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American Journal of Translational Research logoLink to American Journal of Translational Research
. 2016 Jun 15;8(6):2534–2548.

JNK/PI3K/Akt signaling pathway is involved in myocardial ischemia/reperfusion injury in diabetic rats: effects of salvianolic acid A intervention

Qiuping Chen 1,*, Tongda Xu 2,*, Dongye Li 1,2, Defeng Pan 1, Pei Wu 1, Yuanyuan Luo 2, Yanfeng Ma 2, Yang Liu 1
PMCID: PMC4931149  PMID: 27398138

Abstract

Recent studies have demonstrated that diabetes impairs the phosphatidylinositol 3-kinase/Akt (PI3K/Akt) pathway, while insulin resistance syndrome has been associated with alterations of this pathway in diabetic rats after ischemia/reperfusion (I/R), and activation of C-jun N-terminal kinase (JNK) is involved. The present study was designed to investigate whether inhibiting JNK activity would partially restore the PI3K/Akt signaling pathway and protect against myocardial I/R injury in diabetic rats, and to explore the effect of intervention with salvianolic acid A (Sal A). The inhibitor of JNK (SP600125) and Sal A were used in type 2 diabetic (T2D) rats, outcome measures included heart hemodynamic data, myocardial infarct size, the release of lactate dehydrogenase (LDH), SERCA2a activity, cardiomyocyte apotosis, expression levels of Bcl-2, Bax and cleaved caspase-3, and the phosphorylation status of Akt and JNK. The p-Akt levels were increased after myocardial I/R in non-diabetic rats, while there was no change in diabetic rats. Pretreatment with the SP600125 and Sal A decreased the p-JNK levels and increased the p-Akt levels in diabetic rats with I/R, and heart hemodynamic data improved, infarct size and LDH release decreased, SERCA2a activity increased, Bax and cleaved caspase-3 expression levels decreased, and the expression of Bcl-2 and the Bcl-2/Bax ratio increased. Our results suggest that the JNK/PI3K/Akt signaling pathway is involved in myocardial I/R injury in diabetic rats and Sal A exerts an anti-apoptotic effect and improves cardiac function following I/R injury through the JNK/PI3K/Akt signaling pathway in this model.

Keywords: Diabetic rats, ischemia/reperfusion, JNK, PI3K/Akt signaling pathway, Sal A

Introduction

Epidemiological data and clinical studies have demonstrated that diabetes is a major risk factor for cardiovascular events. Statistically, ischemic heart disease is twice as common in diabetic as in non-diabetic patients [1]. Diabetic patients are more liable to myocardial ischemia/reperfusion (I/R) injury than non-diabetic patients [2,3]. Therefore, prevention and treatment of myocardial I/R injury in diabetes has become a global challenge. Animal experiments have confirmed that myocardial apoptosis is higher in diabetic rats than in non-diabetic rats after I/R injury [4,5].

The PI3K/Akt pathway has important biological functions in cell proliferation, survival and apoptosis. Previous studies in our laboratory have revealed that activation of this pathway improves cardiac contractility and decreases cardiomyocyte apoptosis, thus ameliorating I/R injury in non-diabetic rats [6]. It is well known that diabetes could impair PI3K/Akt pathway, and some pharmacological agents and ischemic preconditioning could decrease I/R injury. But they appear to be less effective in diabetes [7-9]. Insulin resistance syndrome has been associated with alterations in Akt phosphorylation in diabetic rats after I/R [10]. Insulin resistance enhances the production of inflammatory factors, endoplasmic reticulum stress and oxidative stress, Both conditions involve in activation of C-jun N-terminal kinase (JNK), a member of the mitogen-activated protein kinase (MAPK) family in the myocardium and other tissues. A large body of literature indicates that JNK is involved in the formation of insulin resistance [11,12]. Activation of JNK increases serine phosphorylation of insulin receptor substrate 1 (IRS-1), decreases tyrosine phosphorylation, and decreases Akt phosphorylation in liver [13,14]. Shah et al. have reported that the novel agent osmotin attenuated glutamate-induced synaptic dysfunction and neurodegeneration via the JNK/PI3K/Akt pathway in postnatal rat brain [15]. The heart is an also insulin-sensitive organ, and its failure to receive normal insulin signals is deleterious. In this context, it is uncertain whether activated JNK impairs the PI3K/Akt pathway after myocardial I/R injury in diabetic rats.

A large body of evidence demonstrate that traditional Chinese medicines rich in salvianolic acid are effective in protecting the myocardium against I/R injury [16-18]. Salvianolic acid A (Sal A, (2R)-3-(3,4-dihydroxyphenyl)-2-[(E)-3-[2-[(E)-2-(3,4-dihydroxyphenyl) ethenyl]-3,4-dihydroxyphenyl] prop-2-enoyl] oxypropanoic acid ) is a water-soluble ingredient of Salvia miltiorrhiza, which has been reported to exert a wide range of pharmacological effects, including anti-inflammatory, anti-platelet aggregation, antioxidant, anticarcinogenic, and anti-fibrotic [19,20]. Investigations in our laboratory have demonstrated that Sal A displays cardioprotective effects in non-diabetic rat hearts following myocardial I/R injury [21]. However, to date, little is known about the effects of Sal A on diabetic rats after myocardial I/R injury.

Therefore, the aims of the current study were (1) to determine whether inhibiting the activity of JNK partially could restore the PI3K/Akt signaling pathway and act against myocardial I/R injury in diabetic rats, and if so, (2) to elucidate whether Sal A intervention exerts cardioprotection against myocardial I/R injury via JNK/PI3K/Akt signaling pathway.

Materials and methods

Experimental animals

This experimental design was reviewed and approved by the Animal Ethics Committee of Xuzhou Medical University. Male Sprague-Dawley rats (160-180 g) were provided by the Experimental Animal Center in Xuzhou Medical University.

Induction of type 2 diabetes (T2D) in rats

T2D was induced by a high-fat diet combined low-dose streptozotocin as previously described [13]. Briefly, the rats were randomly allocated to two dietary regimens by feeding either normal pellet diet (NPD) or high-fat diet (HFD 58% fat, 25% protein and 17% carbohydrate, as a percentage of total kcal) for two weeks. After this dietary manipulation, the HFD rats were injected intraperitoneally with a single dose of STZ (35 mg kg-1), while the normal group rats received an equal volume of the vehicle, citrate buffer (pH 4.4). Seven days after the STZ injection, the animals with non-fasting plasma glucose (PGL) ≥16.7 mmol/L were considered to be T2D rats. In order to maintain the stability of the model, the rats were allowed to continue to feed on their respective diets for two weeks prior to use in the following experiments.

