Anti-inflammatory and protective effects of combined treatment with sitagliptin and melatonin in cardiac ischemia reperfusion injury in obese rats: Involvement of TLR-4/NF-κB pathway

Abstract

Background: Obesity is associated with an augmented risk of myocardial ischemia/reperfusion (I/R) injury. Reduction of I/R injury by effective cardioprotective strategies needs to be investigated in obese subjects. This study aimed to evaluate the combined effects of sitagliptin and melatonin on inflammatory response and TLR4/IκBα/NF-κB signaling following cardiac I/R damage in obese rats. Methods: Sixty-six male Wistar rats (180–200 g) were fed a low fat diet (10% Kcal from lipids) or high fat (45% Kcal from lipids) diets for 12 weeks. High fat-fed (obese) rats experienced 30 min left anterior descending occlusion followed by 24 h reperfusion. Obese rats received sitagliptin (20 mg/kg/day) for 1 month before I/R surgery. Melatonin (10 mg/kg) was injected at early reperfusion. Myocardial infarct size (IS), cTn-I release, pro-inflammatory cytokines, myeloperoxidase (MPO), COX-2 and iNOS, and the protein expressions of TLR4, p-NF-κB/p65, and p-IκBα were evaluated. Results: Monotherapies with sitagliptin-preconditioning or melatonin-postconditioning had no cardioprotective effects in obese rats. However, combined therapy with sitagliptin and melatonin significantly reduced IS, and the release of cTn-I, in comparison to untreated obese rats (p < .01) Moreover, this combination decreased the production of pro-inflammatory cytokines, MPO, COX-2 and iNOS, and the expression of TLR4 and p-NF-κB/p65, while reduced the expression of p-IκBα, in comparison with untreated or monotherapies-received obese rats (p < .01 for all). Conclusion: Combination therapy with sitagliptin and melatonin was a good cardioprotective strategy to modulate the inflammatory responses and TLR4/NF-κB signaling pathway in obese patients with cardiac I/R injury.

Keywords

Cardiac ischemia reperfusion obesity sitagliptin melatonin TLR4/NF-κB pathway

Introduction

Ischemic heart diseases (IHD) are still the main cause of death in the world. The most occurring complication in a patient with IHD is severe cardiac injury induced by ischemia/reperfusion (I/R). Efforts to diminish myocardial I/R damage have limited success.¹ The main obstacle for achieving the highest cardioprotection in patients with myocardial I/R insults is the presence of various comorbidities. It has been implicated that obesity, as a pro-inflammatory state, is a strong risk-factor for ischemic injury development and is related to worse clinical outcomes following I/R damage.² Obesity impacts myocardial function and structure in terms of increased hemodynamic load, impaired ventricular function, and altered left ventricular remodeling, and finally leads to heart failure.³ Therefore, mitigation of the consequences of cardiac I/R injury using the application of more effective and protective approaches should be studied in obese patients.⁴

The pro-inflammatory response and increased production of cytokines and interleukins (IL) such as IL-6, IL-1β, and tumor necrosis factor α (TNF-α) may have a critical involvement in myocardial injury following I/R insult and contributes to the cardiac dysfunction and heart failure.⁵ These mediators lead to capillary vessels obstruction, cytotoxic agents release, and vasoactive substances production.^6,7 In addition, the toll-like receptor 4 (TLR4) is strongly activated during reperfusion injury and participates in neutrophil activation, cytokine releasing, and reactive oxygen species (ROS) overproduction.

It has an important role in the inflammatory response of cardiac I/R injury through There is an association between the TLR4 activation is functionally associated with the upregulation of nuclear factor-κB (NF-κB) signaling pathway, causing the generation of cytokines and other chemokines like cyclooxygenase-2 (COX-2).⁸ Elevated levels of TNF-α and other interleukins also directly activate NF-κB signaling and thus lead to the amplification of the initial inflammatory response.⁹ Of note, finding novel therapeutic options for modulation of TLR4/NF-κB signaling-directed inflammatory reactions to alleviate myocardial I/R injury in obese patients has become an area of growing clinical interest.

