| BackgroundIschemic heart disease is one of most important cause of death worthwhile. With the widely application of Coronary Artery Bypass, thrombolytic therapy, percutaneous transluminal coronary angioplasty (PTCA), it is possible to restore the ischemia area blood supply. However, the restoration of blood flow at the same time can also cause reperfusion injury, so that disease progression. Therefore, more and more attention was paid to the pathomechanism, treatment and prevention measures of myocardial ischemic diseases. Urotensin ? (U ?) is a kind of growth hormone kind nerve cyclic peptide, in mammals, including humans generally exists in a variety of tissues and organs. Many studies have shown that U ? combination with its receptor (UTR) can produce a variety of biological effects and play an important role in the process of various kinds of cardiovascular pathophysiology, but its specific function damage or protection still has a lot of controversy. SB-710411 is one of polypeptide UTR antagonists, which can block human U-?-induced vasoconstriction. However, the effect of SB-710411 against myocardial I/R injury has not been reported. Total flavones of rhododendra flower (TFR) is an effective part extracted from rhododendra flower, our previous studies showed that TFR have obvious vasodilative function and protective effect against myocardial ischemia reperfusion injury, but its mechanism remains poorly understood. In the present study, we investigate the protective effect of SB-710411 on myocardial I/R injury and the underlying mechanism, explore the relationship between UT receptor blocking and myocardial ischemia reperfusion injury, observate the effect of TFR on blood vessel function, myocardial ischemia-reperfusion injury, the role of UTR and its downstream signaling pathways through the application of siRNA in vivo transfection and radioactive ligands competitive combination technique.Purpose:1. To investigate the protective effect of SB-710411 on myocardial ischemia reperfusion injury and its possible mechanism.2. To research the effect of TFR on the vasoconstriction induced by hu-? and its mechanism.3. To investigate whether the protective effect of TFR against myocardial ischemia has relationship with blocking UTR.4. To observe the ability of TFR and its main components competitive binding to UTR.Methods:1 Myocardial I/R injury was induced by occluding the left anterior descending coronary artery in adult male Sprague-Dawley rats. Hemodynamic parameters and electrocardiogram (ECG) were recorded during the experiment. The heart and serum were collected to detect the related indicators.1.1 Sprague-Dawley rats were randomly divided into the following 6 groups: Sham group:The animals' chest were operated, but the coronary ligatures were not tied. Model group:The hearts were subjected to the ischemia/reperfusion (I/R) protocol consisting of 30 min of regional ischemia and 90 min of reperfusion. Verapamil group:Verapamil was intravenously injected daily for 3 days at a dose of 1.6 mg/kg before ischemia, SB-710411 0.5 ?g/kg group:SB-710411 was intravenously injected daily for 3 days at a dose of 0.5 ?g/kg before ischemia, SB-710411 1.0 ?g/kg group:SB-710411 was intravenously injected daily for 3 days at a dose of 1.0 ?g/kg before ischemia. SB-710411 2.0 ?g/kg group:SB-710411 was intravenously injected daily for 3 days at a dose of 2.0 ?g/kg before ischemia.1.2 Electrocardiogram (ECG) and hemodynamics including the date of heart rate (HR), mean arterial blood pressure (MABP), left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP), maximal rate of pressure development for contraction (+dp/dtmax) and maximal rate of pressure development for relaxation (-dp/dtmax) were recorded.1.3 Using TTC and H&E stain to investigate the infarct size and histological alteration respectively. Lactate dehydrogenase (LDH), creatine phosphokinase-MB (CK-MB), cardiac troponin I (cTnI) in serum and RhoA activity, protein expressions of U-II receptor (UTR), ROCK1, ROCK2 in myocardium were evaluated.2 Using the UTR siRNA in vivo transfection technique to reduce the UTR expression in the blood vessels and myocardial tissue of rat and then determine the UTR expression with western-blot analysis.3 The coronary artery, aorta, mesenteric artery, mesenteric vein and abdominal aortic veins were taken from normal and UTR siRNA in vivo transfection rat, using hu-II, U46619 and phenylephrine to contract these vessels respectively and investigate the effect of TFR on the vasoconstriction induced by these vasoconstrictor. The RhoA activity in vessels were investigate by G-LISA way.4 Myocardial I/R injury was induced by ligating and untying left anterior descending coronary artery in normal and UTR siRNA rats. The electrocardiogram was recorded during the experiment, the heart and serum were collected to measure.4.1 The rats were randomized into7 groups:(1) the sham group; (2) the model group; (3) the nifedipine 5.4 mg/kg group; (4) SB-7104112.0 ?g/kg group; (5) the TFR 20 mg/kg group, (6) the TFR 40 mg/kg group (7) the TFR 80 mg/kg group. Each group rats were given throug intragastrical administration corresponding drug daily for 3 days before ischemia. SB-7104112.0 ?g/kg was intravenously injected daily for 3 days before ischemia.4.2 The infarct size and histological alteration were investigated by TTC and H&E stain after experiment. The RhoA activity and the protein expressions of UTR, ROCK1, ROCK2, MLC/p-MLC were evaluated by G-LISA and westen-blot method respectively.5. The myocardial tissue membrane protein receptors were preparaed from left ventricular muscle of normal rats. To observe the inhibiting effect of TFR, hyperoside, rutin on the combination of [125?]