Diabetic cardiomyopathy, especially myocardial ischemia reperfusion (I/R) injury, is a major cause of morbidity and mortality in type 2 diabetic patients. The increasing of basal p38 MAP Kinase (p38 MAPK) activation is a major factor that aggravates cardiac death on diabetic cardiomyopathy. In addition, metformin also shows cardio-protective effects on myocardial ischemia/reperfusion injury. In this study, we investigated the effect of the combination between metformin and p38 MAPK inhibitor (SB203580) in diabetic rats subjected to I/R injury. H9c2 cells were induced into a hyperglycemic condition and treated with metformin, SB203580 or the combination of metformin and SB203580. In addition, cells in both the presence and absence of drug treatment were subjected to simulated ischemia/reperfusion injury. Cell viability and cellular reactive oxygen species (ROS) were determined. Moreover, the Goto-Kakizaki (GK) rats were treated with metformin, SB203580, and the combination of metformin and SB203580 for 4 weeks. Diabetic parameters and cardiac functions were assessed. Finally, rat hearts were induced ischemia/reperfusion injury for the purpose of infarct size analysis and determination of signal transduction. A high-glucose condition did not reduce cell viability but significantly increased ROS production and significantly decreased cell viability after induced sI/R. Treatment using drugs was shown to reduce ROS generation and cardiac cell death. The GK rats displayed diabetic phenotype by increasing diabetic parameters and these parameters were significantly decreased when treated with drugs. Treatment with metformin or SB203580 could significantly reduce the infarct size. Interestingly, the combination of metformin and SB203580 could enhance cardio-protective ability. Myocardial I/R injury significantly increased p38 MAPK phosphorylation, Bax/Bcl-2 ratio and caspase-3 level. Treatment with drugs significantly decreased the p38 MAPK phosphorylation, Bax/Bcl-2 ratio, caspase-3 level and increased Akt phosphorylation. In conclusion, using the combination of metformin and SB203580 shows positive cardio-protective effects on diabetic ischemic cardiomyopathy.
Diabetes is a non-communicable disease, is a major cause of morbidity and mortality in worldwide. Particularly, the International Diabetes Federation (IDF) has reported that cases of type 2 diabetes will increase to 693 million by 2045 (
The major factor of cardiac impairment in diabetic cardiomyopathy is an increasing level of basal p38 mitogen-activated protein kinases (p38 MAPK) activation. p38 MAPK is a serine/threonine protein kinase, which responds to several cellular processes and external stress signaling, such as cell differentiation, cell proliferation, inflammation and cells death (
The combination of controlling blood glucose levels and improving insulin sensitivity is the most effective treatment for diabetic patients. Metformin is one of the anti-diabetic drugs that belongs to the biguanide drug family and is widely used in diabetic treatment around the world. Metformin is not only used to control blood glucose levels, but also shows cardio-protective effects, including improving heart failure progression in animal models (
According to the above information, it is indicated that the aggravation of cardiac death in diabetic cardiomyopathy, especially diabetes complicated with myocardial ischemia/reperfusion disease, may be due to a failure to control blood glucose levels together with an increase of p38 MAPK activation. Although, treatment with metformin itself or p38 MAPK inhibitor itself could decrease cardiac death rates in patients with type 2 diabetes or myocardial ischemia/reperfusion. However, this alone is insufficient to prevent the cardiac death in patients with diabetes complicated with myocardial ischemia/reperfusion injury disease. Therefore, treatment with the combination of metformin and p38 MAPK inhibitor (SB203580) could have a therapeutic potential for patients with diabetes with myocardial ischemia/reperfusion injury disease. However, the effects of this combination of metformin and SB203580 on patients with diabetes complicated with myocardial ischemia/reperfusion injury have not been intensively investigated. So, the objective of this study was to determine the effect of metformin combined with SB203580 in terms of how diabetic parameters can be controlled and cardiac death rates in cardiac cell line (H9c2 cell) and type 2 diabetic models (GK rats) subjected to myocardial ischemia/reperfusion injury condition.
Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum and trypsin-EDTA were purchased from Gibco BRL; Life Technologies Inc. Other purchases included 2′,7′-Dichlorofluorescin Diacetate from Merck, 3-(4,5-dimethyl-2-thiazol)-2,5-diphenyl-2Htetrazolium bromide (MTT) from Ameresco, metformin from MERCK Serono, p38 MAPK inhibitor (SB203580) from Tocris Bioscience. Other chemicals were purchased from Sigma Chemical Company.
The rat cardiac myoblast cell line (H9c2 cell line) was purchased from the American Type Cell Collection (ATCC; no. CRL-1446). The cell was maintained in Dulbecco's modified Eagle's medium (DMEM) (Gibco®) supplemented with 5,000 U/ml of penicillin, 5,000 µg/ml of streptomycin (Gibco®) and 10% fetal bovine serum (FBS) (Gibco®). Cells were cultured at 37°C, 5% CO2 and 95% O2 throughout the experiment. When the cell density was more than 70–80% confluent, the cell was sub-cultured with trypsin.
For an
For cell viability measurement, MTT assay was used based on mitochondria dehydrogenases activity. The H9c2 cells were seeded at a density of 5×103 cells/well in a 96-well plate until 80% confluence was reached. After treatments or at the end of study protocols, the culture medium was discarded and replaced with 0.5 mg/ml MTT reagent and incubate at 37°C for 2 h. After incubation, the excess MTT reagent was removed and the formazan dye was solubilized by adding 100 µl of dimethysulfoxide (DMSO). The optical density (OD) was measured using a spectrophotometric method at λ 490 nm using DMSO as a blank. The relative percentage of cell viability was compared against the control group.
Reactive oxygen species detection was performed by 2′7-dichlorofluorescein diacetate (DCFH-DA) dye. H9c2 cells were cultured in a 96-well plate at the density of 5×103 cell/well at 37°C, 5% CO2, and 95% O2 until 80% confluence was reached. The medium was discarded and cells were washed with PBS solution before being incubated with culture medium or condition medium containing 25 µM DCFH-DA for 24 h at 37°C. The intracellular production of ROS was determined by measuring the fluorescence intensity with an EnSpire Multimode Plate Readers (PerkinElmer). The filter suitable for detecting the signal gave an excitation wavelength of λ 498 nm and emission wavelength of λ 522 nm.
Male type 2 diabetic Goto-Kakizaki (GK) rats (n=40) and age-matched Wistar rats (n=10) weighing approximately 200–250 g were purchased from Nomura Siam International. All animals were maintained under environmentally controlled conditions (22±1°C, 12 h light: Dark cycle) at the Center for Animal Research, Naresuan University, Phitsanulok, Thailand. All protocols used in this study were approved by the committee of the Center for Animal Research, Naresuan University (approval no. NU-AE581023).
All rats were maintained for 4 weeks after being purchased and the glycemic parameters, including fasting blood glucose, hemoglobin A1c levels, and oral glucose tolerance testing (OGTT), were performed for the purposes of confirming the diabetic model. Then, the rats were divided into 2 major groups, including (
The rats were fasted for a period of 12–14 h, and then blood from the rats' tail veins was collected for the measurement of blood glucose levels by glucometer (SD GlucoNavii® GDH; SB Biosensor). The rats' tails were cleaned with 70% alcohol and the blood was drawn using a 1 ml needle, then dropped onto a glucose strip and measured by a glucometer. The amount of electricity is proportional to the glucose content in the sample. The quality control for blood glucose was performed using the quality control material provided by SD Biosensor.
All of the rats were fasted for a period of 12–14 h, and then the blood was collected from rats' tail veins as a baseline. Then, the rats were fed with 40% (w/v) glucose solution by oral gavage at an amount equal to 2 g/kg body weight. The blood was collected from tail veins at 30, 60, 90 and 120 min after the glucose treatment and then analyzed for blood glucose with a glucometer.
