| BackgroundAcute myocardial infarction is a major cause of mortality worldwide. Early and successful reperfusion of the ischemic myocardial is the most effective strategy to reduce infarct size and improve clinical outcome. However, the process of restoring blood flow to the ischemic myocardium can induce myocardial reperfusion injury, which can paradoxically reduce the beneficial effects of myocardial reperfusion. Animal studies suggest that myocardial reperfusion injury accounts for up to 50% of the final infarct size. Therefore, interventions to attenuate myocardial ischemia reperfusion injury (MIRI) are urgently needed.The mechenimsm involved in MIRI are complicated and has not been completely clarified. The possibility mechanisms may including oxidative stress, calcium overload, disorders in myocardial energy metabolism, apoptosis, inflammation, pH paradox and so on. Currently, a lot of cardioprotective strategies are been inveatigated in animal study and clinical trials. However, a number of therapeutic interventions that are verified in animal study failed to translate into clinical setting to prevent MIRI.Hydrogen sulfide (H2S) has been recognized recently as the third therapeutic gaseous signaling molecule, following nitric oxide and carbon monoxide. Growing evidence indicate that H?S is involved in MIRI. H?S is produced by cystathionine-β-synthase (CBS), cystathionine-y-lyase (CSE/CGL) and 3-mercaptopyruvate sulfurtransferase (3-MST) in mammalian cells. Inhibition of endogenous H2S production by knockout CSE significantly increase myocardial infarct size, while cardiac specific CSE overexpression reduced infarct size and improved cardiac function. Additionally, exogenous administration of H2S at the time of reperfusion decreased infarct size and preserved left ventricular function in a rodent model of MIRI. Similar results were observed in a porcine MIRI model. Mechanisms by which H2S exerts its cardioprotective effects may include inhibition of oxidant stress, preservation of mitochondrial structure and function, opening of the KATp channel, alleviation of Ca2+ overload, reduction of cardiomyocyte apoptosis and anti-inflammatory responses. These findings suggest that exogenous administration of H2S could be an attractive treatment for MIRI.H2S is currently administered either by inhalation of gaseous H2S or intravenous /intraperitoneal injection of H2S donors. Inhalation of H2S is poorly tolerated due to the undesirable odor and its irritation of the respiratory tract even at very low concentration. Inorganic donors of H2S, Na2S and NaHS, widely used in the field, have the advantage of rapidly increasing H2S concentration within seconds. However, the effective concentration of H2S may not last long within tissue because of rapid degradation of Na2S and NaHS. In addition, because the sensitivity of organs to H2S differs, systemic delivery of H2S may cause unwanted side effects, including acute change of blood pressure, central neurotoxicity and respiratory depression. Direct delivery of H2S to the myocardium may avoid the unwanted side effects.Microbubble is an ultrasound contrast agent used in clinical practice, which is consisting of organic shell and encapsulating gas core. The microbubbles will oscillate violently and collapse finally when expose to ultrasound with high peak negative pressures, a process known as cavitation. This phenomenon is widely used to deliver bioactive substances, including drugs and genes, to desired sites. Recently, delivery of theraputic gases using ultrasound and microbubbles has been shown to be feasible and of significant therapeutic benefit. For example, using ultrasound and microbubbles loaded with nitric oxide, intramyocardial delivery of nitric oxide enhanced the homing of the mesenchymal stem cells into the infracted myocardium and induced the regional angiogenic response. Delivery of oxygen to hypoxic tumor bed with oxygen-filled microbubbles and ultrasound increased reactive oxygen species generation and result in enhanced sonodynamic effect. Similarly, development of microbubbles encapsulating H2S gas could enable targeted H2S delivery with ultrasound exposure, unfortunately, reports on this assumption has not been found yet.Therefore, we hypothesized that delivery of H2S by ultrasound and microbubble attenuates MIRI and may avoid unwanted side effects.ObjectiveTo prepare stable hs-MB with higher H2S loading capability. To evaluated the cardioprotective effect of hs-MB against MIRI.Methods1. Preparation and characterization of hs-MB.H2S and perfluoropropane were mixed at the ratio of 4:0,1:3,2:2,3:1,0:4 and used to prepared the microbubbles loaded with difference amount of H2S. A microscope was used to characterize the morphology of hs-MB. The amounts of H2S in hs-MB was measured. For stability assessment, the concentrations of the hs-MB were measured at different time points with the Multisizer III Coulter counter.. The optimal ratio of H2S/C3F8 was figured out and used in the following experiment.2. Delivery of H2S by ultrasound and hs-MB① Ultrasound triggered H2S release from hs-MB in vitroUltrasound-triggered H2S release from hs-MB was evaluated with the use of a flow system. PBS at a constant flow rate of 10 mL/min was passed through the system. hs-MB was infused into the tubing at 100μl/min. Ultrasound emitted from an ultrasonic cavitation apparatus with frequency of 1.0 MHz, peak-to-peak pressure of 1.0 MPa and duty cycle of 1.0% at a pulse