| Myocardial cells kept regularly contraction and relaxation,thus,the blood pump function can be effectively realized by the heart.Atherosclerosis,inflammation,embolism,blood pressure and others lead to myocardial ischemia,the heart can not work properly,by thrombolysis,coronal arterial bypass,percutaneous coronary intervention(PCI)and other early recovery of cardiac perfusion may be an effective method to improve the clinical prognosis.Howere,after the recovery of blood flow,the myocardium tends to be aggravated,and myocardial ischemia-reperfusion injury occurs.Therefore,looking for safer and more effective methods and drugs to enhance myocardial contractility and improve myocardial ischemia-reperfusion injury,it is particularly important.Total flavones of rhododendra(TFR)is an active flavonoid extracted from azalea.Our previous study found that TFR has the effect of relaxing blood vessels,protecting cerebral ischemia-reperfusion injury and resisting myocardial hypoxia-reoxygenation injury.However,the effect of TFR on isolated heart ischemia-reperfusion injury and myocardial cell contractility has not been reported yet.Urotensin ?(UII)is a somatostatin-like neuropeptide that is widely expressed in the cardiovascular system.UII binds to its receptor(UTR)and can exert many biological functions.In view of the role of UT receptors in ischemic myocardial injury,and studies have shown that puerarin,a flavonoid widely used in the treatment of cardiovascular and cerebrovascular diseases,can reduce the right ventricular hypertrophy in rats and it's related to the inhibition of the expression of UT receptor in the right ventricle.Does the flavonoid TFR also play the role of anti ischemic myocardium through the UT receptor?RhoA is a small molecule of guanosine three phosphate binding protein,RhoA activates downstream target molecule Rho-related coiled-coil formation of protein kinase(ROCK),involved in the regulation of a variety of physiological functions,UII and UTR binding can activate RhoA-ROCK pathway.The purpose of this study was to investigate whether TFR functions through the UTR-RhoA-ROCK pathway.In this study,a rat model of myocardial ischemia reperfusion injury was established by Langendorff perfusion device.To observe the effect of TFR on myocardial ischemia-reperfusion injury.A paired experimental design was used to explore the mechanism of TFR on myocardial ischemia-reperfusion injury,by measuring the contractility of myocardial cells,we can observe whether TFR can enhance the contractility of myocardial cells and play a better role in myocardial ischemia-reperfusion injury.Purpose:1.To observe the protective effect of TFR on isolated rat heart ischemia-reperfusion injury.2.To observe the effect of TFR on the contractility of myocardial cells.3.To investigate the relationship between the TFR anti rat isolated heart ischemia-reperfusion injury and the UTR-RhoA-ROCK pathway.Methods:1.The Langendorff perfusion device was used to establish the model of isolated rat heart ischemia-reperfusion injury.The changes of hemodynamics in each group during ischemia-reperfusion were observed.The coronary flow was measured at each time point.The infusion fluid was retained before the end of the experiment.After the experiment,the heart tissues of rats were preserved and the corresponding indexes were detected.1.1 The experiment was divided into 9 groups:(1)Sham group;(2)I/R model group;(3)Verapamil 0.02 mg/L group;(4)Y27632 1 ?mol/L group;(5)TFR 3.7 mg/L group;(6)TFR 11.1 mg/L group;(7)TFR 33.3 mg/L group;(8)TFR 100 mg/L group;(9)TFR 300 mg/L group.(Group 3-9 hearts were given perfusion of cardiac perfusion solution containing corresponding drugs 20 minutes before ischemia).1.2 Determination of isolated rat heart balance 20 min,administration 20 min,ischemia 30 min,reperfusion 15 min,30 min,60 min hemodynamic parameters at various time points.1.3 The activity of LDH and CK-MB and the content of cTnl in the perfusion of cardiac perfusion were measured by E-LISA.1.4 The rat heart tissue was taken,and the activity of RhoA in the myocardium was measured by G-LISA.1.5 Western blot method was used to determine the expression of UTR,ROCK1 and ROCK2 protein in myocardium.2.The rat isolated heart was selected by paired experimental design.The UT receptor antagonist SB-706375 and ROCK inhibitor Y27632 were used to block the UT receptor and ROCK pathway respectively.The Langendorff perfusion device was used again to establish the model of ischemia-reperfusion injury.The hemodynamics and coronary flow were measured in each group.The perfusate was collected before the experiment,and the heart tissue was preserved after the experiment.2.1 According to near weight and identical sex,two rats from the same nest were made into one pair.Two hearts from one pair were randomly assigned into the I/R group and the TFR group.Except that control heart was perfused with the normal K-H solution,heart of UTR blockade and heart of ROCK inhibition were respectively perfused with K-H solution containing UTR blocking agent SB-706375 10-6 mol/L and ROCK inhibitor Y27632 1 umol/L.2.2 The activity of LDH and CK-MB and the content of cTnl in the perfusion of cardiac perfusion were measured by E-LISA.2.3 The G-LISA assay was used to determine