Sitagliptin is an inhibitor of the dipeptidyl peptidase 4 (DPP4) enzyme, by which it enhances the levels of glucagon-like peptide-1 (GLP-1) in cells, reduces insulin-resistance, regulates intracellular energy homeostasis and the function of ion channel, and improves mitochondrial function and lipid profile.^10,11 Numerous experiments have revealed the beneficial impacts of sitagliptin in reducing I/R damages in several organs such as the kidney,^12-14 brain,¹⁵ intestine,¹⁶ and heart.¹¹ Additionally, melatonin is an endogenous hormone whose circulating concentration is decreased in patients with different phases of coronary heart disease.¹⁷ Preclinical and clinical studies have demonstrated the involvement of melatonin in normal cardiovascular physiology. The protective features of melatonin in I/R hearts have been reported in numerous studies.^18–20 Due to the anti-inflammatory and anti-oxidant capabilities of melatonin, it has attracted remarkable attention in cardiovascular researches in recent times.²¹

According to the numerous studies demonstrating that both sitagliptin and melatonin have cardioprotective impacts in myocardial I/R injury in no-comorbid settings,^11,19 it seems that their combined administration may diminish the complications of obesity as a main cardiovascular risk factor in patients with myocardial I/R insults. Due to the inhibitory role of obesity and its other complications in cardioprotection, we should increase the potency of individual protective therapies to protect the heart from I/R in obese subjects. Increasing the dose of individual therapy may be associated with other side effects, but using combination therapy is one of the best alternatives in this field. Here, studies suggest that the combined application of two therapeutic modalities, one before ischemia and the other after ischemia (as preconditioning and postconditioning, respectively) has a stronger effect than using them at a fixed time.²² Therefore, the present study aimed to assess the hypothesis that sitagliptin preconditioning in combination with melatonin postconditioning exerts considerable cardioprotection by modulating the inflammatory responses in the I/R heart of obese rats and whether the TLR4/NF-κB pathway is involved in this protection.

Materials and Methods

Animals

Sixty-six male Wistar rats (180–200 g) were attained from the laboratory animal breeding center and randomly assigned to receive a low fat (LF) diet (providing 10% Kcal from fat, 70% from carbohydrates, and 20% from proteins, 3.0 Kcal/g) (n = 6) or a high fat (HF) diet (providing 45% Kcal from fat, 35% from carbohydrates, and 20% from proteins, 4.7 Kcal/g) (n = 60) for 12 weeks (according to the animal grouping and study design in the next section). Rats were housed in standard cages (three in each cage) and kept at 12 h dark/light cycles with the room temperature of 20–24°C and the humidity of 55%, and were freely given food and water. The procedures were performed according to the eighth edition of “Guide for the Care and Use of Laboratory Animals” (NRC, 2011), and validated by the local Ethics Board (Ethical code: 07020018).

Sample size analysis and study design

We planned a sample size calculation study using PS software. Considering an expected mean difference of 20%, normal distribution in the standard deviation of 15%, a power of 85%, and a type I error probability of 0.05 the sample size analysis for the study endpoints revealed a required number of rats being six per group. Accordingly, at the beginning of the experiment, six normal rats were categorized as non-obese rats and fed an LF diet, and 60 rats were categorized as obese rats and fed an HF diet for 12 weeks (6 rats for infarct size study and 6 rats for other endpoints in each group of obese rats). Normal non-obese rats were assigned to compare the obesity-induced biochemical changes and confirm the development of obesity in obese rats. Then, the obese rats were randomly allocated into five experimental groups to receive myocardial I/R injury with/without drug-conditioning, as follows:

(1) Sham: rats did not receive I/R;

(2) I/R: rats received 30 min of regional ischemia and 24 h of reperfusion;

(3) I/R+Sit: firstly rats were intraperitoneally injected with sitagliptin (20 mg/kg/day, Novartis, Switzerland) for four consecutive weeks before I/R surgery, then the rats received 30 min of regional ischemia and 24 h of reperfusion;

(4) I/R+Mel: rats received 30 min of regional ischemia and 24 h of reperfusion plus intraperitoneal injection of melatonin (10 mg/kg, Sigma-Aldrich, USA) at early reperfusion; and

(5) I/R+Sit+Mel: firstly rats were intraperitoneally injected with sitagliptin for 4 weeks before I/R surgery, then received 30 min of regional ischemia and 24 h of reperfusion plus intraperitoneal injection of melatonin at early reperfusion.

The rats in I/R groups were intraperitoneally received normal saline to reduce the vehicle effect. Body weights (BWs) of the non-obese and obese rats were recorded every week and immediately before I/R surgery.

