was then replaced with reoxygenation solution containing NaH2PO4 0.9 mmol/l, NaHCO3 20 mmol/l, CaCl2 1.0 mmol/l, MgSO4 1.2 mmol/l, glucose 5.5 mmol/l, HEPES 20 mmol/l, NaCl 129.5 mmol/l, KCl 5.0 mmol/l, and the pH was adjusted to the reoxygenation condition of 95% oxygen saturation for 3 h.
The present study was divided into five groups according to an experimental scheme. The control group (CN) consisted of myocardial cells cultured under normal conditions. The anoxia-reoxygenation group (AR) was composed of the anoxia/reoxygenation injury model in myocardial cells subjected to anoxia for 3 h and reoxygenation for 3 h. In the proanthocyanidin (Shenfu Inc., Shanxi, China) pretreatment groups (PC), the culture medium was added with a final concentration of 100 mg/l of the proanthocyanidins and the cells were incubated for 2 h prior to exposure to anoxic and reoxygenation conditions. In the LY294002 (blocker of the PIK3/Akt channel) group, culture medium was added containing LY294002 at concentration of 15 μmol/l, proanthocyanidins were added 30 min later, then anoxia and reoxygenation were conducted. In the 5-HD [an inhibitor of mitochondrial ATP-sensitive potassium (mitoKATP) channels] group, the culture medium was added to 5-HD at a concentration of 100 μmol/l, proanthocyanidins were added 30 min later and then anoxia and reoxygenation were conducted.
ROS levels were measured by flow cytometry. Following treatment, cells were trypsinized and centrifuged (2 min at 20,000 × g), and the cell pellet was treated with 2′,7′-dichlorodihydrofluorescein diacetate stain (1:200), resuspended and incubated at 37°C for 20 min in the dark. Cells not treated with 2′,7′-dichlorodihydrofluorescein diacetate were used as negative controls and stained cells treated with 100 μl hydrogen peroxide (30% w/v hydrogen peroxide) incubated for 10 min served as positive controls. ROS levels were measured using a flow cytometer (Becton-Dickinson, Franklin Lakes, NJ, USA) and quantified by determining the mean fluorescence for each treatment.
Potential DNA fragmentation was examined by the TUNEL apoptosis detection kit (Chemicon, Temecula, CA, USA). The specific procedure of the TUNEL assay was performed as previously described (
Binding of annexin V-FITC and uptake of propidium iodide (PI) into the cells were assessed using a FACScan flow cytometer (Becton-Dickinson). Briefly, cells were harvested, resuspended and incubated with Annexin V-FITC and PI (5 μg/ml) in the dark at room temperature for 15 min. Fluorescence was measured through a 530/30 band filter (FL-1) to monitor Annexin V-FITC binding and through a 585/42 band filter (FL-2) to monitor PI uptake.
Western blotting was employed to analyze the expression levels of caspase-3, p-Akt, GSK-3β and p-GSK-3β. The assay was performed as previously described (
Data were expressed as the mean ± standard deviation (SD), variance analysis was used to compare multiple groups and the q test was used to analyze inter-group differences. Analysis was performed using SASS 6.12 statistical software (SPSS, Inc., Chicago, IL, USA) and P<0.01 was considered to indicate a statistically significant difference between values.
Myocardial cells were evidently impaired in the A/R group (41.33±1.45%), and the cell survival rate was significantly reduced as compared with that in the control group (91.95±2.27%; P<0.05). Cell survival rate in the PC group (75.64±2.01%), was significantly higher than that in the A/R group (P<0.05). The cell survival rate in the LY294002 group (46.56±3.3%) was significantly lower than that in the PC group (P<0.05) as compared with that in A/R group, where there were no significant differences. The cell survival rate in the 5-HD group (48.17±1.7%) was significantly lower than that in the PC group (P<0.05), while there was no significant difference from that in the A/R group. There was no significant difference between the cell survival rate in the LY294002 group and that in the 5-HD group (
In the A/R group (fluorescence intensity, 211.69), ROS levels were significantly increased as compared with those in the CN group (27.06; P<0.01). ROS levels were decreased in the PC group (80.11) as compared with those in the A/R group, which were significantly different (P<0.01). ROS levels were significantly increased in the LY294002 group (180.45) as compared with those in the PC group (P<0.01), while there was no significant difference from those in the A/R group. ROS levels increased significantly in the 5-HD group (168.35) as compared with those in the PC group (P<0.01); however, there was no significant difference from those in the A/R group. There were no significant differences between the ROS level of cells in the LY294002 and 5-HD groups (
As compared with the CN group (3.1±0.65%), the percentage of TUNEL-positive cells in the A/R group (25.3±2.64%) was significantly increased (P<0.05). As compared with the A/R group, TUNEL-positive cells in the PC group (10.2±1.59%) were significantly decreased (P<0.01). TUNEL-positive cells in the LY294002 group (18.6+1.79%) were significantly increased as compared with those in the PC group (P<0.01). TUNEL-positive cells in the 5-HD group (19.5±2.03%) were also significantly increased when compared with the PC group (P<0.01). There were no significant differences between the A/R and the 5-HD groups, between the LY294002 and 5-HD groups and between the LY294002 and A/R groups (
Flow cytometric analysis was used to detect the Annexin V/FITC and PI staining and determine apoptotic rates. In the
The levels of activated caspase-3 protein (cleaved caspase-3) reflected the degree of apoptosis in myocardial cells, which showed a positive correlation. From
p-Akt protein expression was moderately increased in the A/R group, but there was no significant difference when compared with the CN group (
p-GSK-3β protein expression was slightly increased in the A/R group, but there was no significant difference when compared with the CN group (
Oxidative stress is important in myocardial anoxia-reoxygenation injury, where excessive ROS production is derived from the mitochondrial electron transport chain, activated neutrophils and the enzyme xanthine oxidase (
When the mitochondrial outer membrane ruptures and the mitochondrial content, including apoptosis inducing factor (AIF) and cytochrome C, is released, this disrupts the electron transfer chain and directly induces cell death (
The PI3K/Akt/GSK-3β signalling pathway is the most important signal transduction pathway in myocardial ischemic pretreatment. The protective effect of ischemic pretreatment on the myocardium is predominantly due to the activation of the PI3K/Akt signal transduction pathway (
In the myocardial cells of the anoxia-reoxygenation model, the addition of diazoxide prior to cell anoxia reduced intracellular calcium overload, mitochondrial membrane potential and oxidative stress induced-apoptosis. By contrast, the addition of the mitoKATP channel blocker 5-HD eliminated this protective effect (
Previous studies have identified that proanthocyanidins have a protective effect on myocardial ischemia reperfusion. Sato
In conclusion, pretreatment with proanthocyanidins had a protective effect on myocardial cell anoxia-reoxygenation injury in rat cells. This effect was associated with the activation of the PI3K/Akt/GSK-3β signaling pathway and the opening of mitoKATP channels, which may have an important role downstream of the PI3K pathway.