Isolated heart perfusion

The hearts of all the rats were isolated and perfused as previously described [22,23]. Briefly, after 20 min of heparinization, the rats were anesthetized with sodium pentobarbital (150 mg/kg) by intraperitoneal injection. The hearts were rapidly excised and immediately mounted and perfused on a Langendorff device. The perfusate was a modified Krebs-Henseleit buffer containing (in mM): 120 NaCl, 4.7 KCl, 1.2 KH2PO4, 1.2 MgSO4, 25 NaHCO3, 11 glucose and 1.25 CaCl2, pH 7.4, saturated with 95% O2/5% CO2 at 37°C. Perfusion was maintained at a constant pressure of 80 mmHg. When all the hearts including those of the control group had equilibrated, they were subjected to 30 min global ischemia and 120 min reperfusion, and then allocated as follows: (1) Ctrl-Sham (n=6); (2) Ctrl-I/R (n=6); (3) Ctrl-Sal A+I/R (n=6); (4) Diab-Sham (n=6); (5) Diab-I/R (n=6); (6) Diab-Sal A+I/R (n=6). In the groups receiving Sal A (groups 3 and 6), Sal A (20 μM) was injected via a side pipe just proximal to the heart cannula, as previously described [21].

To test whether Sal A inhibited JNK phosphorylation and protected the myocardium in diabetic rats hearts, the JNK inhibitor (SP600125) was added to perfusate prior to I/R procedure described above and the rat hearts were divided into five groups: (1) Diab-Sham (n=6); (2) Diab-I/R (n=6); (3) Diab-Sal A+I/R (n=6); (4) Diab-SP+I/R (n=6), (5) Diab-SP+Sal A+I/R (n=6). The hearts were perfused with SP at the concentration of 10 μM for 30 min before administration of Sal A, then subjected to the same course as the Diab-Sal A+I/R group.

Hemodynamic parameters

To determine left ventricular systolic and diastolic function, a fluid-filled latex balloon connected to a Millar transducer (pressure sensor) was inserted into the left ventricle as described previously [24]. The parameters of cardiac function were continuously simultaneously monitored and the entire I/R procedure was recorded using a Biopac system. During the procedure, heart rate (HR), left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP) and ±dp/dtmax were recorded. The left ventricular function parameters were calculated using a hemodynamic analysis system (Chengdu, China).

LDH activity in coronary effluent

Samples were collected from the coronary effluent after 10 min reperfusion. LDH activity was measured spectrophotometrically and calculated using a commercially available assay kit (Jiancheng Bioengineering Institute; Nanjing, China).

Infarct size

Myocardial infarct size was measured by triphenyltetrazolium chloride (TTC, Sigma-Aldrich) staining as previously described [25]. After 2 h of reperfusion, the hearts were removed from the apparatus and the left ventricles were sectioned from apex to base into five to six 1-mm sections. The sections were incubated in 1% TTC in phosphate buffer, pH 7.4, at 37°C for 20 min and then fixed in 10% formalin for 1 h. The normal zone was stained red by TTC while the infarct region (which did not take up the TTC stain) remained gray. The myocardial tissue was weighed and the percentage ratios of infarcted regions in the left ventricles were calculated.

Myocardial apoptosis

A terminal deoxynucleotidyl nick-end labeling (TUNEL) kit (Roche; Switzerland) was used to detect myocardial apoptosis, using at least three hearts in each group. In brief, after the 2 h reperfusion, LV tissues were fixed in formalin and stored at 4°C. The tissues were then embedded in paraffin and cut into 5 μm sections, which were TUNEL-stained according to the manufacturer’s instructions. Then 4’,6-diamidino-2-pheny-lindole (DAPI, Sigma-Aldrich, USA) was used to stain all the cardiomyocyte nuclei. The cells were examined by light microscopy; 10 fields containing at least 50 cells were viewed at ×400 magnification. In each field the nuclei were counted, and the percentage of apoptotic nuclei stained dark brown (TUNEL-positive nuclei) was calculated.

Sarcoplasmic reticulum Ca2+-ATPase (SERCA2a) activity

SERCA2a activities were measured using an ELISA kit (Jiancheng Bioengineering Institute; Nanjing, China), in which sarcoplasmic reticulum calcium pump activity was measured colorimetrically as the amount of inorganic phosphate released from ATP. Myocardial sarcoplasmic reticula were prepared by a modified differential centrifugation method previously described by Kodavanti et al. [26-28]. The sarcoplasmic reticula were separated from the homogenates and the protein concentrations were determined by the Bradford method. Each sarcoplasmic reticulum preparation was suspended in (each component in mmol/L): 50.0 histidine, 3.0 MgCl2, 100.0 KCl, 5.0 NaN3, 3.0 ATP and 50 CaCl2, pH 7.0, and incubated for 10 min at 37°C. Tris-ATP (3 mmol/L) was added to initiate the reaction and the suspension was incubated at 4°C in a water bath for 20 min. The data from three replicate measurements of SERCA2a activity were expressed as μmol inorganic phosphate released per h per mg protein.

Western blotting

Hearts were collected at the end of the 2 h of reperfusion and the left ventricular tissue was frozen in liquid nitrogen before being stored at -80°C. Approximately 100 mg of ventricular tissue was used for protein extraction. After centrifugation at 20,000 g for 20 min at 4°C, the supernatant was collected and protein was quantified using a modified Bradford assay (Bio-Rad, CA, USA). Aliquots of the supernatants containing equal amounts of protein (40 μg) were electrophoresed on SDS-PAGE gels and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were treated with primary antibodies against Bcl-2 and Bax (1:500, Santa Cruz, USA), cleaved caspase-3, phospho-Akt, Akt, phospho-JNK, JNK (1:1000; Cell Signaling Technology, MA, USA), and β-actin (1:1000; Zhongshan; Beijing, China). This step was followed by incubation with the corresponding secondary antibodies (1:1000; Zhongshan; Beijing, China). Protein bands were visualized by nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl-phosphate. The band densities were quantified by the Image J 3.0 system (National Institutes of Health; MD, USA).

Statistical analysis

All values are expressed as means ± SEM. GraphPad Prism 5 software was used for statistical analysis. One-way and two-way analysis of variance (ANOVA) was conducted across all groups, followed by a Bonferroni post-hoc correction for all group comparisons. For the Langendorff data, ANOVA for repeated measurements was used. Values of p<0.05 were considered statistically significant.