Induction of regional myocardial I/R in obese rats

All rats in I/R groups were firstly anesthetized by 50 mg/kg sodium-pentobarbital through a single intraperitoneal injection. Next, an artificial ventilator was attached to the animals and adjusted to generate 2–3 ml of tidal volume and 65–70/min of respiratory rate with positive pressure (Taimeng Technology, China). After surgery and exposing the heart via lateral thoracotomy, a 6.0 silk ligature was placed around the left anterior descending (LAD) coronary artery and tied for 30 min to induce regional ischemia. Myocardial reperfusion was initiated by removing the ligature for 24 h. The same surgery was performed in sham rats but LAD was not ligated.

Measurement of lipid profile and blood parameters

After 12 weeks of the feeding of HF or LF regimens by the rats, the lipid profile and blood parameters including glucose and insulin levels were measured. The concentrations of glucose were obtained rapidly using a glucose glucometer test. Next, the plasma concentrations of insulin, leptin, and adiponectin, as well as lipid profiles of rats, were evaluated using the appropriate specific ELISA kits commercially available (Cayman-Chemical, USA, Wako Pure Chemical Industries, Osaka, Japan), based on the instructions provided by the manufacturers.

Measurement of myocardial infarction

Following 24-h reperfusion, Evans blue and TTC consecutive stainings were performed to measure the area at risk and infarct size of the rat’s heart on 5–6 transverse sections of their left ventricles. After fixation of the sections, the volumes of total left ventricles, the area at risk, and infarct size in each section of each heart were determined using Photoshop image processing software (Adobe System, USA). Areas at risk were calculated as the percentage of total volumes of the left ventricles and the infarct sizes were calculated as the percentage of areas at risk.

Assessment of cardiotroponin-I (cTn-I)

Serum levels of cTn-I were assayed using a specific cTn-I kit (Monobind Inc., California, USA) with a colorimetric method, based on the instructions provided by the manufacturer. The recorded values of cTn-I were reported in ng/ml.

Determination of pro-inflammatory cytokines

After the myocardial samples from areas at risk were being homogenized and centrifuged, the ELISA kits (MyBioSource, San Diego, USA) were used to measure the myocardial levels of pro-inflammatory cytokines including IL-6, IL-1β, and TNF-α, based on the instruction provided by the kits. The protein of samples was calculated according to the Bradford method and the resultant values of the cytokines were normalized to the sample’s protein and expressed as pg/mg of protein.

Determination of myeloperoxidase (MPO), cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS)

The activity of MPO, COX-2, and iNOS were measured in myocardial tissue homogenates using ELISA kits (MPO: Jiancheng Biotechnology Institute, China; COX-2 and iNOS: MyBiosource, San Diego, USA), based on the instruction provided by the kits. The recorded values were reported in U/mg of protein in each sample for MPO, and ng/mg of protein for COX-2 and iNOS.

Western blotting

First, 50 mg fresh cardiac samples were homogenized in a RIPA lysis buffer (Sigma-Aldrich, USA), and the resultant supernatants were collected following subsequent centrifuging (10,000 RCF, 10 min, 4°C). Approximately 20 μg of each sample protein was first separated by the SDS PAGE electrophoresis and then transferred on a nitro-cellulose membrane. Next, the membrane was blocked with 5% non-fat dry milk at room temperature. After 2 h, the primary antibodies against proteins TLR4, phosphorylated and total forms NF-κB/p65 and IκBα, and β-actin (1:1000–1:1500, Cell Signaling Technology, USA) for 24 h at 4°C. Following washing and incubation of the membrane with the HRP-secondary antibody at room temperature, an enhanced-chemiluminescence detection kit (ThermoFisher Scientific, USA) was used to visualize the immunoblotting protein bands. The measured values were presented in arbitrary units (AU).

Statistical analysis

SPSS software (Version 15.0, SPSS Inc, Chicago, USA) was employed to perform all statistical tests. The statistical significances between groups were determined using one-way analysis of variance (ANOVA) and Tukey as post hoc. All values were presented as means±SEM. The significance level was considered when the p-value was less than .05.

Results

General information of the rats

After feeding of rats with HF or LF regimens for 3 months, all obese rats showed a significantly elevated plasma level of insulin (p < .01), leptin and cholesterol (p < .05), and decreased plasma level of adiponectin (p < .05). In addition, HF-fed rats had higher BWs and HWs in comparison to LF-fed animals (p < .05). However, the concentrations of glucose and triglyceride, and the ratio of HW to BW were not significantly different between non-obese and obese rats (Table 1).

Table 1.

General information of non-obese and obese rats.

