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reperfusion rat model
European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
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Cardiovascular pharmacology
Protective effects of sitagliptin on myocardial injury and cardiac function in an ischemia/reperfusion rat model Guanglei Chang, Peng Zhang, Lin Ye, Kai Lu, Ying Wang, Qin Duan, Aihua Zheng, Shu Qin n, Dongying Zhang nn Department of Cardiology, The First Affiliated Hospital of Chongqing Medical University, No.1 Yixueyuan Road, Chongqing 400016, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 20 March 2013 Received in revised form 28 August 2013 Accepted 4 September 2013
The purpose of this study is to investigate the effects and the underlying mechanisms of sitagliptin pretreatment on myocardial injury and cardiac function in myocardial ischemia/reperfusion (I/R) rat model. The rat model of myocardial I/R was constructed by coronary occlusion. Rats were pretreated with sitagliptin (300 mg/kg/day) for 2 weeks, and then subjected to 30 min ischemia and 2 h reperfusion. The release of lactate dehydrogenase (LDH) and creatine kinase-MB (CK-MB), cardiac function and cardiomyocyte apoptosis were evaluated. The levels of malondialdehyde (MDA), glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) in heart and glucagon-like peptide-1 (GLP-1) level in plasma were measured. Western blot analysis was performed to detect the target proteins of sitagliptin. Our results showed that sitagliptin pretreatment decreased LDH and CK-MB release, and MDA level in I/R rats. More importantly, we revealed for the first time that sitagliptin pretreatment decreased cardiomyocyte apoptosis while increased the levels of GSH-Px and SOD in heart. Sitagliptin also increased GLP1 level and enhanced cardiac function in I/R rats. Furthermore, sitagliptin pretreatment up-regulated Aktserine473 and Badserine136 phosphorylation, reduced the ratio of Bax/Bcl-2, and decreased expression levels of cleaved caspase-3 and caspase-3. Interestingly, the above observed effects of sitagliptin were all abolished when co-administered with GLP-1 receptor antagonist exendin-(9-39) or PI3K inhibitor LY294002. Taken together, our data indicate that sitagliptin pretreatment could reduce myocardial injury and improve cardiac function in I/R rats by reducing apoptosis and oxidative damage. The underlying mechanism might be the activation of PI3K/Akt signaling pathway by GLP-1/GLP-1 receptor. Crown Copyright & 2013 Published by Elsevier B.V. All rights reserved.
Keywords: DPP4 inhibitor Sitagliptin Ischemia/reperfusion Myocardial injury Cardiomyocyte apoptosis Cardiac function
1. Introduction Myocardial infarction is a major cause of mortality and morbidity of patients with diabetes mellitus (Acar et al., 2011). In order to prevent the myocardium from further damage, the best therapeutic strategy for myocardial infarction is to reestablish the blood flow as earlier as possible. Nevertheless, ischemia/reperfusion (I/R) injury such as cardiomyocyte apoptosis is inevitable. Cardiomyocyte apoptosis induced by I/R plays an important role in causing a gradual decline of cardiac function (Gottlieb, 2011). Therefore, the exploration of new therapeutic agents that reduce I/R injury of myocardial infarction patients has become very important. Glucagon-like peptide-1 (GLP-1) is secreted by the enteroendocrine L cells of the intestinal mucosa and released in response to nutrient ingestion (Nauck et al., 1993). It exerts insulinotropic and insulinomimetic effects via the G-protein-coupled GLP-1 receptor n
Corresponding author. Tel.: 86 13101345177; fax: 86 2389011562. Corresponding author. Tel.: 86 13650502588; fax: 86 2368055542. E-mail addresses: [email protected] (S. Qin), [email protected] (D. Zhang). nn
(Verge and Lopez, 2010). The therapy based on the functions of GLP1 is currently used as a novel anti-diabetic approach (Doupis and Veves, 2008; Garber, 2012). However, GLP-1 is rapidly degraded by dipeptidyl peptidase-4 (DPP4) enzyme in the blood (Green et al., 2006). The short half life time limited its clinical use. Thus, two classes of drugs, including GLP-1 analogs (Garber, 2012) (i.e. exenatide) and DPP4 inhibitors (Doupis and Veves, 2008) (i.e. sitagliptin), have been recently used for treating type 2 diabetes. Recently, growing evidences have demonstrated the beneficial effects of GLP-1 analogs during I/R injury in both animal models and in clinical studies, such as limiting infarct, improving cardiac function and enhancing myocardial glucose uptake (Bhashyam et al., 2010; Chinda et al., 2012a; Lorber, 2012; Mundil et al., 2012). The mechanisms underlying the cardioprotective effects of GLP-1 analogs may be both GLP-1 receptor dependent and independent pathways (Ban et al., 2008; Chinda et al., 2012a). Unlike GLP-1 analogs, evidences regarding the cardioprotective effects of DPP4 inhibitors are scarce and controversial. Recently, more and more researchers have paid close attention to the cardioprotective effects of DPP4 inhibitors. Chinda, et al. (2012b) reported that DPP4 inhibitor could stabilize cardiac electrophysiology in a myocardial I/R pig model.
0014-2999/$ - see front matter Crown Copyright & 2013 Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejphar.2013.09.007
Please cite this article as: Chang, G., et al., Protective effects of sitagliptin on myocardial injury and cardiac function in an ischemia/ reperfusion rat model. Eur J Pharmacol (2013), http://dx.doi.org/10.1016/j.ejphar.2013.09.007i
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In addition, DPP4 inhibitor has been shown to attenuate the infarct size and improve the left ventricular function during myocardial I/R injury (Chinda et al., 2012a; Jose and Inzucchi, 2012; Lenski et al., 2011; Scheen, 2012). However, the correlation between its cardioprotective effect and cardiomyocyte apoptosis during myocardial I/R is unclear. Hereby, the purpose of this study is to investigate whether the cardioprotective effects of sitagliptin, a DPP4 inhibitor, is relative to its anti-apoptotic function and to explore the underlying mechanism. We hypothesized that sitagliptin played the role of cardioprotection in a myocardial I/R rat model by reducing cardiomyocyte apoptosis. To test this hypothesis, we pretreated rats with sitagliptin for 2 weeks before inducing myocardial I/R. Then the effects of sitagliptin on myocardial injury and cardiomyocyte apoptosis were determined. Finally, we used the GLP-1 receptor antagonist exendin-(9-39) to assess the role of GLP-1 receptor-dependent pathway in the cardioprotective effects of sitagliptin.
after the rats were euthanized. Then the heart was transected parallel to the atrioventricular groove at the center of the ischemia area as previously described (Li et al., 2010). The right ventricle and atria were rapidly removed, and the left ventricle was weighed. The left ventricular weight index was expressed as the ratio of left ventricular weight to body weight. And the blood plasma samples and heart tissue were collected immediately and stored at 80 1C. 2.3. Hemodynamic measurements During the entire I/R period, the right common carotid artery and left femoral artery were isolated. A polystyrene PE-20 catheter was inserted into the left ventricle via right common carotid artery, with one end connected to MPA-2000 multichannel physiologic recorder. The left ventricular end-systolic pressure (LVESP), left ventricular end-diastolic pressure (LVEDP) and the rates of maximum positive and negative left ventricular pressure development (7 LVdp/dt max) were measured.