The HbA1c test was performed by using CLOVER A1c™ Self-analyzer. The blood samples from the rats' tail veins were collected and dropped onto a test cartridge and measured by CLOVER A1c™ Self system. Finally, the percentages of Hemoglobin A1c (HbA1c %) of the blood samples were represented on an LCD screen of CLOVER A1c™ Self machine. The quality control for HbA1c was performed using the quality control material provided by EuriMedix Company.
The plasma insulin levels were determined by Sandwich ELISA (Millipore). The plasma samples were added to a microtiter plate coated with monoclonal mouse anti-rat insulin antibodies. After that, unbound material from the samples was washed away and the immobilized biotinylated antibodies were added. The microtiter plate had the substrate 3,3′,5,5′-tetramethybenzidine added. The enzyme activity was measured by a spectrophotometer (BioTek) with an absorbance at 450 nm, with a corrected wavelength at 590 nm. The plasma insulin concentration in the unknown samples was derived by interpolation from a reference curve generated in the same assay with reference standard of known concentration of rat insulin.
The echocardiography was performed by a veterinary cardiologist before and after drug treatment for 4 weeks. In brief, rats were intraperitoneal injected (IP) with a Ketamine/xylazine cocktail (91 mg/kg Ketamine; 9.1 mg/kg Xylazine) at a concentration of 0.1 ml/100 g (v/w) as an anesthetic. Then, the chest hair was removed, and echocardiography was performed using Mindray M9 (Mindray Medical Co., Ltd.) equipped with 4–10 MHz phased array cardiac probes (Mindray Medical Co., Ltd.) for measuring cardiac function. The cardiac function parameters were analyzed, which included Ejection fraction (EF), End-Diastolic volume (EDV), End-Systolic volume (ESV), Interventricular septum thickness at end-diastole (IVSd), Interventricular septum thickness at end-systole (IVSs), Left ventricular internal dimension at end-diastole (LVIDd), Left ventricular internal dimension at end-systole (LVIDs), Left ventricular posterior wall thickness at end-diastole (LVPWd), Left ventricular posterior wall thickness at end-systole (LVPWs), Cardiac output (CO), Stock volume (SV) and Heart rate (HR).
The
The rats (n=6) were anesthetized and hearts were cannulated. After that, the cannulated hearts were perfused with the K-H buffer for 40 min and then the flow of K-H buffer was halted for 10 min. After 10 min, the hearts were rapidly snapped frozen. 500 µl of homogenization buffer was added to the heart samples for homogenization. The heart samples that were homogenated were centrifuged at 15,366 × g, 4°C for 10 min. Then, the samples were collected and added in equal volume of 2X SDS-PAGE sample buffers; containing 10% (v/v) 2-mercaptoethanol and bromophenol blue dye. The sample was boiled for 10 min. The heart proteins were separated in SDS-PAGE gels at the final amount of protein at 100 µg per lane. Electrophoresis was performed at 120V for 2 h. After separation, proteins were transferred to Polyvinylidene fluoride (PVDF) membranes (Hybond-P; GE Healthcare Life Sciences) using a semi-dry apparatus under an electrical current of 12 mV, for 45 min. Following transfer, membranes were incubated in a blocking solution (5% (w/v) of dried skimmed milk powder, in TBST) for 1 h with gentle shaking at room temperature. The membranes, which transferred protein, were incubated with primary antibodies for phosphorylated-Akt (Santa Cruz Biotechnology, Inc., #sc-7985), total-Akt (Santa Cruz Biotechnology, Inc., #sc-8312), phosphorylated p38 (Santa Cruz Biotechnology, Inc., #sc-17852), total-p38 (Santa Cruz Biotechnology, Inc., #sc-535), Bax (Santa Cruz Biotechnology, Inc., #sc-493), Bcl-2 (Santa Cruz Biotechnology, Inc., #sc-492) caspase 3 (Santa Cruz Biotechnology, Inc., #sc-7148) and GAPDH (Merck, #ABS16) (All of primary antibodies were diluted at 1:1,000 in 1% (w/v) skimmed milk + TBST buffer) and the horseradish peroxidase-conjugated secondary antibody (goat anti-rabbit (Merck, #AP132P) and rabbit anti-goat (Merck, #AP106P), which was diluted at 1:5,000 in 1% (w/v) skimmed milk + TBST, after the heart protein was separated in SDS-PAGE. The band intensity quantitation was determined by Image LabTM 2.0 Software (Bio-Rad Laboratories, Inc.).