Results

Characteristics of the fat-fed/STZ-induced T2D rats

The success of the fat-fed, STZ-induced T2D rats model was confirmed by checking for PGL and plasma insulin (PI) as previously described [13]. PGL was markedly higher in the HFD+STZ-treated group than that of the NPD-treated group (p<0.001). Body weights and PI levels were not significantly different between the groups (Table 1).

Table 1.

Effect of body weight, plasma glucose and plasma insulin in rats

Parameters Weight body (g) PGL (mmol/L) PI (ng/ml)
NPD (n=36) 247.00±5.36 7.95±0.30 2.33±0.25
HFD+STZ (n=60) 255.00±4.36 23.50±0.51*** 2.29±0.11
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mean±S.E.M.

***

P<0.001 versus NPD.

NPD: normal pellet diet; HFD: high-fat diet; STZ: streptozotocin; PGL: plasma glucose; PI: plasma insulin.

Effects of Sal A pretreatment on diabetic and non-diabetic rats

Hemodynamic data

There was no significant difference in HR, LVSP, LVEDP or ±dp/dtmax between the Diab and Ctrl groups at baseline (Figure 1). However, after I/R, HR, LVSP and ±dp/dtmax were decreased and LVEDP was increased significantly in both groups. The hemodynamic values were significantly higher in the Diab than those of the Ctrl group (p<0.05). Pretreatment with Sal A significantly improved cardiac systolic/diastolic function and increased HR before I/R in both groups.

Figure 1.

Figure 1

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Effects of Sal A on the parameters of hemodynamics during a reperfusion period in isolated rat heart preparations (A-E). The results were expressed as the mean ± SEM, n = 6. *P<0.05. Ctrl versus Diab; #P<0.05, ##P<0.01, ###P<0.001 I/R versus Sham; $P<0.05 Sal A (20 μM)+I/R versus I/R.

LDH activity in coronary effluent and Infract size

LDH activity was significantly higher in the Diab than that of the Ctrl group (p<0.05). I/R significantly increased the release of LDH in the Ctrl group (222.00±9.07 i.u.l-1 in Ctrl-I/R vs 51.51±5.56 i.u.l-1 in Ctrl-Sham; p<0.01) and in the Diab group (281.40±7.01 i.u.l-1 in Diab-I/R vs 94.46±3.468 i.u.l-1 Diab-Sham; p<0.01). Sal A significantly decreased LDH activity in the Ctrl group (158.80±5.18 in Ctrl-Sal A+I/R vs 222.00±9.07 in Ctrl-I/R; p<0.05) and the Diab group (224.1±4.47 i.u.l-1 in Diab-Sal A+I/R vs 281.40±7.01 i.u.l-1 in Diab-I/R; p<0.05) (Figure 2A).

Figure 2.

Figure 2

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LDH release and Infarct size quantity in I/R rat hearts. Effects of SalA on release of LDH (A) and Infarct size (B, C) in isolated I/R rat hearts. The results were expressed as the mean ± SEM, n=6. *P<0.05 Ctrl versus Diab; #P<0.05, ##P<0.01, ###P<0.001 I/R versus Sham; $P<0.05 SalA (20 μM)+I/R versus I/R.

Infarct size was expressed as the percentage ratio of infarct area/total area. As illustrated in Figure 2B and 2C, there was a significant difference between the Ctrl and Diab groups (p<0.05). I/R significantly increased infarct size in both the Ctrl (44.96±2.16% in Ctrl-I/R vs 0.00±0.00% in Ctrl-Sham; p<0.01) and Diab (55.42±1.81% in Diab-I/R vs 0.00±0.00% in Diab-Sham; p<0.001) groups. Sal A administration significantly decreased infarct size at 2 h (25.36±0.74% in Ctrl-Sal A+I/R vs 44.96±2.16% in Ctrl-I/R; p<0.05; 35.06±2.02% in Diab-Sal A+I/R vs 55.42±1.81% in Diab-I/R; p<0.05) after I/R.

Apoptosis

The apoptosis rates were significantly higher in the Diab group than those of the Ctrl group (p<0.05). Representative photomicrographs revealed TUNEL-positive cardiomyocytes more frequently in the I/R groups (21.67±2.18% in Ctrl-I/R vs 4.33±2.33% in Ctrl-Sham, 33.67±1.20% in Diab-I/R vs 9.00±1.58% in Diab-Sham; p<0.01). Sal A decreased the number of TUNEL-positive cells (13.67±1.88% in Ctrl-Sal A+I/R vs 21.67±2.82% in Ctrl-I/R; 22.33±3.33% in Diab-Sal A+I/R vs 33.67±3.20% in Diab-I/R; p<0.01) after I/R (Figure 3A and 3B).

Figure 3.

Figure 3

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Cell apoptosis rate and the expression of p-Akt, p-JNK. A. TUNEL assay. Total nuclei labeled by DAPI (blue). Apoptotic nuclei detected by TUNEL staining (brown). B. The rate of apoptosis (%) of each group (n = 6 sections/group). C, E. Representative Western blot for p-JNK and p-Akt. D, F. Bar diagrams showing the increased fold in p-JNK and p-Akt. Data represent the mean ± SEM, n=6. *P<0.05, **P<0.01 Ctrl versus Diab; #P<0.05, ##P<0.01 I/R versus Sham; $P<0.05 SalA+I/R versus I/R.

Akt and JNK phosphorylation

There were no differences in total Akt and JNK levels among the study groups. Therefore, p-Akt and p-JNK levels were expressed as densitometric levels normalized to total protein. The expression level of Akt phosphorylation in the Ctrl group was increased after I/R (p<0.05), and the value of p-Akt was significantly higher after pretreatment with Sal A than that of the I/R group (p<0.05). In contrast, compared with Diab-Sham, I/R had no effect on the expression of Akt. However, pretreatment with Sal A increased the p-Akt levels after I/R (p<0.05) (Figure 3E and 3F). At the same time, the p-JNK levels in the diabetic group were higher than those in the Ctrl group (p<0.05). I/R significantly activated p-JNK expression in both the Ctrl and Diab groups (p<0.01), but this trend was partially reversed by Sal A (p<0.05) (Figure 3C and 3D).