2. Materials and methods 2.4. ELISA assay 2.1. Experimental animals and drugs Male Sprague–Dawley rats aged between 6 and 8 weeks were purchased from the Laboratory Animal Center of Chongqing Medical University [certificate: SCXK (YU) 2007-0001]. Rats were housed under optimal conditions with standard hygiene, temperature, photoperiods (12L: 12D), standard rat chow and water ad libitum. All of these conditions were conformed to the Guidelines for Care and Use of Laboratory Animals. All procedures on animals were approved by the Ethical Committee of the Chongqing Medical University. The DPP4 inhibitor sitagliptin was purchased from Merck Sharp & Dohme Italia SPA. The PI3K inhibitor LY294002 was purchased from Santa Cruz Biotechnology, Inc. The GLP-1 receptor antagonist exendin-(9-39) was purchased from Sigma, St. Louis, MO, USA. 2.2. Establishment of myocardial I/R injury model Forty Male Sprague–Dawley rats were randomly divided into the following five groups (n¼8): the Sham group, the I/R group, the sitagliptinþI/R group (sitagliptin), the sitagliptinþexendin-(939)þI/R group (sitagliptinþE) and the sitagliptinþLY294002þ I/R group (sitagliptinþL). Sitagliptin (300 mg/kg/day) was administrated by intraperitoneal injection for 2 weeks. Exendin-(9-39) (45 μg/kg/3 days) and LY294002 (0.3 mg/kg/3 days) were given by intraperitoneal injection 30 min before sitagliptin injection. Sitagliptin, exendin-(9-39) and LY294002 were all dissolved in dimethyl sulfoxide (DMSO). The Sham group and the I/R group received the same volume of DMSO for 2 weeks. After pretreatment with sitagliptin for 2 weeks, all rats were anesthetized by chloral hydrate (concentration 3.5%, 10 ml/kg). Tracheotomy was carried out for ventilation by a respirator (ALC-V8B, Shanghai Alcott Biotech Co., Ltd.) with a stroke volume of 28 ml/kg, air pressure of 10 mmHg, respiration rate of 1:1 and at a rate of 86 strokes per minute. And the electrocardiogram of lead II was monitored. Thoracotomy was performed and the left anterior descending coronary artery was ligated by 6-0 silk. Then the left anterior descending coronary artery was subjected to 30 min of ischemia followed by reperfusion for 2 h. Rats in the Sham group were subjected to the same surgery process without coronary artery ligation. Glucose levels were measured with a blood glucose monitor (Accu-Checks, Roche, Germany). Body weights of rats were weighted after the establishment of the I/R model. At the end of hemodynamic measurement, the blood plasma samples were collected from the heart using the anticoagulant tube. The hearts were rapidly excised and arrested in diastole in cold diethyl pyrocarbonate water
Levels of active GLP-1 and creatine kinase-MB (CK-MB) in the plasma were detected using ELISA kits according to the instructions provided by the manufacturer (R&D Systems, Minneapolis, MN, USA). Briefly, plasma was centrifuged at 1600g for 10 min at 4 1C. The supernatants were collected for the detection of GLP-1 and CKMB. Then the supernatants were incubated with the regents in kits. Finally, the absorbance values were measured using a microplate reader (Multiskan MK33, Thermolab systems, Helsinki, Finland). The GLP-1 level was expressed as pmol/l. The CK-MB level was expressed as U/l. The experiment of CK-MB was conducted for three times. 2.5. Colorimetry The activity of lactate dehydrogenase (LDH) in plasma and the concentrations of malondialdehyde (MDA), glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) in heart homogenate were determined by colorimetry. The experiment was performed using commercially available kits, according to the manufacturer's instructions (Jiancheng Bioengineering Institute, Nanjing, China). Briefly, plasma was collected as above described. Heart tissues were collected and lysed by cell lysis buffer. Then cell lysates were centrifuged at 1600g for 10 min at 4 1C. The supernatants of plasma and heart cell lysates were collected for the detection of LDH, MDA, GSH-Px and SOD. After incubation with the reagents in kits, the absorbance values at 340 nm, 450 nm, 412 nm and 532 nm were measured using a spectrophotometer (721D, Pudong Shanghai Physical Optical Instrument Factory, Shanghai, China). The LDH level was expressed as U/ml. The SOD and GSH-Px levels were expressed as U/mg protein. The MDA levels were expressed as nmol/mg protein. The experiment of LDH was performed for three times. 2.6. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining TUNEL staining was performed with the TUNEL staining assay kit according to the manufacturer's instructions (Boster Bioengineering Co., Ltd., Wuhan, China). Briefly, after deparaffinization, tissue sections were first treated with hydrogen peroxide (3%) and then digested with proteinase K (20 μg/ml; pH 7.4) at 25 1C. After digestion for 10 min, tissue sections were incubated with the labeling buffer (1:18) at 37 1C. After incubation for 120 min, tissue sections were incubated with biotinylated anti-digoxin antibody (1:100) for 30 min at 37 1C. Then incorporated fluorescein was detected with streptavidin–biotin-peroxidase and subsequently tissue sections were dyed with 3,3′-diaminobenzidine (DAB).
Please cite this article as: Chang, G., et al., Protective effects of sitagliptin on myocardial injury and cardiac function in an ischemia/ reperfusion rat model. Eur J Pharmacol (2013), http://dx.doi.org/10.1016/j.ejphar.2013.09.007i
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This assay detects apoptotic cells by labeling the 3′-OH end DNA fragments with digoxigenin–deoxyuridine triphosphate (Dig–dUTP) using terminal deoxynucleotidyl transferase. The nuclei of apoptotic cells were stained brown and the nuclei of normal cells were stained blue. Apoptotic index was determined as the ratio of brown nuclei number to the total number of nuclei. Nuclei in a total of 10 fields per tissue slice (n¼ 6) were included. 2.7. Flow cytometry analysis Myocardial cells were isolated from heart homogenate by filtration. After washing with ice-cold PBS, cells were double stained with propidium iodide and FITC-coupled annexin V for 20 min. Flow cytometry was performed with a 488 nm laser coupled to a FacsCalibur cell sorter (BD FACSvantage SE, Beckman Coulter, America). Cells stained with both propidium iodide and annexin V were considered necrotic and cells stained only with annexin V were considered apoptotic. 2.8. Western blot analysis Protein samples were isolated from the left ventricular myocardium of I/R rats. Left ventricular myocardium lysates were prepared by homogenization in cell lysis buffer (Beyotime Institute of Biotechnology, China). Lysates were kept on ice for 45 min and total cardiac proteins were isolated by centrifugation at 14,000g for 10 min at 4 1C. Proteins were separated by SDS-PAGE and transferred to membranes. The membranes were blocked in 5% nonfat milk and incubated with primary antibodies. The primary antibodies included anti-AKT antibody (1:1000, Cell Signaling Technology, Inc.), anti-phosphoAKTserine473 antibody (1:1000, Cell Signaling Technology, Inc.), anticleaved caspase-3 antibody (1:1000, Cell Signaling Technology, Inc.), anti-caspase-3 antibody (1:1000, Cell Signaling Technology, Inc.), antiphospho-Badserine136 antibody (1:500, Santa Cruz Biotechnology, Inc.), anti-Bcl-2 antibody (1:1000, Cell Signaling Technology, Inc.), anti-Bax antibody (1:1000, Cell Signaling Technology, Inc.) and anti-GAPDH antibody (1:1000, Beyotime Institute of Biotechnology, China). Then the membranes were incubated with secondary antibodies (Beyotime Institute of Biotechnology, China). The signals were detected with the ECL system (Beyotime Institute of Biotechnology, China). Blots were scanned using Bio-Rad gel imaging system (Bio-Rad Company, USA) and bands were quantified with the Quantity One software. 2.9. Statistical analysis The SPSS 17.0 software was used for statistical analysis. Data were presented as mean 7standard deviation (S.D.). Grouped data were analyzed using a one-way analysis of variance (ANOVA) followed by the Student–Newman–Keuls (SNK) test. When the equal variance test failed, a Mann–Whitney Rank Sum test was used. A P value of less than 0.05 was considered statistically significant.
3. Results 3.1. Sitagliptin does not affect the glucose level, body weight, left ventricular weight and left ventricular weight index In order to roll out the possible side effects of sitagliptin, we measured the basic clinical features of rats after sitagliptin treatment. The basic clinical features included the blood glucose level, body weight, left ventricular weight and left ventricular weight index. The blood glucose levels were measured at 2 weeks before inducing I/R. Statistically, the differences in the blood glucose levels among the five groups were not significant (data not shown). Body weight, left
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ventricular weight and left ventricular weight index were measured after the inducement of I/R model. Similarly there were no significant differences among the five groups (data not shown). 3.2. Sitagliptin upregulates the plasma GLP-1 level after myocardial injury in I/R rats To examine the effect of sitagliptin on plasma GLP-1, we measured the plasma level of GLP-1 after inducing I/R by ELISA assay. Our results (data not shown) were consistent with previous reports. We found that the level of GLP-1 was significantly decreased in I/R group, when compared with that in Sham group (P¼0.006). In contrast, the level of GLP-1 was significantly increased in sitagliptin group compared with that in I/R group (Po0.001). Meanwhile, the levels of GLP-1 in sitagliptinþE group and sitagliptinþ L group were not significantly different from the level of GLP-1 in sitagliptin group (P40.05). 3.3. Sitagliptin reduces the LDH and CK-MB release after myocardial injury in I/R rats LDH and CK-MB are the diagnostic markers of myocardial tissue damage. Thus, we examined the effects of sitagliptin on LDH and CKMB levels in plasma. The changes in LDH and CK-MB levels in this study (data not shown) were also consistent with previous reports. Statistically, the levels of LDH (Po0.001) and CK-MB (Po0.001) in I/R group were significantly higher than those in Sham group. Compared with those in I/R group, the levels of LDH (Po0.001) and CK-MB (Po0.001) in sitagliptin group were significantly lower. In addition, the LDH levels in sitagliptinþ E group (Po0.001) and sitagliptinþ L group (Po0.001) were significantly higher than those in sitagliptin group. Meanwhile, the CK-MB levels in sitagliptinþ E group (Po0.001) and sitagliptinþL group (Po0.001) were also significantly higher than those in sitagliptin group. 3.4. Sitagliptin increases SOD and GSH-Px and decreases MDA after myocardial injury in I/R rats GSH-Px, SOD and catalase are important enzymes of the first line cellular defense against oxidative injury. Therefore, we examined the effects of sitagliptin on levels of SOD, GSH-Px and MDA in myocardial tissue. As shown in Fig. 1A, the concentrations of SOD in Sham group were significantly higher than those in I/R group (P o0.001). Compared with those in I/R group, the concentrations of SOD in sitagliptin group were significantly increased (P o0.001). However, the concentrations of SOD in sitagliptin þE group (P o0.001) and sitagliptin þ L group (P o0.001) were significantly lower than those in sitagliptin group. The effects of sitagliptin on concentrations of GSH-Px are shown in Fig. 1B. Statistically, the concentrations of GSH-Px in I/R group were significantly decreased than those in Sham group (Po 0.001). And compared with those in I/R group, the concentrations of GSH-Px in sitagliptin group were significantly increased (Po 0.001). However, compared with those in sitagliptin group, the concentrations of GSHPx in sitagliptin þ E group (P o0.001) and sitagliptin þL group (P o0.001) were significantly decreased. The effects of sitagliptin on concentrations of MDA are shown in Fig. 1C. Statistically, the concentrations of MDA in I/R group were significantly higher than those in Sham group (P o0.001). And compared with those in I/R group, the concentrations of MDA in sitagliptin group were significantly decreased (Po 0.001). However, compared with sitagliptin group, the concentrations of MDA in sitagliptinþE group (P o0.001) and sitagliptinþL group (P o0.001) were significantly increased.