All values are expressed as the mean ± SEM. All comparisons involving the two groups were determined in terms of significance using Student's t-test and more than one group was assessed in terms of significance using a one-way ANOVA, followed (when appropriate) by the Tukey-Kramer test. P<0.05 was considered to indicate a statistically significant difference.
The H9c2 cells were cultured until the confluency of 80% was reached, and then incubated in a high-glucose condition for 24 h, which was a complete medium supplemented with D-glucose solution to a final concentration of glucose in culture medium at 33 mM. The result showed that the
A hyperglycemic condition showed a slight increase in cell viability (100.00%±0.00 vs. 105.5%±2.60), which was significantly increased when treated with metformin in hyperglycemic condition (100.00%±0.00 vs. 115.6%±3.09) (
The results from cell viability showed that a hyperglycemic condition caused a slight increase in cell viability (100.00%±0.00 vs. 107.9%±1.67), and treatment with SB203580 did not affect cell viability (110.4%±2.54) (
The combination of metformin and SB203580 showed no cytotoxicity to H9c2 cells since there were no significant differences in cell viability in each group compared to the control group (100.00%±0.00 vs. 95.15%±2.70) (
To ensure that the lean type 2 diabetic animal model was valid for study, measurement of several glycemic parameters were performed. To confirm the non-obese or lean animal model, the rats were measured in terms of body weight and the growth rate at 7, 15 and 20 weeks of age. The growth curve was plotted using body weight (
The glycemic measurement showed that the mean FBG level of GK rats was significantly higher than that of Wistar rats (143.6±3.58 vs. 84.00±4.51 mg/dl) (
After 4 weeks of drugs treatment, blood was collected for measuring FBS and HbA1c levels. The result showed that the FBG level of the diabetic group was significantly higher when compared to the control group (153.6±7.27 mg/dl vs. 87.67±3.22 mg/dl; P<0.05). Treatment of metformin (113.3±2.99), or SB203580 (106.9±4.36), and the combination of metformin and SB203580 (113.2±3.79) significantly reduced FBS levels when compared to the diabetic group (
Similar results were observed in HbA1c levels. The result showed that the percentage HbA1c of the diabetic group was significantly higher than the control group (6.70±0.108 vs. 4.38±0.113%; P<0.05). Treatment with metformin significantly reduced the percentage of HbA1c when compared to the diabetic group (5.36±0.224 vs. 6.70±0.108%; P<0.05). However, treatment with SB203580 alone failed to showed any significant reduction in the HbA1c level but not when compared to the diabetic group (6.13±0.159 vs. 6.70±0.108%) (
The results showed that plasma insulin levels in all of diabetic groups were significantly lower than those of the control Wistar rats (
To determine the cardiac function in the type 2 diabetic condition, all rats were measured in terms of cardiac function parameters by using echocardiography after 4 weeks of treatment. The results demonstrated that in the diabetic group, the heart wall thickness was shown to significantly decrease when compared to the control in LVIDd (0.387±0.014 vs. 0.655±0.028 cm, P<0.05), LVIDs (0.248±0.015 vs. 0.432±0.029 cm, P<0.05) and stability in LVPWd, LVPWs. Moreover, the GK rats had a significantly faster heart rate (HR) (354.4±8.21 vs. 494.5±20.75 bpm, P<0.05), lower in EDV (0.694±0.077 vs. 0.152±0.016 ml, P<0.05) and ESV (0.221±0.029 vs. 0.048±0.007 ml, P<0.05), which was caused by a decrease in stroke volume (SV) (0.473±0.055 vs. 0.105±0.009 ml, P<0.05) and cardiac output (CO) (0.169±0.022 vs. 0.050±0.004 l/min, P<0.05;
Treatment with SB203580 could significantly improve heart rate (443.9±30.13 vs. 494.5±20.75 bpm, P<0.05), SV (0.129±0.016 vs. 0.105±0.009 ml, P<0.05), and EF (85.71±2.72 vs. 70.68±2.42%, P<0.05) compared to the diabetic group.