Effects of SP600125 and Sal A on the function of isolated hearts and SERCA2a activity in diabetic rats subjected to I/R

As Figure 4A-E indicated, HR, LVSP and ±dp/dtmax were significantly lower and LVEDP was higher in the I/R than those of the sham group (p<0.001). Consistently, pretreatment with either SP600125 or Sal A before I/R improved myocardial function as evidenced by higher HR, LVSP and ±dp/dtmax and lower LVEDP than those in the I/R group (p<0.05). In addition, administration of SP600125 before Sal A markedly improved these parameters of myocardial function in diabetic rats (p<0.05).

Figure 4.

Figure 4

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SP600125 and Sal A improved Myocardial Function and SERCA2a activity after I/R in diabetic rats. A-E. Effects of SP600125 and Sal A on various parameters of hemodynamics during a reperfusion period in isolated rat heart preparations. F. Effects of SP600125 and Sal A on SERCA2a activity after ischemia/reperfused myocardium in diabetic rats. The results were expressed as the mean ± SEM, n=3. #P<0.05, ##P<0.01, ###P<0.001 versus Diab-Sham; $P<0.05, $$P<0.01 versus Diab-I/R; ΔP<0.05 versus Diab-Sal A (20 μM)+I/R; &P<0.05 versus Diab-SP (10 μM)+I/R.

SERCA2a activity (Figure 4F) was significantly lower in the I/R group than that of the Sham group (5.62±0.16 vs 1.77±0.15; p<0.001). Pretreatment with either SP600125 or Sal A before I/R led to higher SERCA2a activities than in the I/R group (3.47±0.14, 3.49±0.18 vs 1.77±0.15 μmol Pi/mgprot/h; p<0.05). The protective effect of combining Sal A with SP600125 was markedly enhanced, as demonstrated by the comparison between the Sal A+I/R and SP+I/R groups (4.38±0.135 vs 3.49±0.18, 3.47±0.14; p<0.05).

Effects of SP600125 and Sal A on LDH activity in coronary effluent and infarct size in diabetic rats

After pretreatment with SP600125 or Sal A, LDH release was significant lower in the I/R group (210.2±4.17, 224.1±4.47 vs 281.40±7.01; p<0.05). However, co-administration of SP600125 and Sal A before I/R led to a statistically lower release of LDH than in the SP+I/R and Sal A+I/R groups (161.80±5.39vs 210.2±4.17, 224.1±4.47; p<0.05) (Figure 5A).

Figure 5.

Figure 5

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SP600125 and Sal A decreases the LDH release and Infarct size in diabetic rats. Sal A and SP600125 reduced the LDH release (A) and Infarct size (B, C) compared with the I/R group, Administration of SP600125 before SalA markedly alleviated the LDH release and Infarct size compared with Sal A+I/R and SP+I/R. The results were expressed as the mean ± SEM, n=6. #P<0.05, ##P<0.01, ###P<0.001 versus Diab-Sham; $P<0.05 $$P<0.01 versus Diab-I/R; ΔP<0.05 versus Diab-SalA (20 μM)+I/R; &P<0.05 versus Diab-SP (10 μM)+I/R.

Representative images of infarct size as revealed by TTC staining are presented in Figure 5B and 5C. SP600125 or Sal A administration decreased significantly infarct size (31.26±1.53%, 35.06±2.02% vs 55.42±1.81%; p<0.05) after I/R than that in the I/R group, and the infarct size was smaller in the SP+Sal A+I/R group than that of the SP+I/R or Sal A+I/R groups respectively (22.33±1.45% vs 31.26±1.53%, 35.06±2.02%; p<0.05).

Effects of SP600125 and Sal A on cell apoptosis, p-Akt and p-JNK protein expression in diabetic rats

As illustrated in Figure 6A and 6B, there were much more TUNEL-positive cardiomyocytes after I/R (33.67±1.20% vs 9.00±0.58%; p<0.01) than those in the Sham group. However, SP600125 or Sal A decreased I/R-induced cardiomyocyte apoptosis (21.67±1.45%, 22.33±0.33% vs 33.67±1.20%; p<0.05). The combination of SP600125 and Sal A led to a further significant reduction in cardiomyocyte apoptosis (p<0.01). The results of Western blotting demonstrated that the p-JNK levels were increased after I/R (p<0.01), while the p-Akt level had no significant difference. After pretreatment with Sal A, the p-JNK level was lower (p<0.05) and the p-Akt level higher (P<0.05) than those in the I/R group. The p-JNK levels in the SP+I/R group were higher than those in the Sal A+I/R group (p<0.05). However, there was no difference for p-Akt levels between the Sal A+I/R and SP+I/R groups. Pretreatment with both SP600125 and Sal A had no significant effect on the p-JNK levels but p-Akt expression was significantly higher than in the SP+I/R group (p<0.05) (Figure 6C-F).

Figure 6.

Figure 6

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SP600125 and Sal A decreases apoptosis and the expression of p-Akt, p-JNK in diabetic rats with I/R. A, B. Pretreatment with SalA or SP600125 reduced the percentage of apoptotic cells, Administration of SP600125 before SalA markedly alleviated the cell apoptosis compared with Sal A (20 μM)+I/R and SP (10 μM)+I/R. C, E. Representative Western blot for p-JNK and p-Akt. D, F. Bar diagrams showing the increased fold in p-JNK and p-Akt. The results were expressed as the mean ± SEM, n=6. #P<0.05,##P<0.01 versus Diab-Sham; $P<0.05, $$P<0.01 versus Diab-I/R; ΔP<0.05 versus Diab-SalA (20 μM)+I/R; &P<0.05 versus Diab-SP (10 μM)+I/R.

Effects of SP600125 and Sal A on Bcl-2, Bax and cleaved caspase-3

We measured the expression levels of the antiapoptotic protein Bcl-2 and the apoptotic proteins Bax and cleaved caspase-3. As demonstrated in Figure 7A-E, the results of Western blotting also revealed that SP600125 or Sal A pretreatment decreased Bax and cleaved caspase-3 expression, while Bcl-2 expression was increased, resulting in an increased Bcl-2/Bax ratio in cardiac tissue exposed to I/R injury (P<0.05). Pretreatment with SP600125 and Sal A led to a further significant reduction in Bax and cleaved caspase-3 expression, while Bcl-2 expression and the Bcl-2/Bax ratio were increased (p<0.05).

Figure 7.