Please cite this article as: Chang, G., et al., Protective effects of sitagliptin on myocardial injury and cardiac function in an ischemia/ reperfusion rat model. Eur J Pharmacol (2013), http://dx.doi.org/10.1016/j.ejphar.2013.09.007i
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G. Chang et al. / European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
Fig. 1. Effects of sitagliptin on levels of SOD, GSH-Px and MDA in heart homogenate of I/R rats. Levels of SOD, GSH-Px and MDA in heart homogenate of I/R rats were measured by colorimetry. (A) Levels of SOD in heart homogenate of I/R rats. Significance was determined by ANOVA followed by the SNK test, F¼ 63.255. (B) Levels of GSH-Px in heart homogenate of I/R rats. Significance was determined by ANOVA followed by the SNK test, F¼53.189. (C) Levels of MDA in heart homogenate of I/R rats. Significance was determined by ANOVA followed by the SNK test, F¼ 74.287. SOD: superoxide dismutase; GSH-Px: glutathione peroxidase; and MDA: malondialdehyde. Values were expressed as mean7 S.D. Sham: Sham group; I/R: ischemia/reperfusion group; sitagliptin: sitagliptin 300 mg/kg/day pretreatment group; sitagliptinþ E: sitagliptin 300 mg/ kg/day and exendin-(9-39) 45 μg/kg/3 days pretreatment group; sitagliptinþL: sitagliptin 300 mg/kg/day and LY294002 0.3 mg/kg/3 days pretreatment group; N ¼8 in each group; nPo 0.05 vs. Sham group; #Po 0.05 vs. I/R group; and ▲Po 0.05 vs. sitagliptin group.
3.5. Sitagliptin enhances left ventricular function after myocardial injury in I/R rats To determine the effects of sitagliptin on cardiac function in I/R rats, hemodynamic measurements were performed during the entire I/R period. As shown in Fig. 2, compared with those in Sham group, I/R treatment significantly decreased the þ LVdp/dt max (Po0.001), LVdp/dt max (Po0.001), and LVESP (Po0.001) and significantly increased the LVEDP (Po0.001). Compared with those in I/R group, sitagliptin significantly enhanced the þLVdp/dt max (Po0.001), LVdp/dt max (Po0.001), and LVESP (P¼ 0.014) and significantly reduced the LVEDP (P¼0.003). However, the GLP-1 receptor antagonist exendin-(9-39) and the PI3K inhibitor LY294002 abolished the effects of sitagliptin on þLVdp/dt max (Po0.001, Po0.001), LVdp/ dt max (Po0.001, Po0.001), LVESP (P¼0.012, P¼ 0.03) and LVEDP (P¼0.017, P¼ 0.009). 3.6. Sitagliptin inhibits cardiomyocyte apoptosis after myocardial injury in I/R rats We examined the effects of sitagliptin on cell apoptosis in myocardial tissue by TUNEL assay and flow cytometry analysis. The representative graphs of TUNEL assay and flow cytometry analysis are shown in Fig. 3A and C, respectively. The apoptotic index of TUNEL assay is shown in Fig. 3B and the apoptosis ratio of flow cytometry analysis is shown in Fig. 3D. Representative
photomicrograph showed that TUNEL staining positive apoptotic cells were more frequently observed in I/R group, sitagliptinþ E group and sitagliptinþ L group as compared with Sham group and sitagliptin group (Fig. 3A). Statistically, the apoptotic index in sitagliptin group was significantly lower than those in I/R group (Po0.001), sitagliptinþE group (Po0.001) and sitagliptinþ L group (Po0.001). As analyzed by flow cytometry, apoptotic cell ratio in I/R group was significantly increased compared with that in Sham group (P o0.001). However, compared with that in I/R group, apoptosis ratio was significantly decreased in sitagliptin group (Po 0.001). Also apoptosis ratios in sitagliptinþE group (P o0.001) and sitagliptin þ L group (Po 0.001) were significantly increased compared with those in sitagliptin group. 3.7. Sitagliptin increases expression of anti-apoptotic proteins and inhibits expression of pro-apoptotic proteins after myocardial injury in I/R rats The effects of sitagliptin on pho-Aktserine473, pho-Badserine136, caspase-3, cleaved caspase-3, Bax and Bcl-2 in myocardial tissue were analyzed by western blot (Fig. 4). The representative western blot results are shown in Fig. 4A and the quantitative results are shown in Figs. 4B–E, G and H. And the ratio of Bax/Bcl-2 is shown in Fig. 4F. Compared with those in Sham group, I/R treatment significantly decreased levels of pho-Aktserine473 (P¼0.006), pho-Badserine136
Please cite this article as: Chang, G., et al., Protective effects of sitagliptin on myocardial injury and cardiac function in an ischemia/ reperfusion rat model. Eur J Pharmacol (2013), http://dx.doi.org/10.1016/j.ejphar.2013.09.007i
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Fig. 2. Effects of sitagliptin on left ventricular function in I/R rats. The cardiac functions of þLVdp/dt max, LVdp/dt max, LVESP and LVEDP were measured by multichannel physiologic recorder. (A) Values of þ LVdp/dt max (rates of maximum positive left ventricular pressure development). Significance was determined by ANOVA followed by the SNK test, F ¼66.053. (B) Values of LVdp/dt max (rates of maximum negative left ventricular pressure development). Significance was determined by ANOVA followed by a Mann–Whitney Rank Sum test, F¼ 28.161. (C) Values of left ventricular end-systolic pressure (LVESP). Significance was determined by ANOVA followed by the SNK test, F¼ 25.952. (D) Values of left ventricular end-diastolic pressure (LVEDP). Significance was determined by ANOVA followed by the SNK test, F ¼24.460. Values were expressed as mean 7 S.D. Sham: Sham group; I/R: ischemia/reperfusion group; sitagliptin: sitagliptin 300 mg/kg/day pretreatment group; sitagliptin þE: sitagliptin 300 mg/kg/day and exendin-(9-39) 45 μg/kg/3 days pretreatment group; sitagliptinþ L: sitagliptin 300 mg/kg/day and LY294002 0.3 mg/kg/3 days pretreatment group; N ¼ 8 in each group; n P o0.05 vs. Sham group; #Po 0.05 vs. I/R group; and ▲Po 0.05 vs. sitagliptin group.
(P¼0.002) and Bcl-2 (Po0.001) expression. Meanwhile, compared with those in Sham group, I/R treatment significantly increased the expression levels of caspase-3 (Po0.001), cleaved caspase-3 (Po0.001) and Bax (Po0.001). Compared with those in I/R group, sitagliptin significantly enhanced the levels of pho-Aktserine473 (P¼0.01), pho-Badserine136 (Po0.001) and Bcl-2 (Po0.001). At the same time, compared with those in I/R group, sitagliptin significantly reduced levels of caspase-3 (Po0.001), cleaved caspase-3 (P¼0.02) and Bax (Po0.001). Also the Bax/Bcl-2 ratio was significantly decreased in sitagliptin group than that in I/R group (Po0.001). However, the GLP-1 receptor antagonist exendin-(9-39) and the PI3K inhibitor LY294002 attenuated the effects of sitagliptin on antiapoptotic and pro-apoptotic proteins. Statistically, compared with sitagliptin group, exendin-(9-39) and LY294002 administration significantly reduced the levels of pho-Aktserine473 (P¼ 0.011, P¼0.007), pho-Badserine136 (Po0.001, Po0.001) and Bcl-2 (Po0.001, Po0.001). And compared with sitagliptin group, exendin-(9-39) and LY294002 administration significantly increased the expression levels of caspase3 (Po0.001, Po0.001), cleaved caspase-3 (P¼0.001, P¼ 0.001) and Bax (Po0.001, Po0.001).
4. Discussion The major finding of our study was that sitagliptin pretreatment could reduce myocardial injury and improve cardiac function by
inhibiting cardiomyocyte apoptosis in an I/R rat model. Though DPP4 inhibitors were reported to have cardioprotective effects during I/R, this is the first study to examine the effects of sitagliptin on cardiomyocyte apoptosis and oxidative stress induced by myocardial I/R. In addition, our data indicate that sitagliptin could decrease the levels of pro-apoptotic proteins and increase the levels of anti-apoptotic proteins. However, the above observed effects of sitagliptin were all abolished when co-administered with GLP-1 receptor antagonist exendin-(9-39) or PI3K inhibitor LY294002. Thus we speculate that sitagliptin protected the heart in I/R rats from injury through the decrease of apoptosis and oxidative damage and the activation of PI3K/Akt signaling pathway. As reported by previous studies (Khalil et al., 2005; Sun et al., 2012), myocardial I/R impaired cardiac function, increased the release of LDH and CK-MB and the apoptotic rate of cardiomyocyte. We found similar results in this study. Importantly, we found that sitagliptin reduced LDH and CK-MB release in I/R rats. We speculate that this effect might be ascribed to its potential to resist against oxidative stress. Interestingly, we demonstrated that sitagliptin significantly increased the levels of SOD and GSHPx and decreased the level of MDA in myocardial tissues in I/R rats. As we all know, SOD and GSH-Px are key antioxidant enzymes, which constitute first line cellular defense against oxidative injury. MDA is one of the products of oxidative stress, which reflects the damage of cell caused by oxidative stress. To our knowledge, our study demonstrated for the first time that
Please cite this article as: Chang, G., et al., Protective effects of sitagliptin on myocardial injury and cardiac function in an ischemia/ reperfusion rat model. Eur J Pharmacol (2013), http://dx.doi.org/10.1016/j.ejphar.2013.09.007i
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G. Chang et al. / European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
Fig. 3. Effects of sitagliptin on cardiomyocyte apoptosis in I/R rats. Apoptosis were analyzed by TUNEL assay and flow cytometry analysis, respectively. (A) Representative graphics of TUNEL staining. (B) Quantitative results of TUNEL staining (400 ). Apoptotic index was determined as the ratio of brown nuclei number to the total number of nuclei. Nuclei in a total of 10 fields per tissue slice (N ¼ 6) were included. Significance was determined by ANOVA followed by the SNK test, F¼ 69.639. (C) Representative flow cytometry results. (D) Quantitative results of flow cytometry results, N ¼ 8. Apoptosis ratio was determined as the ratio of Annexin V positive and propidium iodide negative cells to total cells analyzed. Significance was determined by ANOVA followed by the SNK test, F¼ 70.412. Values were expressed as mean 7S.D. Sham: Sham group; I/R: ischemia/reperfusion group; sitagliptin: sitagliptin 300 mg/kg/day pretreatment group; sitagliptinþ E: sitagliptin 300 mg/kg/day and exendin-(9-39) 45 μg/kg/3 days pretreatment group; sitagliptinþ L: sitagliptin 300 mg/kg/day and LY294002 0.3 mg/kg/3 days pretreatment group; nPo 0.05 vs. Sham group; #Po 0.05 vs. I/R group; and ▲ Po 0.05 vs. sitagliptin group.