Treatment with the combination of metformin and SB203580 was able to significantly improve some parameters when compared to the diabetic group, including IVSs (0.353±0.015 vs. 0.295±0.004 cm, P<0.05), LVIDd (0.528±0.020 vs. 0.387±0.014 cm, P<0.05), HR (421.6±12.04 vs. 494.5±20.75 bpm, P<0.05), EDV (0.387±0.041 vs. 0.152±0.016 ml, P<0.05), ESV (0.084±0.019 vs. 0.048±0.007 ml, P<0.05), SV (0.304±0.024 vs. 0.105±0.009 ml, P<0.05), CO (0.130±0.011 vs. 0.050±0.004 l/min, P<0.05), and EF (80.09±2.72 vs. 70.68±2.42%, P<0.05). In addition, the combination of metformin and SB203580 was only able to improve LVIDd, SV, and CO, when compared to metformin or SB203580 alone.
The sensitivity to myocardial ischemia/reperfusion injury was performed by an
To determine the effect of the combination of metformin and SB203580 in signal transduction on organ level response to myocardial ischemia/reperfusion injury, Western blotting analysis was performed on homogenated hearts. The results showed that the p38 MAPK activation significantly increased in response to diabetes complicated with ischemia/reperfusion injury when compared to the control group (1.20±0.053 vs. 0.82±0.029; P>0.05,
Diabetic cardiomyopathy is a complication of type 2 diabetic that is known as a major cause of morbidity and mortality in worldwide, especially myocardial ischemia/reperfusion injury. Metformin and SB203580 are also known to have cardio-protective effects on hyperglycaemic condition or hyperglycaemia with ischemia/reperfusion condition. In this study, we proposed that treatment with the combination of metformin and SB203580 could decrease cardiac cell death rates on hyperglycaemia subjected to ischemia/reperfusion condition. Therefore, the simulation of diabetes complicated with myocardial I/R injury condition causing cardiac cell death, as well as the effect of metformin, p38 MAPK inhibitor-SB203580, and a combination of these two drugs, were investigated in this study through carrying out
The major findings in this study showed that a high-glucose condition enhanced the sensitivity to ROS production and ischemia/reperfusion injury. Treatment with drugs could reduce ROS generation and cardiac cell death. Treatment with metformin or SB203580 could significantly reduce the infarct size in
From our
One of the major causes of aggravated cardiac cell death on diabetic cardiomyopathy is an increase in basal p38 MAP Kinase (p38 MAPK) activation in cardiac cells. Therefore, an inhibition of p38 MAPK activation could be a beneficial and therapeutic type of treatment for diabetic cardiomyopathy. In addition, metformin, which is an anti-diabetic drug, has also been demonstrated to have cardio-protective effects on ischemia/reperfusion injury (
The most common cause of diabetes is obesity; however, in actual fact, obese and non-obese people are at risk of type 2 diabetic disease. In particular, there have been several reports that type 2 diabetes was commonly occurs in non-obese people within Asia (
After diabetic diagnosis, the effect of metformin (anti-diabetic drug) or SB203580 (p38 MAPK inhibitor) or a combination of drugs was investigated on diabetic parameters, the plasma insulin level and cardiac function parameters. In this study, we found that treatment with all drugs could significantly decrease diabetic criteria (fasting blood glucose level and HbA1c level). Moreover, treatment with metformin or a combination of drugs could reduce plasma insulin levels. Our findings regarding metformin treatment were similar to those of the reports from previous studies, in which it was found that metformin could increase glucose uptake leading to a decrease in plasma glucose level, decrease in hepatic gluconeogenesis and increase in insulin stimulated peripheral glucose uptake (although there is no change or increase in the insulin secretory responses) (
Previous studies have indicated treatment with SB203580 in diabetic cells could increase glucose uptake (
In this study, the effects of using a combination of metformin and SB203580 on diabetic parameters were assessed. Our findings demonstrated that treatment with combined drugs administered to GK rats (Diabetic condition) could reduce blood glucose levels, reduce HbA1c levels and decrease plasma insulin levels. These results did not provide any further reduction to improve diabetic parameters of metformin and SB203580 combination, and remarkably, metformin or SB203580 did not interfere with each other in relation to improving diabetic parameters.