Groups	Non-obese	Obese
Blood glucose (mg/dl)	84 ± 6	91 ± 5
Plasma insulin (ng/ml)	3.29 ± 0.45	9.11 ± 1.14^**
Plasma leptin (ng/ml)	4.12 ± 0.51	7.81 ± 1.2^*
Plasma adiponectin (ng/ml)	5.96 ± 0.38	4.1 ± 0.29^*
Plasma cholesterol (mg/dl)	81 ± 9	186 ± 19^*
Plasma triglyceride (mg/dl)	107 ± 11	116 ± 19
BW (g)	353 ± 12	467 ± 14^*
HW (g)	1.34 ± 0.06	1.73 ± 0.11^*
HW/BW (%)	0.38 ± 0.00	0.37 ± 0.00

The data were presented as mean±SEM. (^*p < .05 and ^**p < .01 vs. non-obese rats). BW: body weight, and HW: heart weight.

Effect of sitagliptin and melatonin combined-conditioning on myocardial infarction

Figure 1 shows the results of the area at risk and infarct size between experimental rats feeding HF diet. Induction of 30 min ischemic injury followed by 24 h reperfusion caused similar areas at risk between all groups (Figure 1(a)). However, this I/R injury led to the development of infarct size to 56.10 ± 5.9% when compared with the Sham group (p < .001). Sitagliptin preconditioning alone, or melatonin postconditioning alone had no significant limiting effects of infarct size in comparison to I/R rats. Nevertheless, sitagliptin plus melatonin combined-conditioning significantly reduced infarct size to 27.30 ± 2.80% as compared to I/R (56.30 ± 5.90%), I/R+Sit (48.60 ± 5.20%), and I/R+Mel (45.10 ± 3.20%) groups (p < .01) (Figure 1(b)).

Figure 1.

Effects of sitagliptin, melatonin, and their combination on areas at risk (AAR) (A), and infarct sizes (IS) (B) in the myocardium of the obese rats. The data were presented as mean±SEM and n = 6 per group. (^***p < .001 vs. Sham group, ^##p < .01 vs. I/R group, ^$$p < .01 vs. I/R+Sit group, ^@@p < .01 vs. I/R+Mel group). LV: left ventricle; I/R: Ischemia and reperfusion injury; Sit: Sitagliptin, and Mel: Melatonin.

Effect of sitagliptin and melatonin combined-conditioning on cTn-I serum levels

Following induction of I/R injury, cTn-I serum level was elevated as compared to the Sham rats (p < .01). Sitagliptin preconditioning alone, or melatonin postconditioning alone were not able to reduce cTn-I serum levels as compared with those of nontreated rats. But, their combined therapy had a greater effect on cTn-I serum level and significantly lowered it as compared to I/R, I/R+Sit, and I/R+Mel groups (p < .01 for all) (Figure 2).

Figure 2.

Effects of sitagliptin, melatonin, and their combination on cTn-I serum levels in obese rats. The data were presented as mean±SEM and n = 6 per group. (^**p < .01 vs. Sham group, ^##p < .01 vs. I/R group, ^$$p < .01 vs. I/R+Sit group, ^@@p < .01 vs. I/R+Mel group). cTn-I: Cardiotroponin-I; I/R: Ischemia and reperfusion injury; Sit: Sitagliptin, and Mel: Melatonin.

Effect of sitagliptin and melatonin combined-conditioning on pro-inflammatory cytokines in the heart

As shown in Figure 3(a)–(c), the levels of IL-6, IL-1β (p < .001), and TNF-α (p < .01) were increased significantly after induction of I/R in HF-fed rats. Preconditioning with sitagliptin alone could not significantly decrease the myocardial contents of IL-1β, IL-6, and TNF-α, in comparison to the I/R group. Furthermore, postconditioning with melatonin alone significantly diminished the production of IL-1β and IL-6 (p < .05 for both), without having such an effect on TNF-α levels when compared with their levels in nontreated rats. However, the combination of sitagliptin and melatonin significantly showed significant lowering effects on the levels of all cytokines as compared to I/R (p < .01), I/R+Sit (p < .01), and I/R+Mel (p < .05, and p < .01) groups.

Figure 3.

Effects of sitagliptin, melatonin, and their combination on the myocardial contents of IL-1β (A), IL-6 (B), and TNF-α (C) in obese rats. The data were presented as mean±SEM and n = 6 per group. (^**p < .01 and ^***p < .001 vs. Sham group, ^#p < .05 and ^##p < .01 vs. I/R group, ^$$p < .01 vs. I/R+Sit group, ^@p < .05, and ^@@p < .01 vs. I/R+Mel group). I/R: Ischemia and reperfusion injury; Sit: Sitagliptin, and Mel: Melatonin.