pretreatment with sitagliptin could increase the concentrations of antioxidant defense enzymes including GSH-Px and SOD, and decrease the production of MDA in I/R rats. We also found that sitagliptin administration significantly improved the cardiac function via increasing 7LVdp/dt max, LVESP and limiting the increase of LVEDP. Similarly, Sauvé et al. (2010) observed that the left ventricular function was improved in
mice pretreated with sitagliptin for 12 h prior to aortic occlusion and in DPP4 deleted mice after I/R injury. Ku et al. (2011) also reported that DPP4 deficiency could preserve cardiac function in rats subjected to myocardial I/R. In a clinical study, single dose sitagliptin treatment could improve the regional and global left ventricular function in patients with coronary artery disease (Read et al., 2010). However, a recent study demonstrated that in rats
Please cite this article as: Chang, G., et al., Protective effects of sitagliptin on myocardial injury and cardiac function in an ischemia/ reperfusion rat model. Eur J Pharmacol (2013), http://dx.doi.org/10.1016/j.ejphar.2013.09.007i
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Fig. 4. Effects of sitagliptin on expression levels of anti-apoptotic proteins and pro-apoptotic proteins in I/R rats. Expression levels of apoptosis related proteins were analyzed by western blot. (A) Representative western blot results. (B) Ratios of phospho-AKTserine473 to total AKT. Significance was determined by ANOVA followed by a Mann–Whitney Rank Sum test, F¼ 20.546. (C) Ratios of phospho-Badserine136 to GAPDH. Significance was determined by ANOVA followed by the SNK test, F¼ 10.407. (D) Ratios of Bax to GAPDH. Significance was determined by ANOVA followed by a Mann–Whitney Rank Sum test, F¼ 212.404. (E) Ratios of Bcl-2 to GAPDH. Significance was determined by ANOVA followed by a Mann–Whitney Rank Sum test, F¼75.357 (F) Ratios of Bax to Bcl-2. Significance was determined by ANOVA followed by a Mann–Whitney Rank Sum test, F¼ 84.528. (G) Ratios of Caspase-3 to GAPDH. Significance was determined by ANOVA followed by the SNK test, F¼ 70.092. (H) Ratios of cleaved caspase-3 to GAPDH. Significance was determined by ANOVA followed by the SNK test, F¼17.488. Values were expressed as mean7S.D. Sham: Sham group; I/R: ischemia/reperfusion group; sitagliptin: sitagliptin 300 mg/kg/day pretreatment group; sitagliptinþE: sitagliptin 300 mg/kg/day and exendin-(9-39) 45 μg/kg/3 days pretreatment group; sitagliptinþL: sitagliptin 300 mg/kg/day and LY294002 0.3 mg/kg/3 days pretreatment group; N¼ 8 in every group; nPo0.05 vs. Sham group; #Po0.05 vs. I/R group; and ▲Po0.05 vs. sitagliptin group.
with ischemic heart failure, early or late treatment with vildagliptin had no beneficial effect on ventricular performance (Yin et al., 2011). Meanwhile, Sauvé et al. (2010) also reported that acute treatment with sitagliptin for 20 min prior to I/R injury failed to improve ventricular function. The discrepancy in these studies might be attributed to the differences in the duration of treatment time and differences in types of drug administration as well as the differences in study models.
Cardiomyocyte apoptosis is one of the critical reasons of heart failure after myocardial infarction. Many studies have confirmed that blocking the apoptosis process could reduce the loss of cardiomyocyte, minimize myocardial injury and improve ventricular performance induced by I/R (Mughal et al., 2012). Given the significantly improved recovery of cardiac function, we examined the effects of sitagliptin on preventing cardiomyocyte apoptosis in I/R rats. We demonstrated that sitagliptin administration
Please cite this article as: Chang, G., et al., Protective effects of sitagliptin on myocardial injury and cardiac function in an ischemia/ reperfusion rat model. Eur J Pharmacol (2013), http://dx.doi.org/10.1016/j.ejphar.2013.09.007i
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significantly decreased apoptosis ratio of cardiomyocytes as revealed by TUNEL staining and flow cytometry analysis. And sitagliptin administration significantly reduced expression levels of caspase-3 and cleaved caspase-3 in rats subjected to I/R injury. It has been reported that PI3K/Akt signaling pathway activation could inhibit cardiomyocyte apoptosis after I/R injury (Fujio et al., 2000; Matsui et al., 2001; Mullonkal and Toledo-Pereyra, 2007). The mechanisms of the anti-apoptotic effect are acted through various means, such as inhibiting caspase activation, inhibiting death genes expression and regulating the activity of Bcl-2 family. Bcl-2 family, the key regulators of apoptosis, consists of both cell death promoters such as Bax and Bad, and cell death inhibitors including Bcl-2, Bcl-x, etc. It is reported that the high ratio of Bax/ Bcl-2 was associated with great possibility to apoptotic activation (García-Sáez, 2012). In our study, we demonstrated that sitagliptin could increase phosphorylation levels of Akt and Bad and decrease expression levels of caspase-3, cleaved caspase-3 and Bax. Meanwhile, we also found that sitagliptin could up-regulate Bcl-2 expression, resulting in decreased Bax/Bcl-2 ratio in I/R rats. These effects of sitagliptin were correlated with cardiomyocyte apoptosis attenuation. Hence, we next examined whether the sitagliptin exerted its anti-apoptotic action through activation of PI3K/Akt pathway in rats subjected to I/R injury. The PI3K inhibitor LY294002 was employed. We found that co-administration of LY294002 and sitagliptin decreased phosphorylation levels of Akt and Bad and increased expression levels of caspase-3, cleaved caspase-3, Bax, and Bax/Bcl-2 ratio. These findings suggested that LY294002 could abolish the anti-apoptotic effects of sitagliptin. The anti-apoptotic effects of sitagliptin were related to, at least in part, activation of PI3K/Akt signaling pathway. The function of DPP4 inhibitor is to inhibit the proteolytic activity of DPP4 enzyme, postponing the myocardial degradation of GLP-1 (Barnett, 2006). Our data showed that sitagliptin administration resulted in significant accumulation of plasma GLP-1 in I/R rats. This result was similar to previous data reported by Ku et al. (2011) and Ye et al. (2010). Moreover, our results showed that higher levels of plasma GLP-1 were associated with lower levels of cardiac injury markers, higher levels of antioxidant enzymes, lower ratio of cardiomyocyte apoptosis and better ventricular performance in I/R rats. However, these effects were attenuated by the GLP-1 receptor antagonist exendin-(9-39). Exendin-(9-39) has been widely used to estimate the role of receptor-dependent pathway. Thus we speculated that the cardioprotective effects of sitagliptin might be attributed to, at least in part, the GLP-1 receptor-dependent pathway. In summary, sitagliptin exerted its action via up-regulating the level of GLP-1, which activated the PI3K/Akt signaling pathway via binding GLP1 receptor. The prominent finding of this study was that sitagliptin pretreatment could reduce myocardial injury and improve cardiac function in an I/R rat model. The possible mechanisms might be relative to decrease of apoptosis and oxidative damage, up-regulation of GLP-1 level (which activated the PI3K/Akt signaling pathway via binding GLP-1 receptor), increase of Bad phosphorylation and decrease of Bax/Bcl-2 ratio. However, exact cardioprotective mechanisms of DPP4 inhibitors need a further study. To sum up, we conclude that sitagliptin administration has protective effects on myocardial I/R injury. Our findings could provide deeper insights into the treatment of heart disease.
Acknowledgments This work was supported by the National Natural Science Funds for Youths (Grant no. 81100196), Natural Science Foundation
Project of CQ CSTC (Grant no. CSTC, 2011BB5133). Foundation project of Traditional Chinese Medicine of Chongqing Municipal Health Bureau (Grant no. 2012-2-125) and Pfizer pharmaceutical limited competition grants (Grant no. ws1790576). We greatly appreciate Jianyong Wu and Dezhang Zhao (Institute of Life Sciences, Chongqing Medical University) for their excellent technical support for the flow cytometry analysis.
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Ye, Y., Keyes, K.T., Zhang, C., Perez-Polo, J.R., Lin, Y., Birnbaum, Y., 2010. The myocardial infarct size-limiting effect of sitagliptin is PKA-dependent, whereas the protective effect of pioglitazone is partially dependent on PKA. American Journal of Physiology: Heart and Circulatory Physiology 298, H1454–H1465. Yin, M., Silljé, H.H., Meissner, M., van Gilst, W.H., de Boer, R.A., 2011. Early and late effects of the DPP-4 inhibitor vildagliptin in a rat model of post-myocardial infarction heart failure. Cardiovascular Diabetology 10, 85–94.