the activity of RhoA in the myocardium.3.The myocardial cells were cultured and the immunofluorescence method was used to identify the myocardial cells.3.1 Cell contractile frequency was recorded using a synchronized camera system(Olympus).3.2 Measurement of contraction amplitude of cardiac myocytes by computer image analysis system.3.3 The calcium ion fluorescence intensity in myocardial cells was determined by calcium ion imaging system.3.4 The experiment was divided into(1)Control group,(2)TFR 3.7 mg/L group,(3)TFR 11.1 mg/L group,(4)TFR 33.3 mg/L group,(5)TFR 100 mg/L group,(6)TFR 300 mg/L(7)hUII 10 nmol/L group,(8)hUII 100 nmol/L group,(9)hUII 100 nmol/L + TFR group.Results:1.Effect of TFR on isolated rat heart ischemia-reperfusion injury1.1 Before myocardial I/R,hemodynamic parameters(LVSP,LVEDP,+dp/dtmax and-dp/dtmax)and CF were not significantly different among various groups.However,at 60 min of reperfusion,LVSP,+dp/dtmax,-dp/dtmax and CF were decreased,but LVEDP were increased significantly in the I/R group compared to those in the sham group.TFR 100 and 300 mg/L significantly improved the decreases of LVSP,LVEDP +dp/dtmax and-dp/dtmax,CF as well as the increase of LVEDP.Y27632 have similar effects,respectively.These results suggested that TFR could significantly inhibit myocardial I/R-induced cardiac dysfunction.1.2 Compared with I/R group,CK-MB,LDH activity and cTnl content in cardiac perfusate of I/R group were significantly increased,compared with I/R group,CK-MB and LDH activity and cTnl content in TFR 33.3-300 mg/L group decreased significantly,Verapamil and Y27632 had a similar effect.1.3 Compared with sham group,the expression of UTR protein in cardiac tissue of I/R group was significantly increased.Compared with I/R group,the content of UTR protein in isolated heart tissue of TFR 100,300 mg/L group decreased significantly.1.4 Compared with the sham group,the RhoA activity in the heart tissue of the I/R group was significantly increased,and the RhoA activity in the heart tissue decreased significantly after the administration of TFR 33.3-300 mg/L.1.5 Compared with sham group,ROCK,and ROCK2 protein levels in isolated heart tissue were significantly increased in I/R group.Compared with I/R group,ROCK1 and ROCK2 in isolated heart tissue of TFR 100,300 mg/L group protein expression decreased significantly.2.Effects of UTR blockade and ROCK inhibition on TFR improvement of myocardial ischemia-reperfusion injury in isolated rat hearts.2.1 In the control heart,compared with the I/R group,100 mg/L TFR significantly increased CF,LVSP and +dp/dtmax.Compared with the I/R group,in the UTR blockade heart,100 mg/L TFR significantly increased CF,but had no effect on LVSP and +dp/dtmax.Compared with the control heart group,the difference of CF,LVSP and +dp/dtmax decreased significantly in UTR blockade heart group,and the effect of TFR on improving cardiac function decreased significantly under UTR blockade condition.2.2 In the control heart and UTR blockade heart,compared with the I/R group,100 mg/L TFR significantly reduced the activity of CK-MB,LDH and cTnl.However,the results of paired experients showed that the difference of CK-MB,LDH activity and cTnl content in UTR blockade group was significantly lower than that in control heart group.These results showed that the ability of TFR to decrease CK-MB and LDH activity and cTnI content decreased under UTR blockade condition.2.3 In control hearts and UTR blockade hearts,compared with I/R group,TFR can reduce the RhoA activity in myocardial tissue,however,compared with control heart,difference of RhoA activity declined significantly in UTR blockade heart.2.4 The ability of TFR to improve cardiac function(CF,LVSP and +dp/dtmax)and to reduce the content of LDH,CK-MB and cTnl was weakened under the condition of ROCK inhibition.3.The effect of TFR on the contractility of myocardial cells.3.1 Compared with control group,hUII 10,100 nmol/L could accelerate the contraction frequency of myocardial cells.3.2 Compared with control group,hUII 10 nmol/L could increase contraction amplitude of myocardial cells,hUII 100 nmol/L reduce the contraction amplitude of myocardial cells.3.3 Compared with control group,TFR 300 mg/L could reduce the contraction frequency of myocardial cells.3.4 Compared with control group,TFR 300 mg/L could increase contraction amplitude of myocardial cells.3.5 Compared with control group,TFR 33.3-300 mg/L could inhibit the increase of contraction frequency of myocardial cells induced by hUII.3.6 Compared with control group,TFR 33.3-300 mg/L could inhibit the reduce of contraction amplitude of myocardial cells induced by hUII.3.7 Compared with control group,hUII 10,100 nmol/L could increase intracellular Ca2+ concentration.Pretreated by hUII 100 nmol/L,then,the myocardial cells were given TFR 33.3-300 mg/L,the intracellular Ca2+ fluorescence intensity decreased.Conclusions:1.TFR has a protective effect on ischemia-reperfusion injury in isolated rat hearts.2.TFR can improve the contractility of myocardial cells.3.The protective effect of TFR on ischemia-reperfusion injury in isolated rat hearts may be related to blocking the UTR and then inhibiting the RhoA-ROCK signaling pathway. |
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