Effect of sitagliptin and melatonin combined-conditioning on MPO, COX-2 and iNOS levels in the heart

The cardiac levels of MPO, iNOS (p < .001), and COX-2 (p < .01) in I/R treated rats were significantly higher than those of the Sham group (Figure 4(a) and (b)). The levels of MPO and COX-2 were not different between the group receiving sitagliptin alone and nontreated rats. However, postconditioning with melatonin caused a significant reduction of MPO and COX-2 levels in comparison to the I/R group (p < .05). The monotherapies had no significant effect on iNOS levels. Application of sitagliptin preconditioning and melatonin postconditioning significantly decreased the levels of MPO, COX-2, and iNOS as compared to I/R (p < .01), I/R+Sit (p < .01), and I/R+Mel (p < .05) groups.

Figure 4.

Effects of sitagliptin, melatonin, and their combination on MPO (A), COX-2 (B), and iNOS (C) levels in obese rats. The data were presented as mean±SEM and n = 6 per group. (^**p < .01 and ^***p < .001 vs. Sham group, ^#p < .05 and ^##p < .01 vs. I/R group, ^$$p < .01 vs. I/R+Sit group, ^@p < .05 vs. I/R+Mel group). MPO: Myeloperoxidase; COX-2: Cyclooxygenase-2; I/R: Ischemia and reperfusion injury; Sit: Sitagliptin, and Mel: Melatonin.

Effect of sitagliptin and melatonin combined-conditioning on TLR4/NF-κB/IκBα pathway in the heart

The protein expressions of TLR4 and p-NF-κB/p65 were upregulated and that of p-IκBα was downregulated in the I/R group in comparison to the Sham group (p < .001 for both) (Figure 5.). In comparison to the nontreated rats, lone application of sitagliptin preconditioning or melatonin postconditioning did not significantly reverse the I/R-induced expression of these proteins in the inflammatory signaling pathway. Conversely, combined application of sitagliptin plus melatonin significantly downregulated the expression of TLR4 and p-NF-κB/p65 and upregulated the expression of IκBα as compared to I/R, I/R+Sit, and I/R+Mel groups (p < .01) (Figure 5(a)-(d)).

Figure 5.

The representative immunoblots of proteins TLR4, phosphorylated and total forms of NF-κB/p65 and IκBα, and β-actin (A), the expressions of TLR4 (B), p-NF-κB/p65, (C) p-IκBα (D) correlated to the β-actin expression in obese rats. The data were presented as mean±SEM and n = 4 per group. ^(***p < .001 vs. Sham group, ^##p < .01 vs. I/R group, ^$$p < .01 vs. I/R+Sit group, ^@@p < .01 vs. I/R+Mel group). TLR4: Toll-like receptor 4; NF-κB: Nuclear factor-kappa B; IκBα: NF-κB inhibitor alpha; I/R: Ischemia and reperfusion injury; Sit: Sitagliptin, and Mel: Melatonin.

Discussion

The current work showed the potency of combination therapy with sitagliptin and melatonin in cardiac I/R injury in obese rats. Preconditioning with sitagliptin and postconditioning with melatonin caused a substantial reduction of myocardial infarct size and serum level of cTn-I following I/R in comparison with untreated obese rats. The mechanism of the cardioprotective effect of this combination therapy appears to be due, at least in part, to the inhibition of inflammatory reactions via downregulating the activity of TLR4/IκBα/NF-κB inflammatory signaling pathway in obese rats under I/R injury.

The protective effects of sitagliptin and melatonin have been demonstrated in previous studies in animal models without risk factors. Pretreatment of normal rats with sitagliptin reduced myocardial damage and improved heart function by decreasing apoptosis and oxidative injury and upregulating the levels of GLP-1 and the activity of the PI3K/Akt signaling pathway.¹¹ Similarly, pharmacological concentrations of melatonin have powerfully increased cardiac tolerance against I/R injury and restored I/R-induced myocardial dysfunction and cell death through different ways such as inhibiting mitochondrial permeability transition pore opening and cardiolipin peroxidation,²³ attenuating mitochondrial oxidative injury via the upregulating and activating of JAK2/STAT3, and SIRT3-SOD2 signaling pathways.^24,25 However, in the present study, their alone effects were not significant in reducing the infarct size and this is varying with the findings of previous works. It means that their protective effect was minimized or eliminated in obese rats, and the obesity neutralizes cardioprotection caused by these monotherapies. In previous reports, the effects of sitagliptin or melatonin were studied in healthy animals without considering the presence of cardiovascular risk factors, however, it has been verified that cardioprotective effects of single therapies were lost or weakened during the presence of comorbidities.²⁶