-hu-?and UTR and calculate their IC50 respectivly.Results:1. Compared with model group, SB-710411 (1.0,2.0 ?g/kg) could markedly increase the the maximal rate of pressure development for contraction (+dp/dtmax), maximal rate of pressure development for relaxation (-dp/dtmax) and decrease the left ventricular diastolic pressure (LVEDP) in rats, obviously reduce the myocardial infarction area, significantly inhibit the increase of ST segment. SB-710411 (0.5,1.0,2.0 ?g/kg) could reduce serum CK-MB, LDH activity, SB-710411 (1.0,2.0 ?g/kg) could significantly inhibit the incease of cTnI level in serum.2. The myocardium in the model group occurred obvious pathological change such as necrosis, extensive edema, neutrophilic infiltration, mild inflammation, cardiomyocytes arranged irregularly and so on. SB-710411 (1.0,2.0 ?g/kg) could significantly relieved these changes.3. The RhoA activity and UTR, ROCK1, ROCK2 protein expression in myocardial tissue increased significantly after myocardial ischemia reperfusion injury, SB-710411(1.0,2.0 ?g/kg) could obviously inhibit these changes.4. The UTR protein expression of coronary artery, aorta and mesenteric artery UTR siRNA transfected rats decreased obviously compared with that in normal rats. However, the UTR protein expression of mesentery vein in normal and UTR siRNA transfected rats could not be measured.5. TFR could obviously inhibit the vascular contraction response induced by hu-? on the coronary artery, aorta and abdominal aortic veins of normal rats. Hu-? has no vasoconstriction effect on mesenteric artery and vein. TFR also could produce certain relaxation effect on vascular contractile response induced by U46619 and phenylephrine respectively.6. Hu-? has contractile effect on the coronary artery, aorta and abdominal aortic veins of UTR siRNA transfected rats, but the contraction intensity decreased significantly compared with normal rats vessels. TFR could obviously inhibit the vascular contraction response induced by hu-? on the coronary artery, aorta and abdominal aortic veins of UTR siRNA transfected rats. The inhibition rate has no change compared with normal rats vessels.7. The vasoconstriction effects of U46619 and phenylephrine on the coronary artery, aorta, abdominal aortic veins and mesenteric veins of UTR siRNA transfected rats have no obvious change compared to normal rats vessels. TFR could inhibite the vasoconstriction response induced by U46619 and phenylephrine to certain degree, but the inhibition rate significantly declined compared to normal rats blood vessels.8. The RhoA activity of coronary artery, aorta and abdominal aortic vein increased markedly after contracted by hu-?. TFR could obviously inhibite this change.9. TFR could obviously decrease the infarction area and ST segment, relieve myocardium pathological changes after myocardial ischemia-reperfusion injury in normal rats. TFR (40,80 mg/kg) could also obviously reduce the infarction area after myocardial ischemia-reperfusion injury in UTR siRNA transfected rats, but the effect on ST segment and myocardium pathological changes are not obvious. The effect of SB-7104112.0 ?g/kg is similar to TFR. The reduction rate of TFR (40,80 mg/kg) on myocardial infarct size in UTR siRNA transfected rats decreased obviously compared with that in normal rats. The effect of SB-710411 2.0 ?g/kg is similar to TFR.10. The RhoA activity, ROCK1, ROCK2, p-MLC protein expression after myocardial ischemia-reperfusion injury in normal and UTR siRNA transfected rats significantly increased. TFR (40,80 mg/kg) could obviously inhibit these changes. The inhibition rate of TFR (40,80 mg/kg) on the RhoA activity, ROCK1, ROCK2, p-MLC protein expression after myocardial ischemia-reperfusion injury in UTR siRNA transfected rats reduced significantly compared with that in normal rats. The effect of SB-7104112.0 ?g/kg is similar to TFR.11. TFR and hyperoside significantly inhibited the combination of [125 ?]-hu-? and UTR, the IC50 of them were 0.854 mg/l,10-5.104mol/l respectively. Rutin has no effect on the combination of [125?]-hu-? and UTR.Conclusions:1. SB-710411 has a potent protective effect on myocardial I/R injury in rats and the cardioprotection may be associated with the inhibition of UTR/RhoA/ROCK pathway.2. TFR has effect of inhibiting the vascular contraction response induced by hu-II on the coronary artery, aorta and abdominal aortic veins, which may be associated with blocking UTR and inhibiting RhoA activity.3. The cardioprotection of TFR may be associated with inhibition of UTR/RhoA/ROCK pathway.4. TFR has ability of competitive binding to UTR. |
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