| BACKGROUNDWith the change of the people’s eating habits, coronary heart disease becomes a control cardiovascular disease and its incidence is rising year by year. Coronary atherosclerosis induced coronary artery occlusion and local acute myocardial ischemia, leading to some other related symptoms, such as triggering malignant arrhythmia, myocardial infarction, cardiac shock and so on. The main treatment for coronary heart disease is recanalization of coronary artery stenosis or occlusion. Therefore some recanalization therapy such as coronary artery stent implantation, coronary artery thrombolysis and coronary artery bypass surgery have been greatly developed. In cardiac surgery, coronary artery bypass surgery under extracorporeal circulation is a method for the treatment of coronary heart disease. During extracorporeal circulation process, cardiac arrest has to be stopped by the liquid and the heart blood was blocked. After the operation, heart jump was prompted by supplying warm-blooded and the heart blood was restore, which will cause ischemia-reperfusion injury. After extracorporeal circulation, the forward curative effec of the heart disease patients with postoperative recovery process is largely associated with ischemia-reperfusion injury. So how to reduce myocardial ischemia-reperfusion injury has becomed an important goal in the process of cardiac surgeryThe mechanism of myocardial ischemia-reperfusion injury is very complex, mainly includes the theory of reactive oxygen species, inflammation, cell apoptosis, calcium overload and energy metabolic abnormalities, etc. But the first three are the mainly recognized mechanism of myocardial ischemia-reperfusion injury.There are many treatment of myocardial ischemia-reperfusion injury, mainly include drug therapy, physical therapy and gene therapy.Drug treatment is an important therapeutic method of the myocardial ischemia reperfusion injury and has been more conventionally applicated in the clinical practice and been researched by many scholars currently. These kinds of drug include chemical medicine (such as statins, renin-angiotensin system classes, glucocorticoid hormones, etc.), traditional Chinese medicine(such as berberine, Guanxinning, Kudiezi, etc.), polypeptide drugs (such as brain natriuretic peptide) and so on. These drugs could protect myocardial function to a certain extent, but the curative effect is not significant always. Physical therapy including ischemia preconditioning and ischemic postconditioning, remote ischemic conditioning, therapeutic high temperature and low temperature, etc. But because of its unknown mechanism and uncertain curative effect, its clinical application is limited.Gene therapy as a new therapeutic modalities for the treatment of myocardial ischemia-reperfusion injury has opened up a new prospect, but the strict clinical trials about gene therapy for myocardial ischemia-reperfusion injury has not been done. So whether this method could treat myocardial ischemia-reperfusion injury is worth further research and discussing. Although there are a great many prevention methods and treatment for myocardial ischemia-reperfusion injury, the curative effects are not satisfactory. So looking for a safe, reliable and remarkable effective therapeutic method for myocardial ischemia-reperfusion injury, is still one of the difficult and hot issue in the study of the cardiovascular disease.Gas treatment a new therapeutic method for ischemia-reperfusion injury and just emerges in recent years. The main principle of this method is to reduce the generation of reactive oxygen species and inhibit oxidative stress reaction. The gas commonly used gas treatment was NO, CO and H2S, etc. But these gas have a certain toxicity to human body, which restricted their application. Hydrogen(H2) is a non-toxic reducing gas and its antioxidant role in ischemia-reperfusion injury was found by people gradually. At present, the main way of H2 treatment for ischemia-reperfusion injury is direct inhalation and drinking or injecting saline which contain H2. Because H2 is easy to burn, explode and spread, these method of supplying are unsafe. And the production, transportation, preserve of H2 is very difficult and expensive. What’s more, it is often hard to achieve satisfactory results due to the low effective dose of H2 in myocardium.Lipid microbubble, composed of lipid shell and inert gas, has been widely used in clinical as a kind of acoustic contrast agent. Its lipid shell is dense enough to package H2 and inhibit spread. Lipid microbubble can prevent the H2 from going through the blood vessel walls and spreading to other organizations before arriving in myocardial due to micron size.Thus, we put forward:make a kind of lipid microbubble loading H2 for the treatment of myocardial ischemia-reperfusion injury and explore a simple and safe method for the H2 treatment.PURPOSE1. Verify the feasibility of lipid microbubble carrying H2 to make H2 microbubble.2. Verify the feasibility of H2 microbubble reducing myocardialischemia-reperfusion