Figure 7

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Sal A decreases the expression of pro-apoptotic proteins and increases the antiapoptotic proteins in diabetic rats with I/R. (A) Western blot analysis of activated Cleaved caspase-3, Bcl-2, and Bax protein in myocardial tissues. Quantitative analysis of (B) Cleaved caspase-3, (C) Bcl-2, and (D) Bax Western blots. (E) The ratio of Bcl-2/Bax.The results were expressed as the mean ± SEM, n=3. #P<0.05, ##P<0.01, ###P<0.001 versus Diab-Sham; $P<0.05, $$P<0.01 versus Diab-I/R; ΔP<0.05 versus Diab-SalA (20 μM)+I/R; &P<0.05 versus Diab-SP (10 μM)+I/R.

Discussion

The reperfusion injury salvage kinase pathway (RISK) includes the phosphatidylinositol 3-kinase/Akt (PI3K/Akt) and extracellular signal regulated kinases (ERK 1/2), both of which confer protection against I/R injury [29,30]. Several studies have revealed that the PI3K/Akt pathway is impaired in diabetic rats after myocardial I/R. Yin et al. demonstrated that diabetes inhibited the myocardial protective effects mediated by preconditioning and postconditioning during I/R [31]. Miki et al. reported that Akt phosphorylation by EPO and phosphorylation of GSK-3β by ERK were impaired in diabetes [32]. Ghaboura et al. demonstrated that the PI3K/Akt pathway in diabetic rats was altered in insulin resistance and hyperglycemia [8]. Our results demonstrated that the p-Akt level was increased after myocardial I/R in non-diabetic rats, while there was no difference in diabetic rats. Meanwhile, cardiomyocyte apoptosis, LDH leakage, contractile function and infarction size were worser significantly in diabetic than those of non-diabetic rats. These results suggest that an impaired PI3K/Akt signaling pathway made the diabetic myocardium more vulnerable to I/R injury.

Furthermore, the p-JNK expression levels were significantly increased in diabetic cardiomyopathy, especially in myocardial injury induced by I/R. Insulin resistance and hyperglycemia are closely associated with type 2 diabetes. While the molecular mechanisms of insulin resistance are multiple, recent evidence suggests that attenuation of insulin signaling by JNK could be a central part of the pathobiology of insulin resistance [33]. Taniguchi et al. have demonstrated that endoplasmic reticulum stress could inhibit the PI3K/Akt signaling pathway by increasing the activity of JNK in insulin resistance in liver cells [34]. Masharani et al. found that JNK activation could lower the PI3K/Akt activity during insulin resistance in skeletal muscle [35]. Jia et al. have demonstrated that (-)-epigallocatechin-3-gallate (EGCG) inhibited TNF-α/JNK signaling and increased Akt phosphorylation in the hippocampus of APP/PS1 mice [36]. Therefore, we speculates that JNK activation impairs PI3K/Akt signaling in the diabetic myocardium after I/R. To confirm this hypothesis, the JNK inhibitor SP600125 was utilized in our study. The expression of p-JNK was significantly lower and that of p-Akt significantly higher in diabetic rats than those in the I/R group. We also found that SP600125 effectively attenuated I/R-induced cardiomyocyte apoptosis, LDH leakage and infarct size, increased the Bcl-2/Bax ratio and decreased cleaved caspase-3 expression. It also improved the systolic/diastolic function and SERCA2a activity in diabetic rats, which suggested that inhibiting the activation of JNK led to a partial recovery of PI3K/Akt activity to improve cell apoptosis and myocardial contractility in diabetic rats.

Currently there is a lack of effective drugs to inhibit myocardial I/R injury in clinical practice, so the search for a drug conferring resistance to I/R injury is a top priority. Sal A is one of the main active, water-soluble components in Salvia miltiorrhizae, which has been widely used in Asian countries for treating various conditions including cerebrovascular disease, coronary artery disease, and myocardial infarction.

Previous work in our laboratory demonstrated that pretreatment with Sal A could confer protective effects on the myocardium during I/R in non-diabetic rats. To the best of our knowledge, Sal A-induced cardioprotection in diabetic I/R injury has never been tested. Our results demonstrated that the heart hemodynamic data and LDH activity were lower in the diabetic group than those of the Ctrl group after I/R. Infarct size and myocardial apoptosis rate were higher in the Diab group than those of the Ctrl group, while these indexes were partially reversed by Sal A pretreatment in both groups. This suggests that Sal A provides effective cardioprotection against myocardial I/R injury in diabetic rats. As with SP600125, Sal A effectively attenuated I/R-induced cardiomyocyte apoptosis, LDH leakage and infarct size, increased the SERCA2a activity and Bcl-2/Bax ratio, and decreased cleaved caspase-3 expression in diabetic rats. However, the combination of Sal A and SP600125 was more effective than either agent alone on the diabetic myocardium after I/R injury. These data indicated that other signaling molecules could be involved in the protection against myocardial I/R injury by Sal A and additional investigations are required.

Among the numerous signaling pathways involved in regulating cell survival, the PI3K/Akt pathway is crucial for protecting the myocardium from I/R injury [37,38]. SERCA2a is a central regulator of cardiac function, and its overexpression can enhance cardiac contraction and relaxation [39]. Fang et al. demonstrated that luteolin increased the expressions of phospho-Akt, phospho-PLB and SERCA2a, but all three were markedly decreased by pretreatment with the Akt inhibitor LY294002 [6]. Chen et al. demonstrated that endogenous H2S transiently and reversibly inhibits SR Ca2+ uptake in rat heart SR because of the downregulated SERCA2a activity associated with PLB phosphorylation by the PI3K/Akt pathway [40]. Rodent models of both type 1 and type 2 diabetes display reduced SERCA2a function, lengthened relaxation times, and impaired contractility [41], while SERCA2a activity was markedly decreased after myocardial I/R injury [42]. In line with our results concerning the diabetic myocardium, Sal A markedly increased the SERCA 2a activity via JNK/PI3K/Akt signaling and improved systolic/diastolic function in diabetic rats.

The pathogenesis of I/R injury is apparently multifactorial, and myocardial apoptosis is one of the major underlying pathogenic mechanisms [43,44]. Most studies have suggested that Bcl-2 family members are key regulators of physiological and pathological apoptosis. It has been demonstrated that a high Bax/Bcl-2 ratio is associated with greater vulnerability to apoptosis [45,46]. Shi et al. demonstrated that Bilobalide prevents apoptosis through activation of the PI3K/Akt pathway in SH-SY5Y cells [47]. Sun et al. indicated that by suppressing cardiomyocyte apoptosis through Akt-dependent mechanisms, acute ischemic injury in the heart in diabetic rats could be ameliorated [48]. Our present results clearly demonstrate that pretreatment with Sal A, via the JNK/Akt signaling pathway, significantly increased the levels of the anti-apoptotic protein Bcl-2, decreased the pro-apoptotic proteins Bax and cleaved caspase-3, and increased the Bcl-2/Bax ratio against myocardial I/R injury in diabetic rats.