Obesity itself worsens myocardial damage and leads to the impaired mechanical function of the heart during I/R.^27–29 Negative metabolic and intracellular changes following obesity can adversely affect the protective pathways and prevent drugs from activating them. For example, obesity is associated with higher oxidative and inflammatory status, and leptin is the main linker of obesity and the production of pro-inflammatory cytokines in cardiovascular disease.² In this work, obese rats had reduced levels of adiponectin and elevated concentrations of insulin, and leptin as well as the levels of cholesterol, in comparison to normal rats. Contrary to these results, the combined use of sitagliptin and melatonin provided strong protective effects in obese rats. These findings suggest that the combined use of interventions reinforces the protective pathways and overcomes the negative impacts of obesity and its intracellular complications on the beneficial effects of either sitagliptin or melatonin on those pathways. In the meantime, drugs may lead to infarct size reduction and proper cardioprotection by activating different signaling pathways outlined above.^11,23–25 Therefore, conferring to our hypothesis, the use of combination therapy can be considered as an appropriate alternative strategy to overcome the negative influence of comorbidities like obesity on adequate cardioprotection.

Previous studies have shown that TLR4 is critically involved in the inflammatory responses of myocardial I/R injury.^30–32 The main downstream signaling target of TLR4 is the transcription factor NF-κB. This transcription factor is inactive in the cytosol due to the inhibitory effect of an adhering protein called IκBα, and over-activated upon the initiation of reperfusion. Upon activation of TLR4 by various stimuli, including cytokines and other factors produced in I/R, IκBα is phosphorylated and separated from NF-κB-IκBα complex. This activates NF-κB by the phosphorylation of subunit p65 and then translocates it into the nucleus, where it promotes target genes expression and the production of pro-inflammatory cytokines such as TNF-α, IL-6, IL-1, and IL-8 and the mutual oxidative/inflammatory factors such as iNOS, COX-2, and MPO.^33-35 Following these events, the inflammatory response worsens. Postconditioning with melatonin alone decreased IL-1β and IL-6, as well as the levels of MPO, COX-2, and iNOS to some extent, but had no significant effect on TNF-α. Also, single-use of sitagliptin had no significant effect on pro-inflammatory cytokines and chemokines in obese rats. Although the anti-inflammatory effects of sitagliptin and melatonin have been reported in previous studies, here they alone had a modest effect on some indicators and could not overcome the overall pathophysiological events in obese rats. Interestingly, in addition to downregulating the TLR4 and NF-κB, combination therapy also reduced the phosphorylation of IκBα, resulting in the release of NF-κB in the cytosol and its transfer to the nucleus. Consequently, the production and secretion of inflammatory cytokines, as well as iNOS, COX-2, and MPO following combination therapy, were severely and powerfully reduced when compared to those of nontreated obese rats and even monotherapies. Therefore, simultaneous administration of sitagliptin and melatonin in obese rats restored their individual cardioprotective effects by suppressing the activity of the TLR4/IκBα/p65NF-κB/cytokines pathway. Besides, it can be supposed that combined therapy possibly causes co-activation of multiple and diverse cardioprotective mechanisms, which additional studies are needed to clarify their contribution. PI3K/Akt, JAK2/STAT3, and SIRT3/mitochondrial biogenesis are the most likely candidate signaling pathways in this scenario.

Limitation

In this study, the inhibitory effect of the combined intervention on the nuclear translocation of NF-κB was not evaluated, which could further explain the effectiveness and importance of this therapeutic strategy. Also, other chemokines are produced in arachidonic acid metabolism or other related reactions whose involved mutually in inflammation and lipid peroxidation interactions and have not been assayed here due to some limitations. In particular, the involvement of NF-κB in obesity-induced lipid peroxidation and NADPH oxidase system and the effect of combination therapy on it are important challenges that should be evaluated in future studies.

Conclusion

The main finding of the present work was that combined-conditioning with sitagliptin and melatonin had a more powerful cardioprotective effect in obese rats with I/R damage through modulation of inflammatory response. This treatment significantly suppressed the activity of the TLR4/IκBα/NF-κB pathway and subsequent production of pro-inflammatory cytokines and chemokines (Figure 6). Yet, supplementary evaluations should address the contribution of other important survival pathways in the cardioprotection induced by combined sitagliptin and melatonin conditioning in obese rats with I/R injury.

Figure 6.