injury through anti-inflammatory, antioxidant.MATERIALS AND METHODS1. The major experimental material.Experimental apparatus and reagents:DSPC, DSPE-PEG2000, the phospholipid materials of lipid microbubble production with sterile, security and good histocompatibility, were bought from Avanti Company in United States. Perfluorinated propane gas(C3F8) was bought from nuclear industry and physical and chemical engineering institute in Tianjin. H2, with the purity of more than 99%, was bought from Shente industrial gas company in Shenzhen. H2 microelectrode system was bought from Unisense company in Denmark. This system consists of three parts: chemical system, signal processing system and microoperation system. Chemical system is H2 microelectrode. Signal processing system includes a signal exchange equipment and picoammeter. The working principle of H2 microelectrode system was that:when H2 arrived at the silicon membrane of H2 microelectrode by the diffusion effect, H2 was oxidized by platinum anode of sensor, which produced electron flow. In platinum anode of sensor, electron flowed from the anode to internal reference. At the same time, picoammeters acquired a linear pressure signal of H2. The lowest concentration of H2 that the H2 microelectrode system is able to detect in the water is 0.3μM. VEVO2100 small animal ultrasound imaging equipment was bought from VisualSonics company. The left ventricular ejection fraction and short axial shortening fraction of rats were measured in the M-mode with the MS-250 probe (Center frequency is 21 MHz; Broadband frequency is 13MHz-24MHz; resolution 75 microns; Real-time frame rate is more than 500 fps).Experimental animals:Sprague Dawley male rats was bought from Experimental Animal Center of Guangdong province. The body weight of rats were 250～300 grams. All rats were raised in SPF environment.2. The experimental method2.1 Preparation and characterization of H2 microbubbleDSPC and DSPE-PEG 2000 were mixed with the mole ratio of 9:1. H2 microbubble in the different proportion of H2 and C3F8 were prepared with film-vibrating method. The proportion of C3F8 and H2 were 3:0,1:1 and 0:3. The particle size and concentration of different H2 microbubble were measured. The morphology of different H2 microbubble were observed under a microscope. In order to sort out a kind of H2 microbubble with higher H2 content and suitable size distribution for the subsequent experiment, the relative content of H2 in different H2 microbubble were calculated.Detected the curve of H2 releasing from H2 microbubble over time in the PBS and in the left ventricle of rats with the H2 microelectrode systems.Take the ultrasound images of 5×105,5×106,5×107,5×108 and 5×109/ml of H2 microbubbles the VEVO 2100 ultrasound imager. The intensity of echo signal were measured. The 2×1010 H2 microbubbles were injected into rats. The left ventricular angiography were conducted by VEVO 2100 ultrasound imager the time-video intensity curve were recorded.2.2 Experimental group and the preparation of animal modelsAdult male SD rats were randomly divided into three groups. The first group named sham group, was only opened the chest rather than ischemic operation. The second group called control microbubble group, some control microbubble which did not load H2 were injected into caudal vein at 5 min before reperfusion (removal of ligation thread). The third group called H2 microbubble group, some H2 microbubble which loaded H2 were injected into caudal vein at 5 min before reperfusion.After anesthesia, the third or fourth rib at the left side in rats were cutted off. The heart was exposed and the left coronary artery was ligated. Then, myocardial ischemia was appeared. After 30 min, the ligature was loosened and reperfusion was started.To study the relationship between infarct size and hydrogen microbubbles doses we set up high-dose and low dose in control microbubble group and H2 microbubble group. In high dose, each rat was injected 2×1010 microbubbles; In low dose, each rat was injected into the 4X 109 microbubbles.2.3 Measurement of ischemia and infarction area.After 24 hours of reperfusion, thoracotomy was operated and the same ligature as before was tightened. The ischemic and non-ischemic zone were distinguished by injecting Evans blue dye solution into caudal vein. The rats were put to death quickly and hearts were taked out. Hearts were sliced along the short axis. Infarction and non-infarction area were distinguished by TTC dyeing. The infarct area (INF), the area at risk (AAR) and the left ventricle area(LV) of each section were measured. And the ratio of AAR/LV, INF/AAR and INF/LV were calculated.2.4 The cardiac function of rats were determinated by echocardiography.In order to evaluate cardiac function in rats of each group, left ventricular ejection fraction(EF) and shortening fraction(FS) were measured by VEVO 2100 small animal ultrasound imaging equipment after 24 hours of reperfusion.2.5 Myocardial structure in rats of different group were observated by