In this study, we also investigated the cardiac function after treatment and found that the GK rats displayed cardiac hypertrophy at 20-week-old, which was marked by stable left ventricular posterior wall thickness at end-diastole (LVPWd), reduction of cardiac output and a faster heart rate. Our findings were in conflict to previous studies, which have reported that the progression of hyperglycemic condition in GK rats leading to the left ventricle remodeling with marked hypertrophy, increase extracellular matrix deposition, and mild hypertension (
In conclusion, metformin or SB203580 or a combination of metformin and SB203580 exhibited anti-diabetic effects by reducing diabetic parameters and improving insulin sensitivity. In addition, all of the drugs showed that there were improved cardio-protective effects on diabetic condition and diabetes complicated with myocardial ischemia/reperfusion injury condition in which the was a reduction of ROS generation (
The authors would like to thank Professor Joel Nargeot, Institute of Functional Genomics (IGF), CNRS, INSERM, University of Montpellier for his suggestions and experiment planning.
The present study was financially supported by Naresuan University Research endowment fund and National Research Council of Thailand (NRC; grant no. R2560C138), and the Higher Education Research Promotion (HERP)-Office of Higher Education, Ministry of Education (grant no. R2559A017). The present study was also supported by The Royal Golden Jubilee Ph.D. Program-Thailand Research Fund (TRF) for JS (grant no. PHD/0087/2556) and for KK (grant no. PHD/0125/2558), The Naresuan University PhD student scholarship for NN, and Faculty of Allied Health Sciences, Naresuan University for providing research grant for Master degree study and scholarship for PM.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
JS and SK conceived and designed the experiments. JS, EP, PA, NN, PM and KK performed the animal experiments, determined the glycaemic parameters and assisted in the Langendorff protocol. JS and SK analyzed the data, and wrote and prepared the manuscript. AK and SP performed blind echocardiography and data analysis. SLB contributed to the infarct size measurement. JS, SLB and SK analyzed the data, and wrote, prepared, reviewed and corrected the manuscript.
The animal experiments in this study were conducted according to the Guidance on the Operation of the Animals (Scientific Procedures) Act 1986 and the World Health Organization (WHO) Guidelines for Breeding and Care of Laboratory Animals. All the protocols were approved by the committee of the Centre for Animal Research, Naresuan University (approval no. NU AE581023). All procedures were designed to minimise the pain, suffering and distress of the animals involved.
Not applicable.
The authors declare that they have no competing interests.
Effect of high-glucose condition and sI/R condition on H9c2 cells. (A) H9c2 cells were induced with high-glucose condition using 33 mM D-glucose solution for 24 h and the cell viability was determined. *P<0.05; #P<0.05 vs. high-glucose control group by ANOVA. (B) H9c2 cells were incubated in high-glucose solution supplemented with 2′7-dichlorofluorescein diacetate dye for 24 h and the intracellular ROS production was measured. *P<0.05 vs. NG. Each bar graph represents the mean ± SEM. ROS, reactive oxygen species; sI/R, simulated ischemia/reperfusion; NG, normal glucose; HG, high glucose.