This study was supported by a grant from the Independent Innovation Foundation of Shandong University (grant no. 2012TS153).
Cell viability in groups (n=10, mean±standard deviation). *P<0.05 for A/R compared with the CN group; $P<0.05 for PC compared with the A/R group and #P<0.05 for LY294002 (blocker of the PIK3/Akt channel) and 5-HD (inhibitor of mitochondrial ATP-sensitive potassium channels) compared with the PC group. CN, control group; A/R, anoxia-reoxygenation group; PC, the proanthocyanidins pretreatment group; 5-HD, 5-hydroxy decanoic acid; PIK3, phosphatidylinositol-3-kinase.
ROS levels in each group (n=10, ±s). (A) CN group; (B) A/R group; (C) PC group; (D) LY294002 (blocker of the PIK3/Akt channel) group; (E) 5-HD (inhibitor of mitoKATP channels) group. (F) Statistical analysis of the ROS levels in each group. *P<0.05 for A/R compared with the CN group; $P<0.05 for PC compared with the A/R group and #P<0.05 for LY294002 and 5-HD compared with the PC group. CN, control group; A/R, anoxia-reoxygenation group; PC, the proanthocyanidin pretreatment group; 5-HD, 5-hydroxy decanoic acid; PIK3, phosphatidylinositol-3-kinase.
Analysis of TUNEL-positive cells in each group. (A) CN group; (B) A/R group; (C) PC group; (D) LY294002 (blocker of the PIK3/Akt channel) group; (E) 5-HD (inhibitor of mitoKATP channels) group. Magnification, ×200. (F) Statistical analysis of the TUNEL-positive cells. *P<0.05 for A/R compared with the CN group; $P<0.05 for PC compared with the A/R group and #P<0.05 for LY294002 and 5-HD compared with the PC group. CN, control group; A/R, anoxia-reoxygenation group; PC, the proanthocyanidins pretreatment group; 5-HD, 5-hydroxy decanoic acid; PIK3, phosphatidylinositol-3-kinase; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.
Flow cytometric analysis of Annexin V/FITC and PI double staining. (A) CN group; (B). A/R group; (C) PC group; (D) LY294002 (blocker of the PIK3/Akt channel) group; (E) 5-HD (inhibitor of mitoKATP channels) group. (F) Statistical graph of annexin V/FITC and PI staining. *P<0.05 for A/R compared with the CN group; $P<0.05 for PC compared with the A/R group and #P<0.05 for LY294002 and 5-HD compared the with PC group. PI, propidium iodide; CN, control group; A/R, anoxia-reoxygenation group; PC, the proanthocyanidins pretreatment group; 5-HD, 5-hydroxy decanoic acid; PIK3, phosphatidylinositol-3-kinase; FITC, fluorescein isothiocyanate.
Western blot analysis results of cleaved caspase-3 in each group. (A) Western blot analysis. (B) Statistical graph of western blotting bands. The relative value of each preparation is calculated by each gray numerical value of specific product vs that of β-actin. *P<0.05 for A/R compared with the CN group; $P<0.05 for PC compared with the A/R group and #P<0.05 for LY294002 (blocker of the PIK3/Akt channel) and 5-HD (inhibitor of mitoKATP channels) compared with the PC group. CN, control group; A/R, anoxia-reoxygenation group; PC, the proanthocyanidins pretreatment group; 5-HD, 5-hydroxy decanoic acid; PIK3, phosphatidylinositol-3-kinase.
p-Akt and p-GSK-3β expression in each group. (A) p-Akt expression and statistical analysis. (B) p-GSK-3β expression and statistical analysis. The relative value of each preparation was calculated by each gray numerical value of specific product vs that of β-actin. *P<0.05 for A/R compared with the CN group and **P<0.01 for LY294002 (blocker of the PIK3/Akt channel) compared with the PC group. GSK, glycogen synthase kinase; CN, control group; A/R, anoxia-reoxygenation group; PC, the proanthocyanidins pretreatment group; 5-HD, 5-hydroxy decanoic acid; PIK3, phosphatidylinositol-3-kinase.