hematoxylin-eosin staining(H&E).After 24 hours of reperfusion, hearts of rats were took out to do paraffin section, followed by HE staining. The myocardial structure in rats of different group were observed under a microscope.2.6 Detecting apoptosis of myocardial cell by TUNEL.After 24 hours of reperfusion, hearts of rats were took out to do paraffin section. The apoptosis of myocardial cells in rats of each group were detected by TUNEL assay.2.7 Detection of inflammatory cytokines TNF-α and IL-1β.After 40 minutes of reperfusion, hearts of rats were took out to make tissue homogenate. Inflammatory factor TNF-α and IL-1β in myocardial tissue of each rat were detected by enzyme-linked immuno sorbent assay.2.8 Detection of reactive oxygen species (ROS):H2O2, NO·,·OH and O2-·After 40 minutes of reperfusion, hearts of rats were took out to make tissue homogenate. The homogenate were immediately reacted with 5-(and-6)-chloromethy-2’,7’-dichlorodihydroflurescein diacetate, acetyl ester(CM-H2DCFDA), diaminofluorscein-2-diacetate(DAF-2DA),2-[6-(4’-hydroxy)phenoxy-3H-xanthen-3-on-9-yl]benzotate(HPF) and MitoSOX. The fluorescence intensity were measured by microplate-reader and the concentration of H2O2, NO·,·OH and O2-· were calculated.2.9 Toxicity test.H2 microbubbles were injected into three healthy SD rats. Pre-injection, 100μl blood of rats were collected by tail veins. After 3 and 7 days post-injection,100μl blood were taken respectively. And after collecting blood at 7 days, the heart, liver, spleen, lungs and kidneys were took out to conduct paraffin section and HE staining. The organizational structure of these internal organs were observe under a microscope.All of blood samples were examined with an automated hematology analyzer.2.10 Statistical methods.All the results were showed as mean ± standard deviation. The comparison of Myocardial infarction area and left ventricular function was conducted by two independent sample t test. The comparison of myocardial cell apoptosis, inflammation factors and ROS were performed with the one-way analysis of variance, multiple comparisons between groups using the Bonferroni method. All statistical analysis were performed with SPSS 13.0 software. P< 0.05 was defined as the standard of test with significance.RESULT1. H2 microbubbles were obtained successfully with different proportion of H2 and C3F8. The relative H2 content was highest in H2 microbubbles with the ratio of 1:1. The H2 amount, average particle size and concentration of this microbubble were (1.46±0.08)μmol/ml, (0.89 零 0.03)μm and (2.29 ± 0.07)×1010/ml. Under a microscope, This H2 microbubbles were like sphericity with bright center. And the particle size was uniform.2. H2 microbubbles could spontaneously release hydrogen in vitro and in vivo.3. Hydrogen microbubbles could enhance ultrasound imaging in vitro and in vivo.4. Compared with control microbubble group, the myocardial infarction area was significantly decreased in H2 microbubble group(P< 0.05). Compared with the low dose, high doses of hydrogen microbubbles could further reduce myocardial infarct size (P<0.01) and the myocardial protective effect was more significant.5. The number of myocardial cell apoptosis was also significantly reduced in H2 microbubble group(P< 0.05). Under a microscope, myocardial structure in H2 microbubble group was obviously better than control microbubble group. What’s more, EF and FS of H2 microbubble group were significantly higher than control microbubble group(P< 0.05), which illustrated that H2 microbubble could protect myocardial function after ischemia-reperfusion injury.6. Compared with control microbubble group, inflammatory cytokines TNF-α and IL-1βin rats myocardial tissue of H2 microbubble group were reduced obviously, which demonstated that H2 microbubble could significantlyrelieved myocardial inflammatory response.7. Compared with control microbubble group, ·OH in the myocardial tissue ofH2 microbubble group decreased significantly. But NO·, H2O2and O2-· had no significant difference.8. The analysis of blood, including complete blood counts, indicated that the blood count values were within the normal range 3 or 7 days after H2-MB injection by intravenous administration. After 7 days of H2 microbubble injection, the structures of the main visceral organs in rats such as heart, liver, spleen, lung, kidney had no abnormal changes, which indicated that the H2 microbubbles were non-toxic to rats.CONCLUSION1. Lipid microbubble was able to package H2 and could be used as a H2 carrier.2. H2 microbubble could significantly reduce the myocardial infarction area after myocardial ischemia reperfusion injury, inhibit myocardial apoptosis, protect myocardial structure, and improve the myocardial systolic function.3. H2 microbubble could inhibit myocardial inflammatory reaction and production of OH in the process of ischemia reperfusion, which reduced myocardial injury.4. The H2 bubbles have good biocompatibility and were non-toxic to rats. |
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