Determination of the effect of metformin treatment on H9c2 cells in high-glucose and sI/R condition. (A) H9c2 cells were induced with high-glucose condition supplemented with or without 3 mM of metformin for 24 h and the cell viability was determined by an MTT cell survival assay. *P<0.05 vs. NG; #P<0.05 vs. HG determined by ANOVA. (B) H9c2 cells in high-glucose condition treated with metformin for 24 h was simulated ischemia for 40 min followed by 24 h of reperfusion. *P<0.05 vs. NG+sI/R; #P<0.05 vs. HG+sI/R determined by ANOVA. (C) H9c2 cells were induced with a high-glucose condition and treated with metformin supplement with 2′7-dichlorofluorescein diacetate dye for 24 h. Intracellular ROS production was measured. Each bar graph represents the mean ± SEM. *P<0.05 vs. NG; #P<0.05 vs. HG determined by ANOVA. ROS, reactive oxygen species; NG, normal glucose; HG, high glucose; sI/R, simulated ischemia/reperfusion.
Determination of the effect of treatment with SB203580 in H9c2 cells in high-glucose and sI/R condition. The H9c2 cell, which induced high-glucose condition, was treated with or without 10 µM p38 MAPK inhibitor (SB203580) for 24 h. (A) Cell viability was measured by an MTT cell survival assay. The H9c2 cell induced high-glucose was treated with SB203580 for 24 h. (B) Then, the cell was simulated ischemia for 40 min followed by 24 h of reperfusion. *P<0.05 vs. NG+sI/R; #P<0.05 vs. HG+sI/R as determined by ANOVA. (C) The H9c2 cells were induced with high-glucose condition and treatment with SB203580 supplemented with 2′7-dichlorofluorescein diacetate dye for 24 h. Intracellular ROS production was measured. Each bar graph represents the mean ± SEM. *P<0.05 vs. NG; #P<0.05 vs. HG as determined by ANOVA. ROS, reactive oxygen species; NG, normal glucose; HG, high glucose; sI/R, simulated ischemia/reperfusion.
Determination of the effect of combination of treatment with metformin and SB203580 in H9c2 cells on high-glucose and sI/R condition. (A) H9c2 cells was induced high-glucose condition supplement with or without 3 mM of metformin and 10 µM of SB203580 for 24 h, then the cell viability was determined by using MTT cell survival assay. (B) H9c2 cells in high-glucose condition that treatment with combined drugs for 24 h were simulated ischemia for 40 min followed by 24 h of reperfusion. *P<0.05 vs. NG+sI/R; #P<0.05 vs. HG+sI/R as determined by ANOVA. (C) H9c2 cells were induced high-glucose condition that treatment combined drugs supplement with 2′7-dichlorofluorescein diacetate dye for 24 h. Then, the intracellular ROS production was measured. Each bar graph represents the mean ± SEM. *P<0.05 vs. NG and #P<0.05 vs. high-glucose group as determined by ANOVA. ROS, reactive oxygen species; NG, normal glucose; HG, high glucose; sI/R, simulated ischemia/reperfusion.
Determination of non-obese diabetic-like phenotype in animal model. The rats were assessed the non-obese diabetic-like phenotype by using the body weight and the blood samples from tail vein that collected after 12–14 h fasting. (A) Body weight of Wistar rats and GK rats. (B) Fasting blood glucose level of Wistar rats and GK rats before treatment. (C) HbA1C level of Wistar rats and GK rats before treatment. (D) Oral glucose tolerance test of Wistar rats and GK rats before treatment. Each bar graph or line graph represents the mean ± SEM. *P<0.05 vs. control group as determined by a t-test. GK, Goto-Kakizaki; HbA1c, hemoglobin A1C.
Determination of the effect of drugs on diabetic parameters in type 2 diabetic model. The rats were divided into 5 groups and treatment with or without metformin, SB203580, combination of drugs for 4 weeks. After that, the diabetic parameters were performed. (A) Fasting blood glucose level of Wistar rats and GK rats. (B) HbA1C level of Wistar rats and GK rats. (C) Plasma insulin level of Wistar rats and GK rats. Each bar graph represents the mean ± SEM. *P<0.05 vs. control group; #P<0.05 vs. diabetic group as determined by ANOVA. GK, Goto-Kakizaki; HbA1c, hemoglobin A1C.
