Antioxidation Effect Of Omega-3Fatty Acids Postconditioning In Rats Myocardium After Ischemia Reperfusion Injury

1. BackgroundIschemia-reperfusion injury (IRI) refers to ischemic injury of tissue cells in a reversible upon restoration of blood reperfusion, injury aggravated or converted into irreversible damage. Myocardial ischemia-reperfusion injury has become a common clinical problem, and more and more cardiac specialists attention it because the widely carry out of cardiac surgery and percutaneous transluminal coronary angioplasty (PTCA). IRI is a pathophysiological processes involving a variety of complex mechanisms, usually, we believed that the main mechanisms of it are the following:1. radical reaction;2. intracellular calcium overload;3. leukocyte accumulation;4. the lack of high-energy phosphate compound;5.no-reflow phenomenon and so on.1986Murry et al found that the dog’s myocardial injury could be reduced after some short-lived ischemia before a long time reperfusion, then they made the concept of ischemic preconditioning (IPC). They suggested that myocardial IPC could improve myocardial ability to anti IRI, and a large number of experimental studies have confirmed their suggestion. However, to application it was difficult on clinical because it needed to carry out before ischemic. Although myocardial protection has been developed for many years, but most of the existing measures are preventive protection for elective cardiac surgery, and the characteristic of heart specialist is sudden onset and severity (such as acute myocardial infarction or cardiac emergency surgery), for such patients, their cardiac function are poor usually, and surgical treatment will inevitably produce myocardial IRI, leading to further deterioration of heart function, so the significance of myocardial protection is particularly important. But the traditional application of myocardial protection will be limited to be short of opportunity in these conditions. How to mitigate reperfusion injury for ischemic myocardium is a difficult problem about myocardial protection.2003, Zhao et al found that a brief myocardial ischemia and reperfusion before reperfusion also could against subsequent myocardial IRI just like the protective effect of IPC, then their made the concept of ischemic postconditioning (IP). it provides a new way for the prevention and treatment of IRI. Mechanisms of IP involved in reactive oxygen species (ROS)、endogenous activator, mitochondrial ATP-sensitive potassium channels, nitric oxide, reperfusion injury salvage kinases (RISK)^mitochondrial permeability transition pore (MPTP), and so on. It has been found that we can produce similar effects on myocardial protection just like IP when we give amount of drugs to ischemia myocardial at the beginning of reperfusion, which we call the simulation of drug IP. And the drug IP is easy to operate versus the conventional IP obviously, so it has a wider range to applications. Scholars have been applied it to the clinical to protect myocardium and achieved good results. But there are few drugs used to simulate IP have been reported in the literature, such as adenosine, opioids, volatile anesthetics, etc. And because the adverse effects of the drug itself (eg, opioid) or the characteristics of the drug itself (such as the volatile anesthetic drugs), it is limited to application clinically; whereas adenosine produce effect only from a single pathway of improving myocardial energy metabolism. Which has an important clinical value to find more safe and effective drugs for clinical application of IP.ω-3fatty acids are polyunsaturated fatty acids, including a-linolenic acid, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) and so on. In recent years, ω-3fatty acids in cardiac protection attracted more and more attention. In a large-scale clinical trial,500mg to10000mg daily intake of polyunsaturated fatty acids can significantly reduce the risk of cardiovascular events. The results show that, co-3fatty acids have immunomodulatory function, inhibiting inflammation, anti-arrhythmic and antithrombotic effect. As we all know, during heart surgery with cardiopulmonary bypass the blood contact with the surface of foreign directly, surgical wounds, organ ischemia and reperfusion lead to activation of leukocytes and endothelial cells, temperature changes, and intestinal endotoxin release, which may activated systemic inflammatory response syndrome、inflammatory cytokines、 complement and neutrophils, and resulting in impaired cardiac function, systemic dysfunction of vital organs. Therefore, co-3fatty acids can inhibit the systemic and local inflammatory response to protect cardiac function. Further research found that ω-3fatty acids can also play a role in myocardial protection by acting on myocardial cells ion channels、ion pumps、exchangers and the stability and oxidative stress of the cell membrane.ω-3fatty acids in the form of free fatty acids directly to cells in serous phospholipid easily soluble in the hydrophobic inner side of the sensor, can reduce electrical excitability of the cell membrane to some extent play the role of keepping stability of the cell membrane; the content of ω-3fatty acids increases in myocardial membrane phospholipids, the content of ω-6fatty acid reduces, which increases the stability of membrane, and affects a variety of cardiac cell ion channels, exchangers, exchangers, and ultimately reduces the myocardial IRI. Studies have also reported that ω-3fatty acids can reduces the myocardial oxygen consumption, reduces oxidative stress and the loss of K+during myocardial ischemia reperfusion, thereby reducing reperfusion arrhythmias. In clinical observations, ω-3fatty acids can increase the concentration of plasma lipid oxidation compounds and