In the present study, we provided direct ex vivo evidence that SP600125 inhibits JNK activation and partly restores PI3K/Akt signaling against I/R injury in T2D rats, as evidenced by the significantly improved cardiac function and reduced myocardial apoptosis. Our findings also demonstrated that Sal A attenuates myocardial I/R injury in diabetic rats, possibly through the JNK/PI3K/Akt signaling pathway.

Our study has some limitations. First, we employed a high-fat diet combined with low-dose streptozotocin as a model of T2D in rats to investigate the protective effects of Sal A against myocardial I/R injury. The model mimics the metabolic phenotype in patients, but extrapolation to humans requires further studies. Secondly, we employed SP600125 to inhibit p-JNK expression, but the agent not only inhibits this signaling pathway incompletely but also influences the other signaling molecules. Gene-knockout seems to act directly on potential therapeutic targets in animal models, providing the possibility of a direct relationship to human disease.

In conclusion, these results demonstrate that the JNK/PI3K/Akt signaling pathway is involved in myocardial I/R injury in diabetic rats, and Sal A exerts an anti-apoptotic action against myocardial I/R injury and improves cardiac functional recovery following reperfusion through JNK/PI3K/Akt signaling in this model. The present study can provide important insights into the molecular mechanisms involved in cardioprotective effects in diabetic rats that have undergone I/R injury.

Disclosure of conflict of interest

None.

