Myocardial ischemia-reperfusion injury (IRI) is associated with poor outcome in acute myocardial infarction (AMI) patients after revascularization.1 It is known that inflammatory mechanisms play important roles in the development of IRI.2 Myocardial IRI has all the characteristics of acute inflammatory reactions, including upregulated expression of cell adhesion molecules, secretion of cytokines, neutrophil infiltration, and microvascular permeability enhancement.3 Therefore, strategies capable of suppressing the inflammatory response might be beneficial to attenuate the IRI, rescue the damaged myocardium, and lead to the reduction in the infarct size. In fact, recent evidences demonstrated that inhibition of inflammation might be a promising approach for the treatment of AMI including IRI.4
Galectin is known to play a crucial role in the pathogenesis of IRI by targeting cellular homeostasis and inflammatory responses.5 Ticagrelor inhibits uptake of adenosine by inhibiting ENT-1 of erythrocytes and enhances local response of adenosine. Previous study showed that adenosine could inhibit NF-κB activation, downregulate levels of inflammatory factors, and antagonize myocardial ischemic injury in rats.6 Our previous study demonstrated that ticagrelor downregulated the expression of galectin-3.7 Many studies have shown that NF-κB is involved in the regulation of galectin-3 expression.8,9 In this study, we tested the hypothesis that ticagrelor might downregulate galectin-3 expression in the ischemic myocardium partly through inhibiting the activation of NF-κB in the rat model of IRI.
This study was approved by the Animal Care Committees of Wuhan Fourth Hospital. The experimental protocol followed the ARRIVE guidelines and guidelines from the National Institutes of Health for animal care and use (NIH Publications No. 85–23, revised 1996).
Animals and Study Groups
Sprague–Dawley rats (200–250 g) were obtained from the Experimental Animal Center of Huazhong University of Science and Technology (Wuhan, China). IRI was achieved by ligating the left anterior descending artery (LAD) for 45 minutes followed by reperfusion.7,10 All experimental animals were randomly divided into 24-hour, 3-, and 7-day groups according to the time of reperfusion after sham operation or LAD ligation. Each group of rats was treated as follows:
- Sham group (n = 5, each time point): Rats in this group received similar surgical intervention without LAD ligation.
- Placebo group (n = 5, each time point): Immediately after LAD ligation, rats in this group were given equal volume of saline per gavage, once daily till the end of the study.
- Ticagrelor group (n = 5, each time point): Immediately after LAD ligation, rats in this group were given ticagrelor (150 mg/kg11) per gavage, once daily till the end of the study.
- Dextran sodium sulfate (DSS) group (n = 5, each time point): DSS, an agonist of NF-κB,12 was applied to rats in drinking water (20 g/L) at 1 week before IRI and continued till the end of the study.
- DSS + ticagrelor group (n = 5, each time point): After pretreatment with DSS (20 g/L in drinking water) for 7 days, ticagrelor (150 mg/kg) was given per gavage immediately after LAD ligation. DSS (20 g/L in drinking water) was continued, and ticagrelor (150 mg/kg) was given per gavage once daily till the end of study.
Establishment of IRI Model
After anesthetized by 3% pentobarbital sodium with intraperitoneal injection (50 mg/kg), rats were intubated and connected with an animal respirator (ALC-V8S; ALCOTT, Shanghai, China). The IRI model of rats was established as previously described.7,10 Briefly, the LAD of rats was ligated and blood flow was restored after 45 minutes of ischemia. The sham-operated rats received same surgical intervention without LAD ligation.
Determination of the Infarct Area
The LAD was religated at 24 hours, 3 and 7 days after reperfusion. Nonischemic myocardium was stained by 2% Evans blue dye (Fluka, Switzerland), and area at risk (AAR) was delineated. Infarct area (IA) and AAR were determined as reported by Xu et al,13 and a computer-assisted image analysis (NIH Image, developed and maintained by the National Institutes of Health, Bethesda, MD) was used to determine AAR and the degree of myocardial necrosis.
mRNA Expressions of NF-κB, Galectin-3, TNF-α, and IL-6 in the Ischemic Myocardium Detected by Real-Time PCR
Total RNA was extracted from the frozen ischemic myocardial tissue as described by the manufacturer's protocol (Trizol, 15596026; Ambion, TX). Single-strand cDNA was synthesized by reverse transcription (RT) kit PrimeScript RT Master Mix Perfect Real Time (Cat No. 639505; TAKARA, Japan). Quantitative real-time PCR and RT-PCR were performed as reported by Liu et al.7 Quantification of mRNA included normalization to GAPDH levels, and melting curves were used to determine specific PCR products. Primers were shown in Table 1.