Please cite this article as: Chang, G., et al., Protective effects of sitagliptin on myocardial injury and cardiac function in an ischemia/ reperfusion rat model. Eur J Pharmacol (2013), http://dx.doi.org/10.1016/j.ejphar.2013.09.007i
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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Cardiovascular pharmacology
Protective effects of sitagliptin on myocardial injury and cardiac function in an ischemia/reperfusion rat model Guanglei Chang, Peng Zhang, Lin Ye, Kai Lu, Ying Wang, Qin Duan, Aihua Zheng, Shu Qin n, Dongying Zhang nn Department of Cardiology, The First Affiliated Hospital of Chongqing Medical University, No.1 Yixueyuan Road, Chongqing 400016, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 20 March 2013 Received in revised form 28 August 2013 Accepted 4 September 2013
The purpose of this study is to investigate the effects and the underlying mechanisms of sitagliptin pretreatment on myocardial injury and cardiac function in myocardial ischemia/reperfusion (I/R) rat model. The rat model of myocardial I/R was constructed by coronary occlusion. Rats were pretreated with sitagliptin (300 mg/kg/day) for 2 weeks, and then subjected to 30 min ischemia and 2 h reperfusion. The release of lactate dehydrogenase (LDH) and creatine kinase-MB (CK-MB), cardiac function and cardiomyocyte apoptosis were evaluated. The levels of malondialdehyde (MDA), glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) in heart and glucagon-like peptide-1 (GLP-1) level in plasma were measured. Western blot analysis was performed to detect the target proteins of sitagliptin. Our results showed that sitagliptin pretreatment decreased LDH and CK-MB release, and MDA level in I/R rats. More importantly, we revealed for the first time that sitagliptin pretreatment decreased cardiomyocyte apoptosis while increased the levels of GSH-Px and SOD in heart. Sitagliptin also increased GLP1 level and enhanced cardiac function in I/R rats. Furthermore, sitagliptin pretreatment up-regulated Aktserine473 and Badserine136 phosphorylation, reduced the ratio of Bax/Bcl-2, and decreased expression levels of cleaved caspase-3 and caspase-3. Interestingly, the above observed effects of sitagliptin were all abolished when co-administered with GLP-1 receptor antagonist exendin-(9-39) or PI3K inhibitor LY294002. Taken together, our data indicate that sitagliptin pretreatment could reduce myocardial injury and improve cardiac function in I/R rats by reducing apoptosis and oxidative damage. The underlying mechanism might be the activation of PI3K/Akt signaling pathway by GLP-1/GLP-1 receptor. Crown Copyright & 2013 Published by Elsevier B.V. All rights reserved.
Keywords: DPP4 inhibitor Sitagliptin Ischemia/reperfusion Myocardial injury Cardiomyocyte apoptosis Cardiac function
1. Introduction Myocardial infarction is a major cause of mortality and morbidity of patients with diabetes mellitus (Acar et al., 2011). In order to prevent the myocardium from further damage, the best therapeutic strategy for myocardial infarction is to reestablish the blood flow as earlier as possible. Nevertheless, ischemia/reperfusion (I/R) injury such as cardiomyocyte apoptosis is inevitable. Cardiomyocyte apoptosis induced by I/R plays an important role in causing a gradual decline of cardiac function (Gottlieb, 2011). Therefore, the exploration of new therapeutic agents that reduce I/R injury of myocardial infarction patients has become very important. Glucagon-like peptide-1 (GLP-1) is secreted by the enteroendocrine L cells of the intestinal mucosa and released in response to nutrient ingestion (Nauck et al., 1993). It exerts insulinotropic and insulinomimetic effects via the G-protein-coupled GLP-1 receptor n
Corresponding author. Tel.: 86 13101345177; fax: 86 2389011562. Corresponding author. Tel.: 86 13650502588; fax: 86 2368055542. E-mail addresses: [email protected] (S. Qin), [email protected] (D. Zhang). nn
(Verge and Lopez, 2010). The therapy based on the functions of GLP1 is currently used as a novel anti-diabetic approach (Doupis and Veves, 2008; Garber, 2012). However, GLP-1 is rapidly degraded by dipeptidyl peptidase-4 (DPP4) enzyme in the blood (Green et al., 2006). The short half life time limited its clinical use. Thus, two classes of drugs, including GLP-1 analogs (Garber, 2012) (i.e. exenatide) and DPP4 inhibitors (Doupis and Veves, 2008) (i.e. sitagliptin), have been recently used for treating type 2 diabetes. Recently, growing evidences have demonstrated the beneficial effects of GLP-1 analogs during I/R injury in both animal models and in clinical studies, such as limiting infarct, improving cardiac function and enhancing myocardial glucose uptake (Bhashyam et al., 2010; Chinda et al., 2012a; Lorber, 2012; Mundil et al., 2012). The mechanisms underlying the cardioprotective effects of GLP-1 analogs may be both GLP-1 receptor dependent and independent pathways (Ban et al., 2008; Chinda et al., 2012a). Unlike GLP-1 analogs, evidences regarding the cardioprotective effects of DPP4 inhibitors are scarce and controversial. Recently, more and more researchers have paid close attention to the cardioprotective effects of DPP4 inhibitors. Chinda, et al. (2012b) reported that DPP4 inhibitor could stabilize cardiac electrophysiology in a myocardial I/R pig model.
0014-2999/$ - see front matter Crown Copyright & 2013 Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejphar.2013.09.007
Please cite this article as: Chang, G., et al., Protective effects of sitagliptin on myocardial injury and cardiac function in an ischemia/ reperfusion rat model. Eur J Pharmacol (2013), http://dx.doi.org/10.1016/j.ejphar.2013.09.007i
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In addition, DPP4 inhibitor has been shown to attenuate the infarct size and improve the left ventricular function during myocardial I/R injury (Chinda et al., 2012a; Jose and Inzucchi, 2012; Lenski et al., 2011; Scheen, 2012). However, the correlation between its cardioprotective effect and cardiomyocyte apoptosis during myocardial I/R is unclear. Hereby, the purpose of this study is to investigate whether the cardioprotective effects of sitagliptin, a DPP4 inhibitor, is relative to its anti-apoptotic function and to explore the underlying mechanism. We hypothesized that sitagliptin played the role of cardioprotection in a myocardial I/R rat model by reducing cardiomyocyte apoptosis. To test this hypothesis, we pretreated rats with sitagliptin for 2 weeks before inducing myocardial I/R. Then the effects of sitagliptin on myocardial injury and cardiomyocyte apoptosis were determined. Finally, we used the GLP-1 receptor antagonist exendin-(9-39) to assess the role of GLP-1 receptor-dependent pathway in the cardioprotective effects of sitagliptin.
after the rats were euthanized. Then the heart was transected parallel to the atrioventricular groove at the center of the ischemia area as previously described (Li et al., 2010). The right ventricle and atria were rapidly removed, and the left ventricle was weighed. The left ventricular weight index was expressed as the ratio of left ventricular weight to body weight. And the blood plasma samples and heart tissue were collected immediately and stored at 80 1C. 2.3. Hemodynamic measurements During the entire I/R period, the right common carotid artery and left femoral artery were isolated. A polystyrene PE-20 catheter was inserted into the left ventricle via right common carotid artery, with one end connected to MPA-2000 multichannel physiologic recorder. The left ventricular end-systolic pressure (LVESP), left ventricular end-diastolic pressure (LVEDP) and the rates of maximum positive and negative left ventricular pressure development (7 LVdp/dt max) were measured.