References

  • 1.Thom T, Haase N, Rosamond W, Howard VJ, Rumsfeld J, Manolio T, Zheng ZJ, Flegal K, O’Donnell C, Kittner S, Lloyd-Jones D, Goff DC Jr, Hong Y, Adams R, Friday G, Furie K, Gorelick P, Kissela B, Marler J, Meigs J, Roger V, Sidney S, Sorlie P, Steinberger J, Wasserthiel-Smoller S, Wilson M, Wolf P. Heart disease and stroke statistics--2006 update: a report from the American Heart Association Statistics Committee and Stroke Statistics Subcommittee. Circulation. 2006;113:e85–151. doi: 10.1161/CIRCULATIONAHA.105.171600. [DOI] [PubMed] [Google Scholar]
  • 2.Hotta H, Miura T, Miki T, Togashi N, Maeda T, Kim SJ, Tanno M, Yano T, Kuno A, Itoh T, Satoh T, Terashima Y, Ishikawa S, Shimamoto K. Angiotensin II type 1 receptor-mediated upregulation of calcineurin activity underlies impairment of cardioprotective signaling in diabetic hearts. Circ Res. 2010;106:129–132. doi: 10.1161/CIRCRESAHA.109.205385. [DOI] [PubMed] [Google Scholar]
  • 3.Lee MG, Jeong MH, Ahn Y, Chae SC, Hur SH, Hong TJ, Kim YJ, Seong IW, Chae JK, Rhew JY, Chae IH, Cho MC, Bae JH, Rha SW, Kim CJ, Choi D, Jang YS, Yoon J, Chung WS, Cho JG, Seung KB, Park SJ Other Korea Acute Myocardial Infarction Registry Investigators. Comparison of clinical outcomes following acute myocardial infarctions in hypertensive patients with or without diabetes. Korean Circ J. 2009;39:243–250. doi: 10.4070/kcj.2009.39.6.243. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Li H, Liu Z, Wang J, Wong GT, Cheung CW, Zhang L, Chen C, Xia Z, Irwin MG. Susceptibility to myocardial ischemia reperfusion injury at early stage of type 1 diabetes in rats. Cardiovasc Diabetol. 2013;12:133. doi: 10.1186/1475-2840-12-133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Hess K, Marx N, Lehrke M. Cardiovascular disease and diabetes: the vulnerable patient. Eur Heart J Suppl. 2012;14:B4–B13. [Google Scholar]
  • 6.Fang F, Li D, Pan H, Chen D, Qi L, Zhang R, Sun H. Luteolin inhibits apoptosis and improves cardiomyocyte contractile function through the PI3K/Akt pathway in simulated ischemia/reperfusion. Pharmacology. 2011;88:149–158. doi: 10.1159/000330068. [DOI] [PubMed] [Google Scholar]
  • 7.Kristiansen SB, Lofgren B, Stottrup NB, Khatir D, Nielsen-Kudsk JE, Nielsen TT, Botker HE, Flyvbjerg A. Ischaemic preconditioning does not protect the heart in obese and lean animal models of Type 2 diabetes. Diabetologia. 2004;47:1716–1721. doi: 10.1007/s00125-004-1514-4. [DOI] [PubMed] [Google Scholar]
  • 8.Ghaboura N, Tamareille S, Ducluzeau PH, Grimaud L, Loufrani L, Croue A, Tourmen Y, Henrion D, Furber A, Prunier F. Diabetes mellitus abrogates erythropoietin-induced cardioprotection against ischemic-reperfusion injury by alteration of the RISK/GSK-3beta signaling. Basic Res Cardiol. 2011;106:147–162. doi: 10.1007/s00395-010-0130-3. [DOI] [PubMed] [Google Scholar]
  • 9.Song T, Lv LY, Xu J, Tian Z, Cui W, Wang Q, Qu G, Shi X. Diet-induced obesity suppresses sevoflurane preconditioning against myocardial ischemia-reperfusion injury: role of AMP-activated protein kinase pathway. Exp Biol Med (Maywood) 2011;236:1427–1436. doi: 10.1258/ebm.2011.011165. [DOI] [PubMed] [Google Scholar]
  • 10.Yu QJ, Gao F, Ma XL. Insulin says NO to cardiovascular disease. Cardiovasc Res. 2011;89:516–524. doi: 10.1093/cvr/cvq349. [DOI] [PubMed] [Google Scholar]
  • 11.Li HL, Yu XC. Emerging role of JNK in insulin resistance. Curr Diabetes Rev. 2013;9:422–428. doi: 10.2174/15733998113099990074. [DOI] [PubMed] [Google Scholar]
  • 12.Zhang X, Xu A, Chung SK, Cresser JH, Sweeney G, Wong R, Lin A, Lam KS. Selective inactivation of c-Jun NH2-terminal kinase in adipose tissue protects against diet-induced obesity and improves insulin sensitivity in both liver and skeletal muscle in mice. Diabetes. 2011;60:486–495. doi: 10.2337/db10-0650. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Hirosumi J, Tuncman G, Chang L, Görgün CZ, Uysal KT, Maeda K, Karin M, Hotamisligil GS. A central role for JNK in obesity and insulin resistance. Nature. 2002;420:333–6. doi: 10.1038/nature01137. [DOI] [PubMed] [Google Scholar]
  • 14.Taniguchi CM, Aleman JO, Ueki K, Luo J, Asano T, Kaneto H, Stephanopoulos G, Cantley LC, Kahn CR. The p85alpha regulatory subunit of phosphoinositide 3-kinase potentiates c-Jun N-terminal kinase-mediated insulin resistance. Mol Cell Biol. 2007;27:2830–2840. doi: 10.1128/MCB.00079-07. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Shah SA, Lee HY, Bressan RA, Yun DJ, Kim MO. Novel osmotin attenuates glutamate-induced synaptic dysfunction and neurodegeneration via the JNK/PI3K/Akt pathway in postnatal rat brain. Cell Death Dis. 2014;5:e1026. doi: 10.1038/cddis.2013.538. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Qiao ZY, Ma JW, Liu HJ. Evaluation of the antioxidant potential of Salvia miltiorrhiza ethanol extract in a rat model of ischemia-reperfusion injury. Molecules. 2011;16:10002–10012. doi: 10.3390/molecules161210002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Wang XJ, Wang ZB, Xu JX. Effect of salvianic acid A on lipid peroxidation and membrane permeability in mitochondria. J Ethnopharmacol. 2005;97:441–445. doi: 10.1016/j.jep.2004.11.036. [DOI] [PubMed] [Google Scholar]
  • 18.Zhang YQ, Tang Y, Wu AL, Zhu HB. Salvianolic acid A displays cardioprotective effects in in vitro models of heart hypoxia/reoxygenation injury. J Asian Nat Prod Res. 2010;12:899–915. doi: 10.1080/10286020.2010.510115. [DOI] [PubMed] [Google Scholar]
  • 19.Xu H, Li YL, Che X, Tian HC, Fan HY, Liu K. Metabolism of salvianolic acid A and antioxidant activities of its methylated metabolites. Drug Metab Dispos. 2014;42:274–281. doi: 10.1124/dmd.113.053694. [DOI] [PubMed] [Google Scholar]
  • 20.Fan HY, Fu FH, Yang MY, Xu H, Zhang AH, Liu K. Antiplatelet and antithrombotic activities of salvianolic acid A. Thromb Res. 2010;126:e17–22. doi: 10.1016/j.thromres.2010.04.006. [DOI] [PubMed] [Google Scholar]
  • 21.Pan H, Li D, Fang F, Chen D, Qi L, Zhang R, Xu T, Sun H. Salvianolic acid A demonstrates cardioprotective effects in rat hearts and cardiomyocytes after ischemia/reperfusion injury. J Cardiovasc Pharmacol. 2011;58:535–542. doi: 10.1097/FJC.0b013e31822de355. [DOI] [PubMed] [Google Scholar]
  • 22.Morrison LE, Whittaker RJ, Klepper RE, Wawrousek EF, Glembotski CC. Roles for alphaB-crystallin and HSPB2 in protecting the myocardium from ischemia-reperfusion -induced damage in a KO mouse model. Am J Physiol Heart Circ Physiol. 2004;286:H847–855. doi: 10.1152/ajpheart.00715.2003. [DOI] [PubMed] [Google Scholar]
  • 23.Wu X, Xu TD, Li DY, Zhu SS, Chen QP, Hu WJ, Pan D, Zhu H, Sun H. ERK/PP1a/PLB/SERCA2a and JNK pathways are involved in luteolin-mediated protection of rat hearts and cardiomyocytes following ischemia/reperfusion. PLoS One. 2013;8:e82957. doi: 10.1371/journal.pone.0082957. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Qi LL, Pan HJ, Li DY, Fang F, Chen D, Sun H. Luteolin improves contractile function and attenuates apoptosis following ischemia-reperfusion in adult rat cardiomyocytes. Eur J Pharmacol. 2011;668:201–207. doi: 10.1016/j.ejphar.2011.06.020. [DOI] [PubMed] [Google Scholar]