Protein Expression of NF-κB and Galectin-3 in the Ischemic Myocardium Detected by Western Blotting
Protein levels of NF-κB and galectin-3 in the ischemic myocardium were assessed by Western blotting. After the protein contents were determined, the extracted proteins were denatured and separated by SDS/PAGE, and transferred to a nitrocellulose membrane (Pierce, Rockford, IL). The membranes were probed with the appropriate primary antibodies overnight at 4°C after blocked in TBS solution. Membrane-bound primary antibodies were detected using horse radish peroxidase-labeled secondary antibodies for 1 hour at room temperature and colored by electrochemiluminescence. Rabbit anti-NF-κBp65 (Ab 16502, 1:2000; Abcam, Cambridge, United Kingdom), anti-galectin-3 (113486, 1:10000; GENETEX, Irvine, CA), anti-GAPDH (2118, 1:1000; CST, Boston, MA), and Goat Anti-Rabbit IgG (PAB150011, 1:10000; BIOSWAMP, CN) were used to measure the protein expression of NF-κB and galectin-3 in the ischemic myocardium.
Measurements of Plasma cTnI, High-Sensitivity C-Reactive Protein, and NT-proBNP by ELISA
Concentrations of plasma cTnI, high-sensitivity C-reactive protein (hs-CRP), and NT-proBNP were measured with the respective ELISA kit (Immunoway, KE1634, KE1761, KE1457, TX) following the manufacturer's instructions. The absorbance was recorded at 450 nm.
All the parameters among the groups were compared by 2-way analysis of variance (time point and treatment group) following the Tukey honestly significant difference or Games–Howell test as indicated. One-way analysis of variance of the same time point or the same treatment group was performed to evaluate the differences among the groups following the Tukey honestly significant difference or Games–Howell test. All statistical analyses were performed by IBM SPSS, version 22.0 for Windows. P < 0.05 was considered statistically significant.
Infarct Size and Infarct Pattern Among Groups
Figure 1 showed that the IA/AAR ratio was reduced by 12.6% at 24 hours in the ticagrelor group compared with the placebo group (34.5 ± 12.2% vs. 47.1 ± 12.4%, P = 0.144). The IA/AAR ratio was significantly lower in the ticagrelor group compared with the placebo group at 3 days (17.74 ± 9.9% vs. 56.15 ± 9.44%, P < 0.01) and 7 days (14.02 ± 5.08% vs. 48.31 ± 19.01%, P < 0.01), respectively. The IA/AAR ratio was significantly higher in the DSS + ticagrelor group compared with the ticagrelor group at 3 days (49.27 ± 8.73% vs. 17.74 ± 9.9%, P < 0.01) and 7 days (50.66 ± 11.16% vs. 14.02 ± 5.08%, P < 0.01), respectively. The IA/AAR ratio was similar between the DSS+ticagrelor group and placebo group at 24 hours (53.25 ± 9.98% vs. 47.13 ± 12.42%, P = 0.415), 3 days (49.27 ± 8.73% vs. 56.15 ± 9.44%, P = 0.266), and 7 days (50.66 ± 11.16% vs. 48.31 ± 19.01%, P = 0.817), respectively. The IA/AAR ratio was also similar among the same groups at different time points.
Apart from numerical infarct size, the infarct pattern was transmural myocardial infarction in all groups, and there were no differences in infarction pattern among groups.
mRNA Expression of NF-κB, Galectin-3, IL-6, and TNF-α in the Ischemic Myocardium After IRI
The mRNA expressions of NF-κB, galectin-3, IL-6, and TNF-α were significantly upregulated in the ischemic myocardium at 24 hours, 3 and 7 days after IRI in the placebo group, which were significantly downregulated by ticagrelor treatment, and this effect was blocked by pretreatment with DSS (Fig. 2).