2. Materials and methods 2.4. ELISA assay 2.1. Experimental animals and drugs Male Sprague–Dawley rats aged between 6 and 8 weeks were purchased from the Laboratory Animal Center of Chongqing Medical University [certificate: SCXK (YU) 2007-0001]. Rats were housed under optimal conditions with standard hygiene, temperature, photoperiods (12L: 12D), standard rat chow and water ad libitum. All of these conditions were conformed to the Guidelines for Care and Use of Laboratory Animals. All procedures on animals were approved by the Ethical Committee of the Chongqing Medical University. The DPP4 inhibitor sitagliptin was purchased from Merck Sharp & Dohme Italia SPA. The PI3K inhibitor LY294002 was purchased from Santa Cruz Biotechnology, Inc. The GLP-1 receptor antagonist exendin-(9-39) was purchased from Sigma, St. Louis, MO, USA. 2.2. Establishment of myocardial I/R injury model Forty Male Sprague–Dawley rats were randomly divided into the following five groups (n¼8): the Sham group, the I/R group, the sitagliptinþI/R group (sitagliptin), the sitagliptinþexendin-(939)þI/R group (sitagliptinþE) and the sitagliptinþLY294002þ I/R group (sitagliptinþL). Sitagliptin (300 mg/kg/day) was administrated by intraperitoneal injection for 2 weeks. Exendin-(9-39) (45 μg/kg/3 days) and LY294002 (0.3 mg/kg/3 days) were given by intraperitoneal injection 30 min before sitagliptin injection. Sitagliptin, exendin-(9-39) and LY294002 were all dissolved in dimethyl sulfoxide (DMSO). The Sham group and the I/R group received the same volume of DMSO for 2 weeks. After pretreatment with sitagliptin for 2 weeks, all rats were anesthetized by chloral hydrate (concentration 3.5%, 10 ml/kg). Tracheotomy was carried out for ventilation by a respirator (ALC-V8B, Shanghai Alcott Biotech Co., Ltd.) with a stroke volume of 28 ml/kg, air pressure of 10 mmHg, respiration rate of 1:1 and at a rate of 86 strokes per minute. And the electrocardiogram of lead II was monitored. Thoracotomy was performed and the left anterior descending coronary artery was ligated by 6-0 silk. Then the left anterior descending coronary artery was subjected to 30 min of ischemia followed by reperfusion for 2 h. Rats in the Sham group were subjected to the same surgery process without coronary artery ligation. Glucose levels were measured with a blood glucose monitor (Accu-Checks, Roche, Germany). Body weights of rats were weighted after the establishment of the I/R model. At the end of hemodynamic measurement, the blood plasma samples were collected from the heart using the anticoagulant tube. The hearts were rapidly excised and arrested in diastole in cold diethyl pyrocarbonate water
Levels of active GLP-1 and creatine kinase-MB (CK-MB) in the plasma were detected using ELISA kits according to the instructions provided by the manufacturer (R&D Systems, Minneapolis, MN, USA). Briefly, plasma was centrifuged at 1600g for 10 min at 4 1C. The supernatants were collected for the detection of GLP-1 and CKMB. Then the supernatants were incubated with the regents in kits. Finally, the absorbance values were measured using a microplate reader (Multiskan MK33, Thermolab systems, Helsinki, Finland). The GLP-1 level was expressed as pmol/l. The CK-MB level was expressed as U/l. The experiment of CK-MB was conducted for three times. 2.5. Colorimetry The activity of lactate dehydrogenase (LDH) in plasma and the concentrations of malondialdehyde (MDA), glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) in heart homogenate were determined by colorimetry. The experiment was performed using commercially available kits, according to the manufacturer's instructions (Jiancheng Bioengineering Institute, Nanjing, China). Briefly, plasma was collected as above described. Heart tissues were collected and lysed by cell lysis buffer. Then cell lysates were centrifuged at 1600g for 10 min at 4 1C. The supernatants of plasma and heart cell lysates were collected for the detection of LDH, MDA, GSH-Px and SOD. After incubation with the reagents in kits, the absorbance values at 340 nm, 450 nm, 412 nm and 532 nm were measured using a spectrophotometer (721D, Pudong Shanghai Physical Optical Instrument Factory, Shanghai, China). The LDH level was expressed as U/ml. The SOD and GSH-Px levels were expressed as U/mg protein. The MDA levels were expressed as nmol/mg protein. The experiment of LDH was performed for three times. 2.6. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining TUNEL staining was performed with the TUNEL staining assay kit according to the manufacturer's instructions (Boster Bioengineering Co., Ltd., Wuhan, China). Briefly, after deparaffinization, tissue sections were first treated with hydrogen peroxide (3%) and then digested with proteinase K (20 μg/ml; pH 7.4) at 25 1C. After digestion for 10 min, tissue sections were incubated with the labeling buffer (1:18) at 37 1C. After incubation for 120 min, tissue sections were incubated with biotinylated anti-digoxin antibody (1:100) for 30 min at 37 1C. Then incorporated fluorescein was detected with streptavidin–biotin-peroxidase and subsequently tissue sections were dyed with 3,3′-diaminobenzidine (DAB).
Please cite this article as: Chang, G., et al., Protective effects of sitagliptin on myocardial injury and cardiac function in an ischemia/ reperfusion rat model. Eur J Pharmacol (2013), http://dx.doi.org/10.1016/j.ejphar.2013.09.007i
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This assay detects apoptotic cells by labeling the 3′-OH end DNA fragments with digoxigenin–deoxyuridine triphosphate (Dig–dUTP) using terminal deoxynucleotidyl transferase. The nuclei of apoptotic cells were stained brown and the nuclei of normal cells were stained blue. Apoptotic index was determined as the ratio of brown nuclei number to the total number of nuclei. Nuclei in a total of 10 fields per tissue slice (n¼ 6) were included. 2.7. Flow cytometry analysis Myocardial cells were isolated from heart homogenate by filtration. After washing with ice-cold PBS, cells were double stained with propidium iodide and FITC-coupled annexin V for 20 min. Flow cytometry was performed with a 488 nm laser coupled to a FacsCalibur cell sorter (BD FACSvantage SE, Beckman Coulter, America). Cells stained with both propidium iodide and annexin V were considered necrotic and cells stained only with annexin V were considered apoptotic. 2.8. Western blot analysis Protein samples were isolated from the left ventricular myocardium of I/R rats. Left ventricular myocardium lysates were prepared by homogenization in cell lysis buffer (Beyotime Institute of Biotechnology, China). Lysates were kept on ice for 45 min and total cardiac proteins were isolated by centrifugation at 14,000g for 10 min at 4 1C. Proteins were separated by SDS-PAGE and transferred to membranes. The membranes were blocked in 5% nonfat milk and incubated with primary antibodies. The primary antibodies included anti-AKT antibody (1:1000, Cell Signaling Technology, Inc.), anti-phosphoAKTserine473 antibody (1:1000, Cell Signaling Technology, Inc.), anticleaved caspase-3 antibody (1:1000, Cell Signaling Technology, Inc.), anti-caspase-3 antibody (1:1000, Cell Signaling Technology, Inc.), antiphospho-Badserine136 antibody (1:500, Santa Cruz Biotechnology, Inc.), anti-Bcl-2 antibody (1:1000, Cell Signaling Technology, Inc.), anti-Bax antibody (1:1000, Cell Signaling Technology, Inc.) and anti-GAPDH antibody (1:1000, Beyotime Institute of Biotechnology, China). Then the membranes were incubated with secondary antibodies (Beyotime Institute of Biotechnology, China). The signals were detected with the ECL system (Beyotime Institute of Biotechnology, China). Blots were scanned using Bio-Rad gel imaging system (Bio-Rad Company, USA) and bands were quantified with the Quantity One software. 2.9. Statistical analysis The SPSS 17.0 software was used for statistical analysis. Data were presented as mean 7standard deviation (S.D.). Grouped data were analyzed using a one-way analysis of variance (ANOVA) followed by the Student–Newman–Keuls (SNK) test. When the equal variance test failed, a Mann–Whitney Rank Sum test was used. A P value of less than 0.05 was considered statistically significant.
3. Results 3.1. Sitagliptin does not affect the glucose level, body weight, left ventricular weight and left ventricular weight index In order to roll out the possible side effects of sitagliptin, we measured the basic clinical features of rats after sitagliptin treatment. The basic clinical features included the blood glucose level, body weight, left ventricular weight and left ventricular weight index. The blood glucose levels were measured at 2 weeks before inducing I/R. Statistically, the differences in the blood glucose levels among the five groups were not significant (data not shown). Body weight, left
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ventricular weight and left ventricular weight index were measured after the inducement of I/R model. Similarly there were no significant differences among the five groups (data not shown). 3.2. Sitagliptin upregulates the plasma GLP-1 level after myocardial injury in I/R rats To examine the effect of sitagliptin on plasma GLP-1, we measured the plasma level of GLP-1 after inducing I/R by ELISA assay. Our results (data not shown) were consistent with previous reports. We found that the level of GLP-1 was significantly decreased in I/R group, when compared with that in Sham group (P¼0.006). In contrast, the level of GLP-1 was significantly increased in sitagliptin group compared with that in I/R group (Po0.001). Meanwhile, the levels of GLP-1 in sitagliptinþE group and sitagliptinþ L group were not significantly different from the level of GLP-1 in sitagliptin group (P40.05). 3.3. Sitagliptin reduces the LDH and CK-MB release after myocardial injury in I/R rats LDH and CK-MB are the diagnostic markers of myocardial tissue damage. Thus, we examined the effects of sitagliptin on LDH and CKMB levels in plasma. The changes in LDH and CK-MB levels in this study (data not shown) were also consistent with previous reports. Statistically, the levels of LDH (Po0.001) and CK-MB (Po0.001) in I/R group were significantly higher than those in Sham group. Compared with those in I/R group, the levels of LDH (Po0.001) and CK-MB (Po0.001) in sitagliptin group were significantly lower. In addition, the LDH levels in sitagliptinþ E group (Po0.001) and sitagliptinþ L group (Po0.001) were significantly higher than those in sitagliptin group. Meanwhile, the CK-MB levels in sitagliptinþ E group (Po0.001) and sitagliptinþL group (Po0.001) were also significantly higher than those in sitagliptin group. 3.4. Sitagliptin increases SOD and GSH-Px and decreases MDA after myocardial injury in I/R rats GSH-Px, SOD and catalase are important enzymes of the first line cellular defense against oxidative injury. Therefore, we examined the effects of sitagliptin on levels of SOD, GSH-Px and MDA in myocardial tissue. As shown in Fig. 1A, the concentrations of SOD in Sham group were significantly higher than those in I/R group (P o0.001). Compared with those in I/R group, the concentrations of SOD in sitagliptin group were significantly increased (P o0.001). However, the concentrations of SOD in sitagliptin þE group (P o0.001) and sitagliptin þ L group (P o0.001) were significantly lower than those in sitagliptin group. The effects of sitagliptin on concentrations of GSH-Px are shown in Fig. 1B. Statistically, the concentrations of GSH-Px in I/R group were significantly decreased than those in Sham group (Po 0.001). And compared with those in I/R group, the concentrations of GSH-Px in sitagliptin group were significantly increased (Po 0.001). However, compared with those in sitagliptin group, the concentrations of GSHPx in sitagliptin þ E group (P o0.001) and sitagliptin þL group (P o0.001) were significantly decreased. The effects of sitagliptin on concentrations of MDA are shown in Fig. 1C. Statistically, the concentrations of MDA in I/R group were significantly higher than those in Sham group (P o0.001). And compared with those in I/R group, the concentrations of MDA in sitagliptin group were significantly decreased (Po 0.001). However, compared with sitagliptin group, the concentrations of MDA in sitagliptinþE group (P o0.001) and sitagliptinþL group (P o0.001) were significantly increased.