  • 25.Bae SC, Zhang LB. Gender differences in cardioprotection against Ischemia/Reperfusion injury in adult rat hearts: Focus on Akt and protein kinase C signaling. J Pharmacol Exp Ther. 2005;315:1125–1135. doi: 10.1124/jpet.105.090803. [DOI] [PubMed] [Google Scholar]
  • 26.Jones LR. Mg2+ and ATP effects on K+ activation of the Ca2+-transport ATPase of cardiac sarcoplasmic reticulum. Biochim Biophys Acta. 1979;557:230–242. doi: 10.1016/0005-2736(79)90105-6. [DOI] [PubMed] [Google Scholar]
  • 27.Kodavanti PR, Cameron JA, Yallapragada PR, Desaiah D. Effect of chlordecone (Kepone) on calcium transport mechanisms in rat heart sarcoplasmic reticulum. Pharmacol Toxicol. 1990;67:227–234. doi: 10.1111/j.1600-0773.1990.tb00818.x. [DOI] [PubMed] [Google Scholar]
  • 28.Pande M, Cameron JA, Vig PJ, Desaiah D. Phencyclidine block of Ca2+ ATPase in rat heart sarcoplasmic reticulum. Toxicology. 1998;129:95–102. doi: 10.1016/s0300-483x(98)00061-4. [DOI] [PubMed] [Google Scholar]
  • 29.Hausenloy DJ, Yellon DM. New directions for protecting the heart against ischaemia-reperfusion injury: targeting the Reperfusion Injury Salvage Kinase (RISK)-pathway. Cardiovasc Res. 2004;61:448–460. doi: 10.1016/j.cardiores.2003.09.024. [DOI] [PubMed] [Google Scholar]
  • 30.Hausenloy DJ, Tsang A, Yellon DM. The reperfusion injury salvage kinase pathway: A common target for both ischemic preconditioning and postconditioning. Trends Cardiovasc Med. 2005;15:69–75. doi: 10.1016/j.tcm.2005.03.001. [DOI] [PubMed] [Google Scholar]
  • 31.Yin X, Zheng Y, Zhai XJ, Zhao X, Cai L. Diabetic inhibition of preconditioning- and postconditioning-mediated myocardial protection against ischemia/reperfusion injury. Exp Diabetes Res. 2012;2012:198048. doi: 10.1155/2012/198048. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Miki T, Miura T, Hotta H, Tanno M, Yano T, Sato T, Terashima Y, Takada A, Ishikawa S, Shimamoto K. Endoplasmic reticulum stress in diabetic hearts abolishes erythropoietin-induced myocardial protection by impairment of phospho-glycogen synthase kinase-3beta-mediated suppression of mitochondrial permeability transition. Diabetes. 2009;58:2863–2872. doi: 10.2337/db09-0158. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Han MS, Jung DY, Morel C, Lakhani SA, Kim JK, Flavell RA, Davis RJ. JNK expression by macrophages promotes obesity-induced insulin resistance and inflammation. Science. 2013;339:218–222. doi: 10.1126/science.1227568. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Chambers JW, Pachori A, Howard S, Iqbal S, LoGrasso PV. Inhibition of JNK Mitochondrial Localization and Signaling Is Protective against Ischemia/Reperfusion Injury in Rats. J Biol Chem. 2013;288:4000–4011. doi: 10.1074/jbc.M112.406777. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Masharani UB, Maddux BA, Li XJ, Sakkas GK, Mulligan K, Schambelan M, Goldfine ID, Youngren JF. Insulin Resistance in Non-Obese Subjects Is Associated with Activation of the JNK Pathway and Impaired Insulin Signaling in Skeletal Muscle. PLoS One. 2011;6:e19878. doi: 10.1371/journal.pone.0019878. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Jia N, Han K, Kong JJ, Zhang XM, Sha S, Ren GR, Cao YP. (-)-Epigallocatechin-3-gallate alleviates spatial memory impairment in APP/PS1 mice by restoring IRS-1 signaling defects in the hippocampus. Mol Cell Biochem. 2013;380:211–218. doi: 10.1007/s11010-013-1675-x. [DOI] [PubMed] [Google Scholar]
  • 37.Lovell MJ, Yasin M, Lee KL, Cheung KK, Shintani Y, Collino M, Sivarajah A, Leung KY, Takahashi K, Kapoor A, Yaqoob MM, Suzuki K, Lythgoe MF, Martin J, Munroe PB, Thiemermann C, Mathur A. Bone marrow mononuclear cells reduce myocardial reperfusion injury by activating the PI3K/Akt survival pathway. Atherosclerosis. 2010;213:67–76. doi: 10.1016/j.atherosclerosis.2010.07.045. [DOI] [PubMed] [Google Scholar]
  • 38.Granata R, Trovato L, Gallo MP, Destefanis S, Settanni F, Scarlatti F, Brero A, Ramella R, Volante M, Isgaard J, Levi R, Papotti M, Alloatti G, Ghigo E. Growth hormone-releasing hormone promotes survival of cardiac myocytes in vitro and protects against ischaemia-reperfusion injury in rat heart. Cardiovasc Res. 2009;83:303–312. doi: 10.1093/cvr/cvp090. [DOI] [PubMed] [Google Scholar]
  • 39.Anwar A, Schluter KD, Heger J, Piper HM, Euler G. Enhanced SERCA2A expression improves contractile performance of ventricular cardiomyocytes of rat under adrenergic stimulation. Pflugers Arch. 2008;457:485–491. doi: 10.1007/s00424-008-0520-7. [DOI] [PubMed] [Google Scholar]
  • 40.Chen Y, Zhao J, Du JB, Xu GH, Tang CS, Geng B. Hydrogen sulfide regulates cardiac sarcoplasmic reticulum Ca(2+) uptake via K(ATP) channel and PI3K/Akt pathway. Life Sci. 2012;91:271–278. doi: 10.1016/j.lfs.2012.07.026. [DOI] [PubMed] [Google Scholar]
  • 41.Kralik PM, Ye G, Metreveli NS, Shem X, Epstein PN. Cardiomyocyte dysfunction in models of type 1 and type 2 diabetes. Cardiovasc Toxicol. 2005;5:285–292. doi: 10.1385/ct:5:3:285. [DOI] [PubMed] [Google Scholar]
  • 42.Zhang CM, Gao L, Zheng YJ, Yang HT. Berbamine protects the heart from ischemia/reperfusion injury by maintaining cytosolic Ca(2+) homeostasis and preventing calpain activation. Circ J. 2012;76:1993–2002. doi: 10.1253/circj.cj-11-1431. [DOI] [PubMed] [Google Scholar]
  • 43.Zhao ZQ, Vinten-Johansen J. Myocardial apoptosis and ischemic preconditioning. Cardiovasc Res. 2002;55:438–455. doi: 10.1016/s0008-6363(02)00442-x. [DOI] [PubMed] [Google Scholar]
  • 44.Stephanou A, Scarabelli TM, Townsend PA, Bell R, Yellon D, Knight RA, Latchman DS. The carboxyl-terminal activation domain of the STAT-1 transcription factor enhances ischemia/reperfusion-induced apoptosis in cardiac myocytes. FASEB J. 2002;16:1841–1843. doi: 10.1096/fj.02-0150fje. [DOI] [PubMed] [Google Scholar]
  • 45.Chae IH, Park KW, Kim HS, Oh BH. Nitric oxide-induced apoptosis is mediated by Bax/Bcl-2 gene expression, transition of cytochrome c, and activation of caspase-3 in rat vascular smooth muscle cells. Clin Chim Acta. 2004;341:83–91. doi: 10.1016/j.cccn.2003.11.009. [DOI] [PubMed] [Google Scholar]
  • 46.Dong JW, Zhu HF, Zhu WZ, Ding HL, Ma TM, Zhou ZN. Intermittent hypoxia attenuates ischemia/reperfusion induced apoptosis in cardiac myocytes via regulating Bcl-2/Bax expression. Cell Res. 2003;13:385–391. doi: 10.1038/sj.cr.7290184. [DOI] [PubMed] [Google Scholar]
  • 47.Shi C, Wu F, Yew DT, Xu J, Zhu Y. Bilobalide prevents apoptosis through activation of the PI3K/Akt pathway in SH-SY5Y cells. Apoptosis. 2010;15:715–727. doi: 10.1007/s10495-010-0492-x. [DOI] [PubMed] [Google Scholar]
  • 48.Sun DD, Huang J, Zhang Z, Gao HK, Li JY, Shen M, Cao F, Wang H. Luteolin limits infarct size and improves cardiac function after myocardium ischemia/reperfusion injury in diabetic rats. PLoS One. 2012;7:e33491. doi: 10.1371/journal.pone.0033491. [DOI] [PMC free article] [PubMed] [Google Scholar]

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