| Ischemic cardiovascular disease, the most common heart disease is the main reason of mortality and morbidity worldwide. Coronary arteries and their branches supply oxygen-rich blood to myocardium. Stopping of myocardial blood leads to myocardial ischemic injury. Restoration of blood supply is critical to prevent the irreversible injury. However, sudden blood flow returning to ischemic myocardium may paradoxically augment myocardial injury. This is referred to myocardial ischemia/reperfusion(I/R) injury, characterized by myocardial inflammatory responses, metabolic disorder, cardiac dysfunction and subsequent myocardial cell death. So many studies focus on the pathomechanism, treatment and prevention measures of myocardial ischemic diseases.Total flavones of rhododendra flower(TFR), an effective part extracted from rhododendra flower, is comprised of flavones such as quercetin, hyperin, rutin and other flavonoids. Our previous studies have shown that TFR has significant protective effects against myocardial ischemic injuries in rat and mice. However, its mechanism of cardioprotection remains poorly understood. Therefore, in the present study, the protective effect of TFR on myocardial A/R injury was investigated, and the roles of ROCK, H2 S and K+ channels in the cardioprotection of TFR were explored.Purpose:1. To observe the protective effect of the TFR on I/R and anoxia/reoxygenation(A/R) injures in mice and rat.2. To explore the mechanism of TFR against myocardial I/R injury, especially focuson the role of H2S-mediated inhibition of Rho associated coiled coil-forming kinase Rho(ROCK)pathway in the cardioprotection of TFR.3. To study the effect of TFR on K+ channel.Methods:1. Rat myocardial I/R injury was induced by ligating and untying the left anterior descending coronary artery. The ST segment change of electrocardiogram(ECG) were recorded. The activities of lactate dehydrogenase( LDH) and creatinine kinase(CK) in blood serum were measured. Infarct size(IS), as a percentage of the area at risk(AAR),was determined by Evans blue and TTC double staining. The histopathologic changes of myocardium in rats were examined with HE dying method. The expression of ROCK1 and ROCK2 protein was detected by Western blotting method.2. The primary culture of neonatal rat cardiomyocytes was used to make A/R injury model. Myocardial damage are judged by the measurement of LDH, c Tn T and MDA. Using Western blot, the proteins ROCK1 and ROCK2 in the myocardial cells were detected using Western blotting.3. Myocardial ischemic model was made by using subcutaneous injection of Iso in mice. Change of ST segment of electrocardiogram( ECG) was observed at 5, 10 and 15 min. The activities of LDH and CK-MB were measured, and histopathologic changes of myocardium were examined with HE staining. The expressions of ROCK1, ROCK2 and MLC1 proteins were detected by Western blotting method.4. Whole cell patch-clamp recording. K+ outward and inward currents in single rat cardiomyocyte was recorded, and the current was normalized through dividing current by cell capacitance to get current density(p A/p F). Changes in the currents were observed before and after administration of TFR,Ba Cl2,Apamin or IBTX.Results:1. The ST segment of ECG markedly elevated during the process of ischemiareperfusion, while TFR and Y27632 could markedly inhibit the elevation of ST segment.2. The LDH and CK activitiesmarkedly elevated in I/R model group. 20,40,80 mg/kg TFR or 1.6mg/kg verapamil or Y27632 could significantly inhibit the elevation of LDH and CK activities in serum.3. There are significant infarction of the I/R model group after ischemia 30 min and reperfusion 90 min, the IS/AAR were 46.3% ± 6.2%. 20,40,80mg/kg TFR could also significantly reduce IS/AAR as compared to I/R model hearts. Verapamil 1.6 mg/kg and Y27632 30 mg/kg have same effect.4. Morphological observation by HE staining showed that the I/R-induced morphological alteration of myocardium was improved by administration of TFR 40 and 80 mg/kg, as well as verapamil or Y27632.5. I/R can markedly increase the expressions of the ROCK1 and ROCK2 protein. TFR 20, 40 and 80 mg/kg or Y27632 can remarkably suppress I/R-increased ROCK1 and ROCK2 expressions.6. A/R injury induced marked increment of LDH, c Tn T and MDA levels in the supernatant of primary culture of neonatal rat cardiomyocytes. In the range of 3.7- 300 mg/L TFR could markedly inhibit above elevations.7. A/R can markly increased the expressions of the ROCK1 and ROCK2 protein in the neonatal rat cardiomyocytes, TFR33.3,100 and 300 mg/L or Y27632 can remarkably inhibit the expression of the ROCK1 and ROCK2.8. In the myocardium the inward current could be blocked by Ba Cl2(100 μmol/L), a specific Kir channel blocker, that showed the inward current was carried by the Kir channel. This inward K+ current were significantly increased by 300 mg/L TFR, the result showed that TFR can evoke the Kir. The outward K+ current were increased by 300 mg/L TFR, and could be blocked by Apamin(300 nmol/L), a specific SKca channel blocker, that showed the outward current was carried by the SKca channel. BKCa blocker IBTX(100 nmol/L), did not cause significant inhibition on myocardial K+ current, that showed there were not BKca channel on the myocardium.9. Myocardial ischemic injury induced more elevation of ST segment of ECG in CSE knock out mice than that in wild type mice. TFR 120 mg/kg markedly inhibited the elevation of ST segment of ECG in wild type mice rather than that in CSE knock out mice.10. Myocardial ischemia-induced elevations of serum LDH and CK-MB activities were more obviously in CSE KO mice than that in wild type mice. TFR 120 mg/kg markedly inhibited the elevations of serum LDH and CK-MB activities in wild type mice rather than that in CSE knock out mice.11. TFR 120 mg/kg markedly improve myocardial ischemia-induced morphological alteration of myocardium in wild type mice, but have no effect on that in CSE KO mice.12. The increases of ROCK1, ROCK2 and MLC1 protein expressions induced by myocardial ischemia were more greatly in CSE KO mice than that in WT mice. TFR120 mg/kg can remarkably suppress the expressions of the ROCK1, ROCK2 and MLC1, but TFR have no effect on suppression the increases of ROCK1, ROCK2 and MLC1 expression in WT mice, but TFR had no obvious effect in CSE KO mice.Conclusion:1. TFR have significant protective effect against myocardial I/R injury.2. H2S-mediated inhibition of ROCK pathway may involve in the protective effect of TFR on myocardial I/R injury.3. TFR could activate Kir channel and SKca channel to cause K+ currents in rat cardiomyocytes, this may be one of the action mechanism of TFR. |
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