Introduction
Myocardial ischemia/reperfusion injury (MIRI) often occurs in the processes of extracorporeal circulation, thrombolysis, coronary artery bypass grafting, and heart transplantation. MIRI may seriously influence the therapeutic effects and prognosis of cardiac patients by aggravating myocardial injury and causing arrhythmia. The process of MIRI includes alterations in levels of cytokines, neutrophil activation and infiltration, oxygen free radicals, calcium overload, myocardial energy metabolic dysfunction, vascular endothelial cells, and apoptosis [
1]. Oxidative stress, calcium overload, and inflammation induce endoplasmic reticulum stress (ERS), while the excessive ERS-mediated myocardial dysfunction and apoptosis further exacerbate MIRI [
2‐
4].
Lipoxins (LXs) are arachidonic acid metabolites formed during inflammation via transcellular biosynthetic pathways. LXs are the first class of lipid mediators that are “switched on” in the resolution phase of an inflammatory response and function as “braking signals” in inflammation. LXs, with dual functions of anti-inflammation and pro-resolution [
5], have obvious protective effects on ischemia/reperfusion (I/R) injury of lung, stomach, brain, and kidney [
6‐
9].
It was recently found that atorvastatin and losartan have cardiovascular protective effects on rat heart and aorta [
10]. Erythropoietin has recently been shown to confer cardioprotective effects via angiogenesis and antiapoptosis in porcine myocardial injury [
11]. Sulfur dioxide can decrease the activated ERS in rats with myocardial injury [
12]. Aminobenzoic acid hydrazide, a myeloperoxidase inhibitor, may have protective functions by reducing neutrophil adhesion [
13]. In the present study, we tested a hypothesis that administration of LXA
4 pre-MIRI or post-MIRI attenuates MIRI through the ERS apoptotic pathway in rats.
Materials and methods
Rat model of myocardial ischemia–reperfusion injury
The animal experimental procedures were approved by Wenzhou Medical University Animal Care and Use Committee, which is certified by the Chinese Association of Accreditation of Laboratory Animal Care (SYXK, Zhejiang 2010-0150). Sprague–Dawley (SD) male rats (8 weeks old, 200–250 g) were fed a standard diet and maintained in the controlled environment of the animal center at 25 ± 1 °C under a 12 h light–dark cycle, and were used in this study.
In brief, rats were anesthetized by an intraperitoneal injection of 10 % chloral hydrate (300 mg/kg body weight) and placed in a supine position. Blood was collected by femoral venipuncture. Next, the animals were intubated for artificial ventilation with 100 % oxygen using a small animal breathing machine (Tidal volume 5 ml, frequency 70/min) and electrocardiogram (ECG) monitor. Thoracotomy was performed between the sternum and left costa, then the pericardium was gently opened. Myocardial ischemia was induced by ligating the left anterior descending coronary artery (LAD) using a 3-0 silk suture with a section of polyethylene tubing placed over the LAD and 1 mm from the tip of the normally positioned left atrium. The coronary artery was occluded by pulling on the suture tightly 10 min later. After 30 min of myocardial ischemia, reperfusion started by releasing the ligature and removing the tube for 120 min. The chest wall was closed, the animal was extubated, and the body temperature was maintained using a 37 °C warming plate.
The indications of successful LAD occlusion included ST-segment elevation of 0.1 mV or sharp rise in T wave, wider and higher QRS wave of the ECG, and visual cyanosis of myocardial discoloration. The indications of successful reperfusion were ST-segment depression (≥1/2) of the ECG and the myocardial color being normal (pink).
Animal grouping and treatments
Healthy adult male SD rats were randomly divided into six groups as follows. (1) Using LXA4 before I/R (LX1 group): LXA4 (100 μg/kg) was injected by femoral vein before thoracotomy. After 30 min of myocardial ischemia, reperfusion was performed by releasing the ligature and removing the tube for 120 min. (2) Using LXA4 after I/R (LX2 group): After 30 min of myocardial ischemia, reperfusion lasted for 30 min, then LXA4 (100 μg/kg) was injected by femoral vein. Thereafter, reperfusion was initiated again for 90 min. (3) MIRI group 1 (I/R1 group): Normal saline (2 ml/kg) was injected by femoral vein before thoracotomy, with the remaining treatment the same as for the LX1 group. (4) MIRI group 2 (I/R2 group): Normal saline (2 ml/kg) was injected by femoral vein after I/R, with the remaining treatment the same as for the LX2 group. (5) Sham group 1 (C1 group): Rats underwent a similar operation without myocardial I/R, with the remaining treatment the same as for the I/R1 group. (6) Sham group 2 (C2 group): Rats underwent a similar operation without myocardial I/R, with the remaining treatment the same as for the I/R2 group.