Please cite this article as: Chang, G., et al., Protective effects of sitagliptin on myocardial injury and cardiac function in an ischemia/ reperfusion rat model. Eur J Pharmacol (2013), http://dx.doi.org/10.1016/j.ejphar.2013.09.007i
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G. Chang et al. / European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
Fig. 1. Effects of sitagliptin on levels of SOD, GSH-Px and MDA in heart homogenate of I/R rats. Levels of SOD, GSH-Px and MDA in heart homogenate of I/R rats were measured by colorimetry. (A) Levels of SOD in heart homogenate of I/R rats. Significance was determined by ANOVA followed by the SNK test, F¼ 63.255. (B) Levels of GSH-Px in heart homogenate of I/R rats. Significance was determined by ANOVA followed by the SNK test, F¼53.189. (C) Levels of MDA in heart homogenate of I/R rats. Significance was determined by ANOVA followed by the SNK test, F¼ 74.287. SOD: superoxide dismutase; GSH-Px: glutathione peroxidase; and MDA: malondialdehyde. Values were expressed as mean7 S.D. Sham: Sham group; I/R: ischemia/reperfusion group; sitagliptin: sitagliptin 300 mg/kg/day pretreatment group; sitagliptinþ E: sitagliptin 300 mg/ kg/day and exendin-(9-39) 45 μg/kg/3 days pretreatment group; sitagliptinþL: sitagliptin 300 mg/kg/day and LY294002 0.3 mg/kg/3 days pretreatment group; N ¼8 in each group; nPo 0.05 vs. Sham group; #Po 0.05 vs. I/R group; and ▲Po 0.05 vs. sitagliptin group.
3.5. Sitagliptin enhances left ventricular function after myocardial injury in I/R rats To determine the effects of sitagliptin on cardiac function in I/R rats, hemodynamic measurements were performed during the entire I/R period. As shown in Fig. 2, compared with those in Sham group, I/R treatment significantly decreased the þ LVdp/dt max (Po0.001), LVdp/dt max (Po0.001), and LVESP (Po0.001) and significantly increased the LVEDP (Po0.001). Compared with those in I/R group, sitagliptin significantly enhanced the þLVdp/dt max (Po0.001), LVdp/dt max (Po0.001), and LVESP (P¼ 0.014) and significantly reduced the LVEDP (P¼0.003). However, the GLP-1 receptor antagonist exendin-(9-39) and the PI3K inhibitor LY294002 abolished the effects of sitagliptin on þLVdp/dt max (Po0.001, Po0.001), LVdp/ dt max (Po0.001, Po0.001), LVESP (P¼0.012, P¼ 0.03) and LVEDP (P¼0.017, P¼ 0.009). 3.6. Sitagliptin inhibits cardiomyocyte apoptosis after myocardial injury in I/R rats We examined the effects of sitagliptin on cell apoptosis in myocardial tissue by TUNEL assay and flow cytometry analysis. The representative graphs of TUNEL assay and flow cytometry analysis are shown in Fig. 3A and C, respectively. The apoptotic index of TUNEL assay is shown in Fig. 3B and the apoptosis ratio of flow cytometry analysis is shown in Fig. 3D. Representative
photomicrograph showed that TUNEL staining positive apoptotic cells were more frequently observed in I/R group, sitagliptinþ E group and sitagliptinþ L group as compared with Sham group and sitagliptin group (Fig. 3A). Statistically, the apoptotic index in sitagliptin group was significantly lower than those in I/R group (Po0.001), sitagliptinþE group (Po0.001) and sitagliptinþ L group (Po0.001). As analyzed by flow cytometry, apoptotic cell ratio in I/R group was significantly increased compared with that in Sham group (P o0.001). However, compared with that in I/R group, apoptosis ratio was significantly decreased in sitagliptin group (Po 0.001). Also apoptosis ratios in sitagliptinþE group (P o0.001) and sitagliptin þ L group (Po 0.001) were significantly increased compared with those in sitagliptin group. 3.7. Sitagliptin increases expression of anti-apoptotic proteins and inhibits expression of pro-apoptotic proteins after myocardial injury in I/R rats The effects of sitagliptin on pho-Aktserine473, pho-Badserine136, caspase-3, cleaved caspase-3, Bax and Bcl-2 in myocardial tissue were analyzed by western blot (Fig. 4). The representative western blot results are shown in Fig. 4A and the quantitative results are shown in Figs. 4B–E, G and H. And the ratio of Bax/Bcl-2 is shown in Fig. 4F. Compared with those in Sham group, I/R treatment significantly decreased levels of pho-Aktserine473 (P¼0.006), pho-Badserine136
Please cite this article as: Chang, G., et al., Protective effects of sitagliptin on myocardial injury and cardiac function in an ischemia/ reperfusion rat model. Eur J Pharmacol (2013), http://dx.doi.org/10.1016/j.ejphar.2013.09.007i
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Fig. 2. Effects of sitagliptin on left ventricular function in I/R rats. The cardiac functions of þLVdp/dt max, LVdp/dt max, LVESP and LVEDP were measured by multichannel physiologic recorder. (A) Values of þ LVdp/dt max (rates of maximum positive left ventricular pressure development). Significance was determined by ANOVA followed by the SNK test, F ¼66.053. (B) Values of LVdp/dt max (rates of maximum negative left ventricular pressure development). Significance was determined by ANOVA followed by a Mann–Whitney Rank Sum test, F¼ 28.161. (C) Values of left ventricular end-systolic pressure (LVESP). Significance was determined by ANOVA followed by the SNK test, F¼ 25.952. (D) Values of left ventricular end-diastolic pressure (LVEDP). Significance was determined by ANOVA followed by the SNK test, F ¼24.460. Values were expressed as mean 7 S.D. Sham: Sham group; I/R: ischemia/reperfusion group; sitagliptin: sitagliptin 300 mg/kg/day pretreatment group; sitagliptin þE: sitagliptin 300 mg/kg/day and exendin-(9-39) 45 μg/kg/3 days pretreatment group; sitagliptinþ L: sitagliptin 300 mg/kg/day and LY294002 0.3 mg/kg/3 days pretreatment group; N ¼ 8 in each group; n P o0.05 vs. Sham group; #Po 0.05 vs. I/R group; and ▲Po 0.05 vs. sitagliptin group.
(P¼0.002) and Bcl-2 (Po0.001) expression. Meanwhile, compared with those in Sham group, I/R treatment significantly increased the expression levels of caspase-3 (Po0.001), cleaved caspase-3 (Po0.001) and Bax (Po0.001). Compared with those in I/R group, sitagliptin significantly enhanced the levels of pho-Aktserine473 (P¼0.01), pho-Badserine136 (Po0.001) and Bcl-2 (Po0.001). At the same time, compared with those in I/R group, sitagliptin significantly reduced levels of caspase-3 (Po0.001), cleaved caspase-3 (P¼0.02) and Bax (Po0.001). Also the Bax/Bcl-2 ratio was significantly decreased in sitagliptin group than that in I/R group (Po0.001). However, the GLP-1 receptor antagonist exendin-(9-39) and the PI3K inhibitor LY294002 attenuated the effects of sitagliptin on antiapoptotic and pro-apoptotic proteins. Statistically, compared with sitagliptin group, exendin-(9-39) and LY294002 administration significantly reduced the levels of pho-Aktserine473 (P¼ 0.011, P¼0.007), pho-Badserine136 (Po0.001, Po0.001) and Bcl-2 (Po0.001, Po0.001). And compared with sitagliptin group, exendin-(9-39) and LY294002 administration significantly increased the expression levels of caspase3 (Po0.001, Po0.001), cleaved caspase-3 (P¼0.001, P¼ 0.001) and Bax (Po0.001, Po0.001).
4. Discussion The major finding of our study was that sitagliptin pretreatment could reduce myocardial injury and improve cardiac function by
inhibiting cardiomyocyte apoptosis in an I/R rat model. Though DPP4 inhibitors were reported to have cardioprotective effects during I/R, this is the first study to examine the effects of sitagliptin on cardiomyocyte apoptosis and oxidative stress induced by myocardial I/R. In addition, our data indicate that sitagliptin could decrease the levels of pro-apoptotic proteins and increase the levels of anti-apoptotic proteins. However, the above observed effects of sitagliptin were all abolished when co-administered with GLP-1 receptor antagonist exendin-(9-39) or PI3K inhibitor LY294002. Thus we speculate that sitagliptin protected the heart in I/R rats from injury through the decrease of apoptosis and oxidative damage and the activation of PI3K/Akt signaling pathway. As reported by previous studies (Khalil et al., 2005; Sun et al., 2012), myocardial I/R impaired cardiac function, increased the release of LDH and CK-MB and the apoptotic rate of cardiomyocyte. We found similar results in this study. Importantly, we found that sitagliptin reduced LDH and CK-MB release in I/R rats. We speculate that this effect might be ascribed to its potential to resist against oxidative stress. Interestingly, we demonstrated that sitagliptin significantly increased the levels of SOD and GSHPx and decreased the level of MDA in myocardial tissues in I/R rats. As we all know, SOD and GSH-Px are key antioxidant enzymes, which constitute first line cellular defense against oxidative injury. MDA is one of the products of oxidative stress, which reflects the damage of cell caused by oxidative stress. To our knowledge, our study demonstrated for the first time that
Please cite this article as: Chang, G., et al., Protective effects of sitagliptin on myocardial injury and cardiac function in an ischemia/ reperfusion rat model. Eur J Pharmacol (2013), http://dx.doi.org/10.1016/j.ejphar.2013.09.007i
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Fig. 3. Effects of sitagliptin on cardiomyocyte apoptosis in I/R rats. Apoptosis were analyzed by TUNEL assay and flow cytometry analysis, respectively. (A) Representative graphics of TUNEL staining. (B) Quantitative results of TUNEL staining (400 ). Apoptotic index was determined as the ratio of brown nuclei number to the total number of nuclei. Nuclei in a total of 10 fields per tissue slice (N ¼ 6) were included. Significance was determined by ANOVA followed by the SNK test, F¼ 69.639. (C) Representative flow cytometry results. (D) Quantitative results of flow cytometry results, N ¼ 8. Apoptosis ratio was determined as the ratio of Annexin V positive and propidium iodide negative cells to total cells analyzed. Significance was determined by ANOVA followed by the SNK test, F¼ 70.412. Values were expressed as mean 7S.D. Sham: Sham group; I/R: ischemia/reperfusion group; sitagliptin: sitagliptin 300 mg/kg/day pretreatment group; sitagliptinþ E: sitagliptin 300 mg/kg/day and exendin-(9-39) 45 μg/kg/3 days pretreatment group; sitagliptinþ L: sitagliptin 300 mg/kg/day and LY294002 0.3 mg/kg/3 days pretreatment group; nPo 0.05 vs. Sham group; #Po 0.05 vs. I/R group; and ▲ Po 0.05 vs. sitagliptin group.