Protein Expression of Galectin-3 and NF-κB in the Ischemic Myocardium of Rats After IRI
Compared with respective expressions of galectin-3 and NF-κB in the nonischemic region of the sham-operated group, expressions of galectin-3 and NF-κB in the ischemic myocardium were significantly increased in the placebo group from 24 hours to 7 days after IRI. Compared with 24 hours after IRI, the protein expression of galectin-3 was further increased at 3 and 7 days after IRI in the placebo group. Compared with the placebo group, expression of galectin-3 protein in the ischemic region was significantly downregulated in the ticagrelor group at 24 hours, 3 and 7 days after IRI. This effect was blocked by pretreatment with DSS (Figs. 3A, B). Similar to galectin-3, protein expression of NF-κB in the ischemic myocardium was also significantly increased from 24 hours to 7 days in the placebo group as compared to the respective nonischemic region of the sham group, which could be significantly downregulated by ticagrelor, while pretreatment with DSS blocked the effect of ticagrelor (Figs. 3A, C).
Changes in Plasma cTnI, hs-CRP, and NT-proBNP Levels in Rats After IRI
The plasma levels of cTnI were significantly increased at 24 hours, 3 and 7 days after IRI in the placebo group compared with the sham group, which were significantly decreased by ticagrelor treatment at 24 hours and 3 days, and this effect could be blocked by pretreatment with DSS at 3 and 7 days (Fig. 4A). Compared with the placebo group, the plasma levels of hs-CRP were significantly reduced in the ticagrelor group at 24 hours and 3 days after IRI. This effect was blocked by pretreatment with DSS (Fig. 4B). Similar to cTnI, the plasma levels of NT-proBNP were also significantly increased from 24 hours to 7 days in the placebo group compared with the sham group, which could be significantly decreased by ticagrelor, while pretreatment with DSS blocked the effect of ticagrelor (Fig. 4C).
The main results of this study were as follows: (1) Treatment with ticagrelor immediately after LAD ligation significantly reduced the IA/AAR ratio in this rat model of IRI. (2) Ticagrelor also significantly downregulated the expression of NF-κB, galectin-3, IL-6, and TNF-α in the ischemic region and significantly decreased plasma levels of cTnI, NT-proBNP, and hs-CRP. (3) Above beneficial effects of ticagrelor could be blocked by pretreatment with NF-κB agonist DSS. Our results thus indicate that the cardioprotective effect of ticagrelor might be partly mediated by inhibiting the activation of NF-κB in the ischemic myocardium of this IRI rat model.
In previous studies, we and others found that the use of P2Y12 receptor antagonist ticagrelor significantly reduced the IA/AAR ratio and area of AMI in rats with IRI.7,11 Our experiments further showed that galectin-3 was participated in the process of IRI, and the expression of galectin-3 in the ischemic myocardium peaked at 7 days after IRI.7 Galectin-3 is mainly produced by macrophages and expressed in epithelial cells, dendritic cells, and various inflammatory cells.14 Previous study15 also observed the expression of galectin-3 in myocardial tissue after AMI in rats, and rats were killed at 1, 2, 4, 12, and 24 weeks after AMI; subsequent observations revealed that mRNA expression of galectin-3 increased in the infarct zone and peaked at 1 week after AMI and gradually decreased thereafter. Hashmi et al16 demonstrated that protein and mRNA expressions of galectin-3 were significantly elevated at 60 minutes and 24 hours after AMI. Our previous finding showed that ticagrelor treatment applied immediately after LAD ligation could downregulate galectin-3 expression in the ischemic myocardium,7 which might be one of the working mechanisms contributing to the observed cardioprotective effect of ticagrelor in the rat model of IRI.
Previous study showed that troponin release was positively correlated with myocardial infarct size.17 Hallén et al18 found that cTnI is an effective indicator of early estimation of myocardial IA. The results of our study were consistent with above studies, and the changes in plasma levels of cTnI were correlated with the IA/AAR ratio in our study. Marrero et al12 found that the degree of activation of NF-κB was also significantly correlated with the severity of the inflammatory response. Our study results demonstrated that the levels of plasma hs-CRP were consistent with the change of NF-κB in ischemic myocardial tissue, indicating that the activation of NF-κB might aggravate the inflammatory response in IRI rats. Morrow et al19 found that circulating levels of BNP were significantly elevated within 1–4 days after AMI, and our study revealed that levels of plasma NT-proBNP peaked within 24 hours after IRI and ticagrelor treatment applied immediately after LAD ligation could decrease the plasma levels of NT-proBNP, which indicated that ticagrelor treatment could attenuate myocardial injury after IRI.