In the non-MIRI experiments, the rats were randomly divided into six groups (Sham1, Sham2, Sham3, Sham4, Sham5, and Sham6). In the Sham1 group, the rats were kept for 150 min and then anesthetized, followed by bloodletting. In the Sham2 group, the rats were treated as the same as in the Sham1 group, except that the time before anesthesia was 90 min. In the Sham3 group, rats were injected with 2 ml/kg normal saline. After 150 min, the rats were anesthetized and bloodletting was performed. In the Sham4 group, the rats were treated the same as in Sham3 group, except that the time before anesthesia was 90 min. In the Sham5 group, rats were injected with 100 μg/kg LXA4 in 2 ml/kg normal saline. After 150 min, the rats were anesthetized and bloodletting was performed. In the Sham6 group, the rats were treated the same as in the Sham5 group, except that the time before anesthesia was 90 min. The myocardial tissues were collected. The 150- and 90-min intervals before anesthesia mimicked the pretreatment and postprocessing times in the above MIRI experiments.
Blood collection and tissue harvest
Blood samples were collected in each group immediately before thoracotomy and after anesthetization (T
1) or after the experiments (T
2). In groups C1 and C2, T
2 was obtained after 150 min of placing surgical suture under LAD. For all the other groups, T
2 blood samples were obtained after 120 min of reperfusion. The heart was removed after obtaining blood samples (T
2) and a portion of myocardial tissue was fixed in 4 % formalin. Pathologic examinations of paraffin-embedded sections were performed. A separate portion of myocardial tissue was frozen in liquid nitrogen and kept in the freezer at −70 °C.
Myocardial tissue hematoxylin–eosin staining
Myocardial tissue slices were baked at a high temperature of 55–65 °C for 1–2 h, followed by xylene dewaxing, alcohol rinsing, and hematoxylin staining for 5 min. The slices were then treated with hydrochloric acid alcohol, added with the bluing agent (Blue Season Sci & Tech Development, Shanghai, China) to promote bluing of the sample, followed by eosin staining for 20 s. Finally, the slices were mounted with neutral gum after dehydration with alcohol and treatment with xylene.
Transmission electron microscopy (TEM)
For TEM examination, samples containing a 2-mm portion from the edge of the incision were immediately fixed for 4 h in 0.1 M phosphate buffer containing 2.5 % glutaraldehyde and 2 % paraformaldehyde. The samples were then fixed with 1 % osmium tetroxide for 2 h, dehydrated with ethanol, and embedded in epoxy resin. Resin-embedded blocks were cut into 60–80-nm ultrathin sections with an ultra-microtome (PT-XL, RMC Boeckeler, Tucson, AZ, USA). The ultrathin sections were placed on carbon-coated nickel grids and examined with an H-7500 transmission electron microscope (Hitachi, Tokyo, Japan) operated at 80 kV.
TUNEL assay
Apoptosis was determined by TUNEL assay according to manufacturer’s instructions. Cells with apoptotic morphologic features as well as with tan or brown nuclei were judged to be apoptotic cells. The five fields of view were automatically selected by the Image-Pro Plus version 5.1 image analysis software. The percentage of apoptosis-positive cells was calculated for each field of view. The mean was calculated to obtain the percentage of apoptotic cells, and expressed as apoptotic index. Apoptosis index (%) = (apoptotic nuclei count/total nucleus count) × 100 %.
Cytokine and cardiac troponin I (cTnI) levels
For cytokine immunoassay, blood samples were collected by femoral venipuncture at the indicated time points before thoracotomy (T
1) and after reperfusion (T
2). The serum levels of interleukin (IL)-1β, IL-6, IL-10, tumor necrosis factor (TNF)-α, and cTnI were measured using a rat enzyme-linked immunosorbent assay (ELISA) kit (Shanghai Boyun Biotech, China) in accordance with the manufacturer’s instructions. Cytokine and cTnI levels were expressed as ng/l.
Myeloperoxidase (MPO) and superoxide dismutase (SOD) activity
The myocardial MPO activity was determined on frozen tissue by use of colorimetry assay kits (Nanjing Jiancheng Bioengineering Institute, China), and expressed as units per gram tissue wet weight. The myocardial SOD activity was determined on frozen tissue using Xanthine Oxidase assay kits (Nanjing Jiancheng Bioengineering Institute, China), and expressed as units per milligram protein.
Malondialdehyde (MDA) determination
The MDA content was determined on frozen myocardial tissue by use of the thiobarbituric acid assay kit (Nanjing Jiancheng Bioengineering Institute). The level of MDA was expressed as nanomoles per milligram protein.