repetition frequency of 100 Hz was used to fragment the hs-MB. The concentration of H2S was measured with free radical analyzer. ② In vivo delivery of H2S by ultrasound targeted hs-MB destruction9 SD rats were randomly divided into 3 groups:Control, hs-MB and hs-MB+US. Rats in hs-MB group received intravenously hs-MB for 30min. Rats in hs-MB+US received hs-MB and ultrasound simultaneously. The enhancing acoustic effects and the destruction of the hs-MB were observed using contrast enhanceing ultrasound. The blood pressure, heart rate and respiratory rate were observed during the experiment. The concentration of H2S in various organs were measured.3. Ultrasound targeted hs-MB destruction attenuated MIRI72 rats were randomly divided into 4 groups (n=18 in each group):1) SHAM:the suture was not tightened after operation and each rat received 6ml/(kg·h) saline via tail vein injection.2) MIRI:the suture was tightened for 30 minutes and each rat received 6ml/(kg-h) saline via tail vein injection.3) c-MB+US:the suture was tightened for 30 minutes and each rat received 6×109/(kg·h) control microbubble (c-MB) and ultrasound emitted from an ultrasonic cavitation apparatus was applied to fragment the hs-MB.4) hs-MB+US:the suture was tightened for 30 minutes and rats received hs-MB and ultrasound as in group 3.Treatments were performed five minutes before reperfusion and lasted for 30 minutes. At 4h of reperfusion,6 rats in each group were sacrificed for hematoxylin-eosin and TUNEL staining and 6 rats for MDA and SOD measurement. At 24h of reperfusion, echocardiography was performed and hearts were harvested for Evans Blue/TTC staining.4. Statistical analysis.Statistical analysis was performed with SPSS 19.0 software. All values are presented as mean±SD. Comparisons between multiple groups were performed by one-way ANOVA followed by Bonferroni post hoc test. Data of the stability assessment of hs-MB were analyzed using repeated-measures ANOVA. Statistical significance was set at P<0.05.Results1. hs-MB prepared with the H2S/C3F8 ratios of 2/2 possessing excellent stability and highest H2S encapsulation, exhibited a concentration of (1.01±0.19)×109/mL and a mean microbubble diameter of 2.26±0.17μm,ranging from 0-9μm.2. Ultrasound triggered H2S release from hs-MB. There was a slightly increased in H2S level when hs-MB was infusion into the flow system. However, the H2S level was significantly increased when ultrasound and hs-MB were applied.3. In vivo delivery of H2S by ultrasound targeted hs-MB destruction. hs-MB possessing excellent contrast enhancing acoustic effects and could be destryed by ultrasound. After hs-MB infusion and ultrasound sonication, the H2S concentration in the heart and lung were significantly increased (both P<0.05), but not in the kidny and liver (both P>0.05).4. hs-MB+US reduce myocardial infarct size. Compared with SHAM group, MIRI caused a significant increase in infarct size (P<0.01). No difference in IS/AAR between MIRI group and c-MB+US group was observed (P>0.05). Treatment with hs-MB and ultrasound caused a significant reduction in INF/AAR as compared with c-MB+US (P<0.05). H&E stained sections revealed that irregular or ruptured myocardial fibers, extensive edema, and mass inflammatory cell infiltration were detectable in the MIRI group and c-MB+US group. In the hs-MB+US group, slight edema and a small amount of inflammatory cells was observed between the wavy myocardial fibers and around blood vessels.5. hs-MB+US preserved left ventricular function. MIRI caused an increase in EDd (P<0.05) and ESd (P<0.05) and decrease in LVFS (P<0.01) and LVEF (P<0.01). No significant differences in ESd, EDd, LVFS or LVEF were observed between c-MB+US group and MIRI group (both P>0.05). Attenuation of the increased ESd (P<0.05) but not the EDd (P>0.05) was observed in hs-MB+US group compared with c-MB+US group. LVFS and LVEF were improved in hs-MB+US group compared with c-MB+US group (both P<0.01).6. hs-MB+US alleviated MIRI induce apoptosis. In response to MIRI, total TUNEL positive nuclei were significantly increased compared to the SHAM group (P<0.01). There was no difference in TUNEL labeling nuclei between c-MB+US and MIRI group (P>0.05). Significant reduction in TUNEL labeling nuclei was noted in hs-MB+US group compared with c-MB+US group (P<0.01).7. hs-MB+US attenuated MIRI induce oxidative stress. MIR significantly increased the MDA level and reduced the SOD level in myocardium when compared with the SHAM group (P<0.01). No difference was observed in MDA and SOD between c-MB+US and MIRI group (both P>0.05). There was a marked decreased in MDA (P<0.05) and increase in SODin hs-MB+US group in comparison with the c-MB+US group (P<0.01).8. The blood pressure, heart rate and respiratory rate were not change during hs-MB+US treatment.ConclusionThe hs-MB prepared at the H2S/C3F8 ratios of 2/2 possessing excellent stability and carrying highest H2S amount. Ultrasound targeted hs-MB destruction triggered H2S releasing and increased the H2S concentration in myocardium. hs-MB and ultrasound reduced myocardial injury and improved cardiac function in a rodent model of MIRI, which may result from inhibition of oxidant stress and cardiomyocyte apoptosis. No changes in blood pressure, heart rate and respiratory rate when hs-MB administration. Microbubbles and ultrasound may be a useful method for site-specific delivery of H2S to avoid unwanted side effects. |
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