Activation of NF-κB might aggravate myocardial IRI.20 Our study demonstrated that the protective effect of ticagrelor was blocked by pretreatment with DSS, which was in line with previous reports suggestive of the protective effect of NF-κB antagonists in myocardial IRI.21–24 Izumi et al21 showed the protective effects of NF-κB antagonist on myocardial IRI, and Han et al20 found that the activation of NF-κB aggravated myocardial IRI. Our results demonstrated that the expression of NF-κB and other inflammatory factors was increased after DSS pretreatment. However, there was no significant change in the myocardial infarct size after DSS intervention. The main reasons might be related to the following factors: (1) Both positive and negative feedback might affect the regulation of NF-κB activation. Positive feedback regulation mainly occurs extracellularly. TNF-α stimulates the production of anti-inflammatory factors such as IL-10 and IL-13, which could inhibit the transcription and expression of proinflammatory factors. The production of anti-inflammatory factors might somewhat reduce the myocardium damage caused by the excessive increase of inflammatory factors. (2) Myocardial IRI is a complex process. It might also be related to the action of free radicals and the role of intracellular calcium overload in addition to inflammation.
NF-κB is a key transcription factor that mediates the release of inflammatory factors.25,26 NF-κB is presented in cardiomyocytes, vascular endothelial and smooth muscle cells, and is closely related to the initiation and progression of various cardiovascular diseases.27 In vivo studies showed that NF-κB played an important role in the inflammatory response after AMI28; NF-κB could be activated by phosphorylation, transferred to the nucleus, and further promoted the transcription of target genes, thereby enhancing a variety of inflammatory responses after AMI.29–31 Moreover, literature reports also elucidated the role of other inflammation-related factors such as TNF-α, IL-6, and IL-1 in the pathological process of AMI, and they were all effector target genes of NF-κB.32–34 It is to note that NF-κB is also a known regulator of galectin-3 expression.8 Our results showed that pretreatment with NF-κB agonist DSS blocked the cardioprotective effects of ticagrelor and upregulated galectin-3 in the ischemic myocardium, suggesting that one of the major therapeutic working mechanisms of ticagrelor on reducing the IA/AAR ratio in this rat model of IRI might be mediated by downregulating NF-κB expression in the ischemic myocardium. Li et al6 previously confirmed that adenosine could inhibit the activity of NF-κB, downregulate the levels of inflammatory cytokines, and antagonize myocardial ischemic injury in rats, indicating that regulating NF-κB activity and inhibiting inflammatory factors might be important therapeutic options for attenuating myocardial insult after myocardial injury.
There are several study limitations of our experiments.
- Previous study reported that the effect of ticagrelor might be mediated at least through 2 pathways related to the process activated by adenosine and COX-2–dependent process.10 Ticagrelor could inhibit the uptake of adenosine in cardiomyocytes, which could subsequently inhibit NF-κB expression. This study showed that NF-κB agonist DSS blocked the beneficial effects of ticagrelor; it is therefore possible that part of the beneficial effects of ticagrelor achieved in this model might be mediated through modulating adenosine signaling and other NF-κB–independent mechanisms. Future studies are warranted to explore the protective mechanisms of ticagrelor focusing on the COX-2 dependent process.
- Cautions are also needed regarding the specificity of DSS on NF-κB signaling and subsequent cardioprotection mechanisms. DSS is currently considered as one of the few agonists of NF-κB. However, it is possible that DSS could generally antagonize cardioprotection by promoting inflammation because inflammatory pathway activation is a common process after IRI, and DSS could induce excessive inflammatory responses in various circumferences.35 If it would be the case, it is reasonable but not peculiar that DSS might antagonize the cardioprotection induced by ticagrelor by enhancing the general inflammatory responses independent of its activating role in NF-κB. Because it is shown that galectin-3 is downregulated by ticagrelor, it could be readily speculative that the NF-κB pathway is involved in the downstream process. Future studies are thus warranted to explore this issue with the help of knock-out and knock-in animal models. Ideally, the role ticagrelor could be tested in terms of ischemia in NF-κB knock-out and knock-in animal models, but these experiments could not be performed in our laboratory because of technical and financial reasons. Additional experiments with various signaling antagonists/agonists are also needed to clarify the cardioprotective issue of ticagrelor, but these contents are beyond the experimental focus of this study.
Our study indicates that the beneficial cardioprotective effects of ticagrelor might be partly mediated by downregulating the NF-κB–dependent pathway in this rat model of IRI.
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