Real-time quantitative polymerase chain reaction (RT-qPCR) analysis
Total RNAs of the tissues were extracted using Trizol Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Total RNA concentrations were quantified using a spectrophotometer (UV-2000; UNICO, Shanghai, China). Subsequently, 1 ng of total RNA was reverse-transcribed via the cDNA synthesis kit (Invitrogen). RT-PCR was performed using the SYBR green system (Bio-Rad, Hercules, CA, USA). Amplifications for cDNA samples were carried out using a PCR machine, Roto-Gene 3000 (Corbett Robotics, Brisbane, Australia). The primers used in this study are given in Table
1. The relative quantification of target gene was normalized to glyceraldehyd-3-phosphate dehydrogenase (GAPDH), and calculated using the absolute quantification standard curve method. The melting curves were produced at the end of each PCR to confirm the specific amplification. Each sample was analyzed in triplicate.
Table 1
Real-time PCR primer sequences
GRP-78 | 5′-CCTGTTGCTGGACTCTGTGA-3′ | 5′-GAATACACCGACGCAGGAAT-3′ | 204 |
Caspase-12 | 5′-GCTGCCAAGAGAACACATGA-3′ | 5′-GGTTCTCAGCTTTGCTCAGG-3′ | 169 |
GAPDH | 5′-GAGTCAACGGATTTGGTCGT-3′ | 5′-TTGATTTTGGAGGGATCTCG-3′ | 238 |
Western blot analysis
Equal amounts of protein (50 μg) were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Proteins were then transferred to polyvinylidene fluoride membrane. Membrane was blocked with 5 % nonfat milk in Tris-buffered saline, 0.1 % Tween 20 (Sigma, St Louis, MO, USA). The immunoblotting was performed using rabbit antirat GRP-78 (1:500; Cell Signaling Technology, Danvers, MA, USA) and caspase-12 (1:1000; Cell Signaling Technology) as described by the manufacturer. Anti-β-actin was analyzed with the antibody (1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA). Blots were then developed by incubation with biotinylated antirabbit antibody (1:2000; Vector Laboratories, Burlingame, CA, USA), followed by incubation with the ABC reagent (GE, Fairfield, CT, USA). Signal was detected using an ECL luminescence kit (GE) and X-ray film.
Immunofluorescence
Rat cardiac tissues were collected and cut into 5-μm slices for immunofluorescence examination. The samples were rinsed with phosphate buffer solution (PBS) for three times and fixed with 4 % (w/v) paraformaldehyde at 4 °C for 2 h. Samples were then permeabilized with dimethylbenzene and blocked with goat serum for 1 h at 37 °C, followed by incubation at 4 °C overnight in a primary antibody solution containing rabbit anti-IL-10 or rabbit anti-GRP-78 BiP antibody (1:70; Cell Signaling Technology). Samples were rinsed with PBS, then incubated with goat anti-rabbit fluorescein isothiocyanate-biotinylated immunoglobulin G secondary antibody (Blue Season Sci & Tech Development). Samples were then observed with a fluorescence microscope (IX71; Olympus, Tokyo, Japan) equipped with ISCapture software, and images were taken with a CCD camera (Discovery C15; Olympus, Tokyo, Japan). For each sample, 10 random microscopic fields were captured and integral optical density (IOD) was calculated.
Statistical analysis
All data were expressed as mean ± standard deviation. Significant differences were evaluated by one-way analysis of variance to compare more than two groups, or with paired t test to compare two groups. The significance level was set at P < 0.05. All statistical analyses were performed using SPSS version 17.0 (SPSS, Chicago, IL, USA).
Discussion
The ER is a highly dynamic organelle that plays a central role in lipid and protein biosynthesis. The ER has multiple cellular functions in the synthesis of integral membrane proteins, proper folding and oligomerization of proteins, and Ca2+ storage and signaling. ERS is a subcellular pathologic process of imbalance in ER homeostasis, which can be caused by the disturbances in redox regulation, calcium regulation, and glucose deprivation. ER can be very sensitive to various stresses. Through ERS, cells can be automatically self-protected to a certain extent.
ERS can be induced by MIRI, leading to the imbalance of ER homeostasis. However, different stages of ERS determine divergent cell fates [
14]. In the early stage of ERS, the misfolded proteins accumulate and trigger the release of GRP-78 and then launch the unfolded protein response (UPR). UPR is an evolutionarily conserved response that can be activated to enhance the protein-folding capacity of the ER and promote ER-associated protein degradation to remove the misfolded proteins. Therefore, the release of GRP-78 is critical in the early protective response, which is an upstream gene in the ERS signaling and can also be seen as a marker in the early ERS [
15]. In our study, the increased mRNA and protein expression of GRP-78 induced by I/R demonstrated the process of ERS, which can protect the ER and further maintain ER homeostasis. Immunofluorescence also confirmed that the expression of GRP-78 in the cytoplasm increased significantly after MIRI. However, persistent existence of excessive stress inhibits the re-establishment of ER homeostasis and triggers cell death, usually via inducing apoptosis. The chaperone GRP-78 will then bind to other proteins. In rodents, caspase-12 separated from GRP-78 complex will be activated and will trigger ERS apoptosis [
16,
17]. It was also observed that compared with the control group, caspase-12 expression was upregulated and the incidence of myocardial apoptosis increased, suggesting that MIRI induces myocardial apoptosis through the ERS apoptotic pathways.