The diagram of concept map representing the pathway and findings of the study. P: Phosphorylation.

Footnotes

Authors’ contributions

HS and ZY conceived and designed the experiment; all authors collected the data and contributed to data analysis and interpretation; HX and ZY wrote the first draft of the manuscript. All authors critically reviewed, revised, and approved the manuscript.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was financially granted by the Key Research and development project of Anhui Province (No. 202004j070200180).

Availability of data and material

The authors confirm that the data supporting the findings of this study are available upon request from the corresponding author.

Ethics approval

Ethical approval for this study was obtained from the Animal Ethical Committee of Cheeloo College of Medicine, Anhui Provincial Hospital, Shandong University, China (Ethical code: 07020018).

Animal welfare

The present study followed international, and institutional guidelines for humane animal treatment and complied with relevant legislation.

ORCID iD

Zhongya Yan

References

Hausenloy

Yellon

. Myocardial ischemia-reperfusion injury: a neglected therapeutic target. J Clin Invest 2013; 123(1): 92–100.

Rajendran

Devarajan

Ganesan

, et al. Obesity, inflammation and acute myocardial infarction - expression of leptin, IL-6 and high sensitivity-CRP in Chennai based population. Thromb J 2012; 10(1): 13–16.

Thakker

Frangogiannis

Bujak

, et al. Effects of diet-induced obesity on inflammation and remodeling after myocardial infarction. Am J Physiology-Heart Circulatory Physiol 2006; 291(5): H2504–H2514.

Khodeer

Bilasy

Farag

, et al. Sitagliptin protects diabetic rats with acute myocardial infarction through induction of angiogenesis: role of IGF-1 and VEGF. Can J Physiol Pharmacol 2019; 97(11): 1053–1063.

Chao

Yin

Yao

Y-Y

, et al. Novel role of Kallistatin in protection against myocardial ischemia-reperfusion injury by preventing apoptosis and inflammation. Hum Gene Ther 2006; 17(12): 1201–1213.

Guo

Wang

S-B

Yuan

T-Y

, et al. Coptisine protects rat heart against myocardial ischemia/reperfusion injury by suppressing myocardial apoptosis and inflammation. Atherosclerosis 2013; 231(2): 384–391.

Liu

Shen

Jin

, et al. Recombinant human erythropoietin (rhEPO) preconditioning on nuclear factor-kappa B (NF-kB) activation & proinflammatory cytokines induced by myocardial ischaemia-reperfusion. Indian J Med Res 2006; 124(3): 343–354.

Oyama

J-i

Blais

Jr Liu

, et al. Reduced myocardial ischemia-reperfusion injury in toll-like receptor 4-deficient mice. Circulation 2004; 109(6): 784–789.

Zhang

Sun

Liu

, et al. A novel naturally occurring salicylic acid analogue acts as an anti-inflammatory agent by inhibiting nuclear factor-kappaB activity in RAW264.7 macrophages. Mol Pharmaceutics 2012; 9(3): 671–677.

10.

Patel

Pramanik

Rathwa

, et al 112-LB: Melatonin and DPP-IV Inhibitor: A Novel Combinatorial Approach for ß-Cell Regeneration. Arlington, VA: American Diabetes Association, 2019.

11.

Chang

Zhang

, et al Protective effects of sitagliptin on myocardial injury and cardiac function in an ischemia/reperfusion rat model. Eur J Pharmacol 2013; 718(1–3): 105–113.

12.

Chang

M-w

Chen

C-h

Chen

Y-c

, et al. Sitagliptin protects rat kidneys from acute ischemia-reperfusion injury via upregulation of GLP-1 and GLP-1 receptors. Acta Pharmacologica Sinica 2015; 36(1): 119–130.

13.

Vaghasiya

Sheth

Bhalodia

, et al. Sitagliptin protects renal ischemia reperfusion induced renal damage in diabetes. Regul Peptides 2011; 166(1–3): 48–54.

14.

Nuransoy

Beytur

Polat

, et al. Protective effect of sitagliptin against renal ischemia reperfusion injury in rats. Ren Fail 2015; 37(4): 687–693.

15.

El-Sahar

Safar

Zaki

, et al. Sitagliptin attenuates transient cerebral ischemia/reperfusion injury in diabetic rats: implication of the oxidative-inflammatory-apoptotic pathway. Life Sci 2015; 126: 81–86.

16.

Khedr

Ahmed

AAE

Kamel

, et al. Sitagliptin attenuates intestinal ischemia/reperfusion injury via cAMP/PKA, PI3K/Akt pathway in a glucagon-like peptide 1 receptor-dependent manner. Life Sci 2018; 211: 31–39.