ROS, including free radicals and peroxidase, some scholars think that this is equivalent to activated the pathway of survival signal transduction of the human body to achieve the effect of IPC, which effectively reduces the occurrence of myocardial IRI after myocardial infarction or cardiopulmonary bypass. The study also found that co-3fatty acids can regulate neonatal rat myocardial cells, which regulate downstream gene expression related to inflammation, cell matrix remodeling, release of calcium ions and production of ROS, which maybe is an important mechanism that ω-3fatty acids protects myocardium. With the model of myocardial ischemia reperfusion in New Zealand white rabbit, Mc Guinness et al found that co-3fatty acids preconditioning can also reduced myocardial infarct size by induced a great expression of heat shock protein (HSP). However, the studys about ω-3fatty acids present were almost focused on its preconditioning of myocardial protection. Linked to the several pathogenesis of myocardial IRI, we see that, ω-3fatty acids in all these aspects may play a role in reducing myocardial IRI, there is a hope that ω-3fatty acids as a safe and effective drugs for IP used in clinical to prevent and treat myocardial IRI.2. ObjectivesWe want to use isolated rat heart ischemia-reperfusion model to research whether ω-3fatty acids postconditioning can improve the antioxidant capacity of isolated rat heart during ischemia and reperfusion, and to get the message whether ω-3fatty acids postconditioning can protect myocardium through comparing the myocardial infarct size and the degree of the ultrastructural cell damage, and we want to investigate the possible mechanisms and to provide theoretical and experimental basis to prevent and treat myocardial IRI with a safe and effective drug clinically.3. Materials and methods3.1Establish Langendorff model with isolated rat heart of ischemia-reperfusion3.2Animal groups:60SPF-class male SD rats were randomly divided into4groups, n=15in each group3.2.1Infusion group (group A):continuous perfusion150min;3.2.2Ischemia-reperfusion group (group B):infusion30min, reperfusion90min after30min ischemia;3.2.3IP group (group C):infusion30min, reperfusion90min after30min ischemia,4cycles of reperfusion20s/ischemia20s before the beginning of reperfusion;3.2.4ω-3fatty acids postconditioning group (group D):infusion30min,30min ischemia followed by reperfusion90min, before the start of reperfusion infusion15min with ω-3fatty acids in intravenous fat emulsion preparation (ω-3fish oil fat emulsion). ω-3fish oil fat emulsion has a final concentration1:400(v/V).3.3Methods and indicators for observing3.3.15rats’ hearts in each group will be taken detection of left ventricular free wall myocardial oxidative damage indicators and antioxidant indicators after perfusion:Coomassie brilliant blue method to measure protein content, superoxide dismutase (SOD) by xanthine oxidase method, malondialdehyde(MDA) using thiobarbituric acid assay, lactate dehydrogenase (LDH) using visible light spectrophotometer;3.3.25rats’ hearts in each group will be cutted from the apex to the bottom into lmm thick slices.Viable myocardium with TTC staining will be brick-red and infarct area will be grey. Then these slices will be placed on glass slides, photographed to calculate the percentage of myocardial infarct size with the Image J1.37software;3.3.35rats’ hearts apex organization in each group will be taken to process after perfusion to observe mitochondrial morphology and ultrastructure injury with electron microscopy.4. Results4.1After myocardial ischemia and reperfusion, compared group A with B、C、D, the LDH and MDA in cardiomyocytes increased (P<0.05), and SOD decreased (P<0.05). Although SOD increased and MDA reduced in group C than group B, but there was no significant difference between the two groups(P=0.165. P=0.056); and SOD was significantly higher in group D(P=0.049), at the same time LDH and MDA significantly lower (P=0.000、P=0.049), compared with group B.4.2The percentage of myocardial infarct size:group A (11.14±3.09)%, group B (34.15±3.06)%, group C (26.94±2.73)%, group D (23.28±2.60)%. Compared with group B, the results of myocardial infarct size in group A、C、D are reduced statistically significantly (P=0.000, P=0.007, P=0.000), and there is no significant difference between group C and D (P=0.368).4.3The different groups of rats myocardial tissue were placed and observated under the transmission electron microscopy in11500times magnification. We could see that the volume of rat heart mitochondrias were normal in group A, encapsulated, crest neat and continuous, matrix density was normal, no muscle fiber edema or disrupted; there were a lot of swelling and deformation of mitochondrias in group B, the number of ridges reduced significantly, many of them were disrupt and dissolved into vacuoles, mitochondrial matrix electron density decreases, part of the mitochondrial matrix condensation, the electron density to enhance and the volume of mitochondria smaller, muscle fiber breakage and dissolution; compared with group B,mitochondrial structure and lesions had improved in group C, but there are still some mitochondria swell, crest fracture, and part of the mitochondrial vacuolation within; the damage was further reduced in group D, most of the mitochondrias were normal, and mild swelling could been seen in individual mitochondrias with reduced the number of ridges, but without fracture and dissolution obviously.5. Conclusions:ω-3fatty acids postconditioning after myocardial ischemia and reperfusion can rise SOD level of isolated rat hearts efficiently, improve myocardial antioxidant capacity, reduce mitochondrial injury, reduce myocardial infarct size significantly, and thus relieves myocardial IRI.