Myocardial cell apoptosis is one of characteristics of MIRI damage, and plays a crucial role in MIRI. Several pathways linking ERS to cell death have been reported, although the related mechanisms have not been clearly identified. However, in rodents the activation of ERS apoptotic pathways specific for protein caspase-12 in ER membrane has been observed during apoptosis [
18]. Lee et al. found that caspase-12 was particularly activated in ERS but that no activation of caspase-12 was detected in non-ERS-mediated apoptosis [
19]. Wang et al. reported that cardiac muscles can be protected by inhibiting the ERS and ERS-related apoptosis [
20]. It was shown in this study that the downregulated myocardial cell apoptosis and cTnI expression, as well as the reduced expressions of GRP-78 and caspase-12 after using LXA
4, indicate that the protective effective of LXA
4 on cardiac muscle may be related to the ERS. LXA
4 can inhibit excessive ERS and maintain the function of ER, resulting in reduced expression of GRP-78 and caspase-12. Consequently, myocardial cell apoptosis can be alleviated, which also provides new insight into the prevention and treatment of MIRI.
However, the mechanism underlying the effect of LXA
4 on ERS is still not clear. Hayashi et al. considered that ER is sensitive to oxidative stress and that brain I/R injury-produced active oxygen may lead to ER damage [
21]. Kumar and Sitasawad discovered that ER could be the possible target of active oxygen [
22]. LXs are newly discovered bioactive products derived from arachidonic acid that have a number of proinflammatory and anti-inflammatory functions. LXs also inhibit neutrophil granulocyte infiltration and the production of reactive oxygen. LXA
4 can inhibit the production of NADPH oxidase-mediated reactive oxygen in BV2 cells [
23] and the recruitment of neutrophils to the inflammatory sites [
24]. The MPO activity in lung tissue after I/R can also be reduced by LXA
4 [
6]. Meanwhile, LXA
4 can suppress the expression of IL-6, TNF-α, and IL-8, and upregulate the IL-10 expression of vascular endothelial growth factor-stimulated inflammation in human umbilical vein endothelial cells [
25]. In vivo study also showed that LXA
4 can restrain TNF-α-induced I/R injury through upregulation of IL-10 in IL-10(–/–) mice [
26].
In this study, with the treatments of LXA
4, the expression of proinflammatory cytokines (IL-1β, IL-6, IL-8, and TNF-α) were reduced while the anti-inflammatory cytokine IL-10 were upregulated, leading to the balance of pro-inflammatory/anti-inflammatory cytokines and a less inflammatory response. The increased expression of MPO, SOD, and MDA in the I/R groups indicates that peroxidative stress has been induced after I/R, suggesting that the decreased scavenging ability of free radicals may further lead to myocardial injury. After the treatments of rats with LXA
4, the SOD activity increased significantly and the expression of MPO and MDA was markedly reduced, suggesting that the reduced MIRI may be due to the decreased expression of the lipid peroxidation caused by oxygen free radicals. The attenuated neutrophil infiltration may also play a role in restoring the oxidant/antioxidant balance after I/R injury. LXA
4 can protect the ultrastructure of ER and mitochondria. It has been reported that LXA
4 can remarkably inhibit the release of mitochondria-mediated apoptosis proteins and the activation of caspase [
27]. Research showed that antioxidants can alleviate ERS injury through eliminating the oxygen free radicals and decreasing the production of GRP-78 and caspase-12 [
28]. The proinflammatory cytokines are able to cause cardiac injury through cell apoptosis and may influence the remodeling of the extracellular matrix [
29]. Therefore, we assume that the attenuated excessive ERS and cell apoptosis by LXA
4 may be caused by the restraint of neutrophil activation and further inflammatory injury.
In conclusion, the application of LXA4 pre-MIRI and post-MIRI apparently inhibited the activation of neutrophils, alleviated oxidative damage of cardiac muscle, reduced the expression of GRP-78 and caspase-12, prevented the excessive ERS-mediated cell apoptosis, and therefore effectively protected the cardiac muscles. Although both pharmacologic preconditioning and postconditioning with LXA4 can protect cardiac muscle from MIRI, the preconditioning has been largely restrained because of the poor prediction of MIRI in clinical terms, implicating that the postcondition treatment of LXA4 should have great potential for clinical application in MIRI in the future. Further studies to optimize the dose, timing, and drug administration of LXA4 are warranted.