pretreatment with sitagliptin could increase the concentrations of antioxidant defense enzymes including GSH-Px and SOD, and decrease the production of MDA in I/R rats. We also found that sitagliptin administration significantly improved the cardiac function via increasing 7LVdp/dt max, LVESP and limiting the increase of LVEDP. Similarly, Sauvé et al. (2010) observed that the left ventricular function was improved in
mice pretreated with sitagliptin for 12 h prior to aortic occlusion and in DPP4 deleted mice after I/R injury. Ku et al. (2011) also reported that DPP4 deficiency could preserve cardiac function in rats subjected to myocardial I/R. In a clinical study, single dose sitagliptin treatment could improve the regional and global left ventricular function in patients with coronary artery disease (Read et al., 2010). However, a recent study demonstrated that in rats
Please cite this article as: Chang, G., et al., Protective effects of sitagliptin on myocardial injury and cardiac function in an ischemia/ reperfusion rat model. Eur J Pharmacol (2013), http://dx.doi.org/10.1016/j.ejphar.2013.09.007i
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Fig. 4. Effects of sitagliptin on expression levels of anti-apoptotic proteins and pro-apoptotic proteins in I/R rats. Expression levels of apoptosis related proteins were analyzed by western blot. (A) Representative western blot results. (B) Ratios of phospho-AKTserine473 to total AKT. Significance was determined by ANOVA followed by a Mann–Whitney Rank Sum test, F¼ 20.546. (C) Ratios of phospho-Badserine136 to GAPDH. Significance was determined by ANOVA followed by the SNK test, F¼ 10.407. (D) Ratios of Bax to GAPDH. Significance was determined by ANOVA followed by a Mann–Whitney Rank Sum test, F¼ 212.404. (E) Ratios of Bcl-2 to GAPDH. Significance was determined by ANOVA followed by a Mann–Whitney Rank Sum test, F¼75.357 (F) Ratios of Bax to Bcl-2. Significance was determined by ANOVA followed by a Mann–Whitney Rank Sum test, F¼ 84.528. (G) Ratios of Caspase-3 to GAPDH. Significance was determined by ANOVA followed by the SNK test, F¼ 70.092. (H) Ratios of cleaved caspase-3 to GAPDH. Significance was determined by ANOVA followed by the SNK test, F¼17.488. Values were expressed as mean7S.D. Sham: Sham group; I/R: ischemia/reperfusion group; sitagliptin: sitagliptin 300 mg/kg/day pretreatment group; sitagliptinþE: sitagliptin 300 mg/kg/day and exendin-(9-39) 45 μg/kg/3 days pretreatment group; sitagliptinþL: sitagliptin 300 mg/kg/day and LY294002 0.3 mg/kg/3 days pretreatment group; N¼ 8 in every group; nPo0.05 vs. Sham group; #Po0.05 vs. I/R group; and ▲Po0.05 vs. sitagliptin group.
with ischemic heart failure, early or late treatment with vildagliptin had no beneficial effect on ventricular performance (Yin et al., 2011). Meanwhile, Sauvé et al. (2010) also reported that acute treatment with sitagliptin for 20 min prior to I/R injury failed to improve ventricular function. The discrepancy in these studies might be attributed to the differences in the duration of treatment time and differences in types of drug administration as well as the differences in study models.
Cardiomyocyte apoptosis is one of the critical reasons of heart failure after myocardial infarction. Many studies have confirmed that blocking the apoptosis process could reduce the loss of cardiomyocyte, minimize myocardial injury and improve ventricular performance induced by I/R (Mughal et al., 2012). Given the significantly improved recovery of cardiac function, we examined the effects of sitagliptin on preventing cardiomyocyte apoptosis in I/R rats. We demonstrated that sitagliptin administration
Please cite this article as: Chang, G., et al., Protective effects of sitagliptin on myocardial injury and cardiac function in an ischemia/ reperfusion rat model. Eur J Pharmacol (2013), http://dx.doi.org/10.1016/j.ejphar.2013.09.007i
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significantly decreased apoptosis ratio of cardiomyocytes as revealed by TUNEL staining and flow cytometry analysis. And sitagliptin administration significantly reduced expression levels of caspase-3 and cleaved caspase-3 in rats subjected to I/R injury. It has been reported that PI3K/Akt signaling pathway activation could inhibit cardiomyocyte apoptosis after I/R injury (Fujio et al., 2000; Matsui et al., 2001; Mullonkal and Toledo-Pereyra, 2007). The mechanisms of the anti-apoptotic effect are acted through various means, such as inhibiting caspase activation, inhibiting death genes expression and regulating the activity of Bcl-2 family. Bcl-2 family, the key regulators of apoptosis, consists of both cell death promoters such as Bax and Bad, and cell death inhibitors including Bcl-2, Bcl-x, etc. It is reported that the high ratio of Bax/ Bcl-2 was associated with great possibility to apoptotic activation (García-Sáez, 2012). In our study, we demonstrated that sitagliptin could increase phosphorylation levels of Akt and Bad and decrease expression levels of caspase-3, cleaved caspase-3 and Bax. Meanwhile, we also found that sitagliptin could up-regulate Bcl-2 expression, resulting in decreased Bax/Bcl-2 ratio in I/R rats. These effects of sitagliptin were correlated with cardiomyocyte apoptosis attenuation. Hence, we next examined whether the sitagliptin exerted its anti-apoptotic action through activation of PI3K/Akt pathway in rats subjected to I/R injury. The PI3K inhibitor LY294002 was employed. We found that co-administration of LY294002 and sitagliptin decreased phosphorylation levels of Akt and Bad and increased expression levels of caspase-3, cleaved caspase-3, Bax, and Bax/Bcl-2 ratio. These findings suggested that LY294002 could abolish the anti-apoptotic effects of sitagliptin. The anti-apoptotic effects of sitagliptin were related to, at least in part, activation of PI3K/Akt signaling pathway. The function of DPP4 inhibitor is to inhibit the proteolytic activity of DPP4 enzyme, postponing the myocardial degradation of GLP-1 (Barnett, 2006). Our data showed that sitagliptin administration resulted in significant accumulation of plasma GLP-1 in I/R rats. This result was similar to previous data reported by Ku et al. (2011) and Ye et al. (2010). Moreover, our results showed that higher levels of plasma GLP-1 were associated with lower levels of cardiac injury markers, higher levels of antioxidant enzymes, lower ratio of cardiomyocyte apoptosis and better ventricular performance in I/R rats. However, these effects were attenuated by the GLP-1 receptor antagonist exendin-(9-39). Exendin-(9-39) has been widely used to estimate the role of receptor-dependent pathway. Thus we speculated that the cardioprotective effects of sitagliptin might be attributed to, at least in part, the GLP-1 receptor-dependent pathway. In summary, sitagliptin exerted its action via up-regulating the level of GLP-1, which activated the PI3K/Akt signaling pathway via binding GLP1 receptor. The prominent finding of this study was that sitagliptin pretreatment could reduce myocardial injury and improve cardiac function in an I/R rat model. The possible mechanisms might be relative to decrease of apoptosis and oxidative damage, up-regulation of GLP-1 level (which activated the PI3K/Akt signaling pathway via binding GLP-1 receptor), increase of Bad phosphorylation and decrease of Bax/Bcl-2 ratio. However, exact cardioprotective mechanisms of DPP4 inhibitors need a further study. To sum up, we conclude that sitagliptin administration has protective effects on myocardial I/R injury. Our findings could provide deeper insights into the treatment of heart disease.
Acknowledgments This work was supported by the National Natural Science Funds for Youths (Grant no. 81100196), Natural Science Foundation
Project of CQ CSTC (Grant no. CSTC, 2011BB5133). Foundation project of Traditional Chinese Medicine of Chongqing Municipal Health Bureau (Grant no. 2012-2-125) and Pfizer pharmaceutical limited competition grants (Grant no. ws1790576). We greatly appreciate Jianyong Wu and Dezhang Zhao (Institute of Life Sciences, Chongqing Medical University) for their excellent technical support for the flow cytometry analysis.
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Please cite this article as: Chang, G., et al., Protective effects of sitagliptin on myocardial injury and cardiac function in an ischemia/ reperfusion rat model. Eur J Pharmacol (2013), http://dx.doi.org/10.1016/j.ejphar.2013.09.007i