Determination of the effect of drugs on myocardial ischemia/reperfusion that caused complications in a type 2 diabetic model. After 4 weeks of treatment, all rats were subjected to myocardial ischemia/reperfusion injury using Langendorff Systems. The percentage infarct size was measured by triphenyltetrazolium chloride staining. Each bar graph represents the mean ± SEM. *P<0.05 vs. control group; #P<0.05 vs. diabetes group; &P<0.05 vs. SB203580 as determined by ANOVA.
Determination of the effect of drugs on signaling transduction in the organ level response to myocardial ischemia/reperfusion that caused complications in a type 2 diabetic model. After 4 weeks of treatment, all rats were subjected to myocardial ischemia/reperfusion injury by using Langendorff Systems. Then, the expression protein was assessed by western blot analysis. (A) p38 MAPK activation through phosphorylation. (B) Akt activation through phosphorylation. (C) Expression of caspase-3. (D) Expression of Bax and Bcl-2. Each bar graph represents the mean ± SEM. *P<0.05 vs. control group; #P<0.05 vs. diabetic group as determined by ANOVA. p, phosphorylated; t, total.
Determination of cardiac function parameters after treatment in type 2 diabetic model.
Groups | |||||
---|---|---|---|---|---|
Parameters | Control | Diabetes | Metformin | SB203580 | Combination |
IVSd (cm) | 0.273±0.010 | 0.258±0.006 | 0.222±0.011 |
0.249±0.014 | 0.246±0.014 |
IVSs (cm) | 0.327±0.014 | 0.295±0.004 | 0.287±0.013 | 0.329±0.020 | 0.353±0.015 |
LVIDd (cm) | 0.655±0.028 | 0.387±0.014 |
0.440±0.029 |
0.371±0.015 |
0.528±0.020 |
LVIDs (cm) | 0.432±0.029 | 0.248±0.015 |
0.284±0.030 |
0.183±0.011 |
0.286±0.023 |
LVPWd (cm) | 0.285±0.016 | 0.265±0.004 | 0.256±0.017 | 0.272±0.015 | 0.262±0.016 |
LVPWs (cm) | 0.319±0.014 | 0.329±0.033 | 0.305±0.019 | 0.304±0.016 | 0.305±0.016 |
HR (Bpm) | 354.4±8.21 | 494.5±20.75 |
463.0±11.43 |
443.9±30.13 |
421.6±12.04 |
EDV (ml) | 0.694±0.077 | 0.152±0.016 |
0.246±0.040 |
0.142±0.015 |
0.387±0.041 |
ESV (ml) | 0.221±0.029 | 0.048±0.007 |
0.087±0.023 |
0.020±0.004 |
0.084±0.019 |
SV (ml) | 0.473±0.055 | 0.105±0.009 |
0.161±0.027 |
0.129±0.016 |
0.304±0.024 |
CO (l/min) | 0.169±0.022 | 0.050±0.004 |
0.069±0.015 |
0.055±0.007 |
0.130±0.011 |
EF (%) | 67.78±2.43 | 70.68±2.42 | 67.98±4.93 | 85.71±2.72 |
80.09±2.72 |
The parameters of cardiac function of all the rats were assessed by echocardiography after treatment. Each parameter represents the mean ± SEM.
P<0.05 vs. control group
P<0.05 vs. diabetic group
P<0.05 vs. SB203580 group by ANOVA. IVSd, interventricular septum thickness at end-diastole; IVSs, interventricular septum thickness at end-systole; LVIDd, left ventricular internal dimension at end-diastole; LVIDs, left ventricular internal dimension at end-systole; LVPWd, left ventricular posterior wall thickness at end-diastole; LVPWs, left ventricular posterior wall thickness at end-systole; HR, heart rate; EDV, end-diastolic volume; ESV, end-systolic volume; SV, stock volume; CO, cardiac output; EF, ejection fraction.