17.

Dominguez-Rodriguez

Abreu-Gonzalez

. Myocardial ischemia-reperfusion injury: possible role of melatonin. World J Cardiol 2010; 2(8): 233.

18.

Sahna

Acet

Kaya Ozer

, et al. Myocardial ischemia-reperfusion in rats: reduction of infarct size by either supplemental physiological or pharmacological doses of melatonin. J Pineal Res 2002; 33(4): 234–238.

19.

Chen

Liu

Chen

, et al. Melatonin attenuates myocardial ischemia/reperfusion injury by inhibiting autophagy via an AMPK/mTOR signaling pathway. Cell Physiol Biochem 2018; 47(5): 2067–2076.

20.

Lee

Y-M

Chen

H-R

Hsiao

, et al. Protective effects of melatonin on myocardial ischemia/reperfusion injury in vivo. J Pineal Res 2002; 33(2): 72–80.

21.

Imenshahidi

Karimi

Hosseinzadeh

. Effects of melatonin on cardiovascular risk factors and metabolic syndrome: a comprehensive review. Naunyn-Schmiedeberg's Arch Pharmacol 2020; 393(4): 521–536.

22.

Davidson

Ferdinandy

Andreadou

, et al. Multitarget strategies to reduce myocardial ischemia/reperfusion injury. J Am Coll Cardiol 2019; 73(1): 89–99.

23.

Petrosillo

Colantuono

Moro

, et al. Melatonin protects against heart ischemia-reperfusion injury by inhibiting mitochondrial permeability transition pore opening. Am J Physiology-Heart Circulatory Physiol 2009; 297(4): H1487–H1493.

24.

Yang

Duan

Jin

, et al. JAK2/STAT3 activation by melatonin attenuates the mitochondrial oxidative damage induced by myocardial ischemia/reperfusion injury. J Pineal Res 2013; 55(3): 275–286.

25.

Zhai

Duan

, et al. Melatonin ameliorates myocardial ischemia reperfusion injury through SIRT3-dependent regulation of oxidative stress and apoptosis. J Pineal Res 2017; 63(2): e12419.

26.

Bozkurt

Aguilar

Deswal

, et al. Contributory risk and management of comorbidities of hypertension, obesity, diabetes mellitus, hyperlipidemia, and metabolic syndrome in chronic heart failure: a scientific statement from the American Heart Association. Circulation 2016; 134(23): e535–e578.

27.

Song

L-Y

, et al. Diet-induced obesity suppresses sevoflurane preconditioning against myocardial ischemia-reperfusion injury: role of AMP-activated protein kinase pathway. Exp Biol Med 2011; 236(12): 1427–1436.

28.

Maarman

Marais

Lochner

, et al. Effect of chronic CPT-1 inhibition on myocardial ischemia-reperfusion injury (I/R) in a model of diet-induced obesity. Cardiovasc Drugs Ther 2012; 26(3): 205–216.

29.

Liu

Lloyd

. High-fat, low-carbohydrate diet alters myocardial oxidative stress and impairs recovery of cardiac function after ischemia and reperfusion in obese rats. Nutr Res 2013; 33(4): 311–321.

30.

Chen

Zhang

R-Q

Wei

X-G

, et al. Mechanism of TLR-4/NF-κB pathway in myocardial ischemia reperfusion injury of mouse. Asian Pac J Trop Med 2016; 9(5): 503–507.

31.

Hernandez-Resendiz

Chinda

Ong

S-B

, et al. The role of redox dysregulation in the inflammatory response to acute myocardial ischaemia-reperfusion injury - adding fuel to the fire. Curr Med Chem 2018; 25(11): 1275–1293.

32.

Vilahur

Badimon

. Ischemia/reperfusion activates myocardial innate immune response: the key role of the toll-like receptor. Front Physiol 2014; 5: 496.

33.

Kleinbongard

Schulz

Heusch

. TNFα in myocardial ischemia/reperfusion, remodeling and heart failure. Heart Fail Rev 2011; 16(1): 49–69.

34.

Cha

Wang

, et al. Cytokines link toll-like receptor 4 signaling to cardiac dysfunction after global myocardial ischemia. Ann Thorac Surg 2008; 85(5): 1678–1685.

35.

Saini

Zhang

, et al. Role of tumour necrosis factor-alpha and other cytokines in ischemia-reperfusion-induced injury in the heart. Exp Clin Cardiol 2005; 10(4): 213–222.