| The occurance and development of myocardial ischemia reperfusion injury (MIRI) are multiple cascade associated with various factors, for example, free radical injury, calcium overload, microvascular injury and the effects of leucocytes. The most recent researches indicate that comlements, selectins, endothelin and apoptosis are involved in the pathophysiological process of MIRI. Obviously MIRI is a complex pathological process which multiple factors interact and improve each other. MIRI is a troublesome problem of mycardial ischemia for long time.The natural resources of Chinese medicinal materials is very rich in China. It is of great significance to search for the safe and effective herbs for the prevention and treatment of MIRI, particularly for the active components in these herbs and the study of their pharmacodynamics and mechanisms on anti-MIRI, which can help to develop our country's herbal resources and to make the modernization of Traditional Chinese Medicine.For the last more than ten years, we have gone in for exploring the rich ethnic herbal resources in Guangxi. After screening a large number of herbs, two better effective anti-MIRI active site of the ethnic medical herbs—Yulangsan flavonoids (YLSF) and persimmon leaf flavonoids (PLF) were found. Our preliminary studies showed that: (1) YLSF preconditioning could significantly decrease the activitis of AST, LDH and LDH1, the contents of MDA, and the myocardial infarct size, and increase the activity of SOD. The results indicated that YLSF had a significant protective effect on myocardial ischemia reperfusion injury (MIRI) in rats. In addition, the two flavone morphons had good effects on hypoxia/reoxygenation injury in cultured neonatal rat cardiomyocytes, which was related with scavenging free radicals and inhibiting Ca2+-overloading. The further study showed that the two flavone morphons also had better antioxidation, anti-hypoxia,anticoagulation, and scavenging of free radicals.(2) PLF could obviously recover cardiac function, reduce the releases of CK, CK-MB, LDH, and LDH-1 from rat hearts of MIRI, increase the activities of SOD, Na+-K+-ATPase and Ca2+-ATPase, and decrease the MDA product. It were suggested that PLF may offer myocardial protective effect against MIRI,the mechanism might be related to attenuating free radicals. Other, the study of antihypertensive effect of PLF showed that PLF could decrease blood pressur and heart rat in normal rats and L-NAME induced hypertention in rats, and might adjust the imbalance of cardiovascular active substances by increasing the release of endogenous vasodilators and reducing the release of endogenous vasoconstrictors, resulting in antihypertention.In short, our prior studies indicated that YLSF and PLF had significant protective effects on MIRI in rats, which are related with many kinds of factors,such as antioxidation and scavenging free radicals. To understand further the influence of YLSF and PLF on MIRI in big animal (rhesus monkeys or dogs), and to accomplish their pre-clinical studies, the rhesus monkey or dog model was used to research the effects of YLSF and PLF on MIRI. The experiments contained two parts as the following:Part I Effect and action mechanism of Yulangsan flavonoids on myocardial ischemic reperfusion in rhesus monkeysObjective: To study the effect and action mechanism of Yulangsan flavonoids (YLSF) on myocardial ischemic reperfusion injury (MIRI) in rhesus monkeys.Methods: Rhesus monkeys were randomly divided into 5 groups: sham operation (SHAM) group, myocardial ischemia reperfusion injury (MIRI) group, YLSF low dose (YLSFL) group, YLSF high dose(YLSFH) group, Diltiazem(DIL) group, and there were 4 animals at each group. Different doses of drug were administered into duodenum 30 min before ligation of the left anterior descending coronary artery (LAD) for 60 min and followed with reperfusion for 180 min to establish MIRI model. Determination of the following indexes: (1) MPA-cardiac function acquisition and analytical system (MPA-CFS) was used to record HR, LVSP, Lvedp, +dp/dtmax, and -dp/dtmax. (2) Myocardial ischemic tissues were taken to make histological section and electron microscopic section S.A. for observing the changes of histopathology and ultrastructure of myocardium under optical microscope or transmission electron microscope respectively, and taking the pictures with camera simultaneously. (3) The blood was collected via the femoral vein, centrifuged, and the upper serum was taken for detecting activities of AST, LDH, LDH1, CK, CK-MB, SOD, GSH-Px, TNOS, MDA and NEFA in each Beagle dog. The myocardial ischemic tissues were taken for determining the activities of ATPase. (4) The myocardial ischemic tissues were taken for determining Bcl-2, Bax and NF-κB protein expressions, cardiac muscle cell apoptosis, ANT1 and Caspase 3 mRNA expressions.Results: (1). Compared with MIRI group, at 180min after reperfusion, YLSFH could significantly increased the LVSP, while could not have effect on HR, LVDEP, +dp/dtmax and -dp/dtmax。(2) Compared with MIRI group, YLSF could significantly inhibited the increasing of myocardial enzyme activity and the content of MDA and NEFA, and increase the activities of SOD, GSH-Px, Na+K+-ATPase and Ca2+Mg2+-ATPase. (3) Histopathological examination confirmed that the degree of myocardial injury in YLSF groups were obviously lessened when compared with model group. (4) The high dosage of YLSF could downregulate the expression of Bax and significantly increase the ratio of Bcl-2/Bax. The high dosage of YLSF could increase the expression of Bcl-2, but the low dosages of YLSF could not have effect on Bcl-2. YLSF could downregulate the expression of NF-κBp65 and the percentage of positive cell. (5) The high dosage of YLSF could lessen the degree of the cardiac muscle cell apoptosis. (6) Compared with MIRI group, expression of ANT1 mRNA increased in YLSFH and YLSFL, expression of Caspase 3 mRNA decreased in YLSFH significantly (P<0.05). (7) YLSF could reduce the injury of the myocardial ultrastructure.Conclusion: Yulangsan flavonoids (YLSF) may offer myocardial protective effects against ischemia reperfusion injury, which may be related to their effects of improving hemodynamics of heart, anti-oxidation, downregulating the expression of Bax protein, increasing the Bcl-2/Bax ratio, downregulating the expression of NF-κBp65, lessening the degree of the cardiac muscle cell apoptosis, and up-regulation of ANT1 mRNA expression and down-regulation of Caspase 3 mRNA expression.Part II The Protective Effect and Mechanisms of Persimmon leaf Flavonid on Myocardial Ischemia Reperfusion Injury in DogsObjective: To study the protective effect of persimmon leaf flavonoid on on myocardial ischemia reperfusion injury in dogs and its related mechanisms.Methods: Healthy Beagle dogs were picked out and randomly divided into 4 groups with 6 dogs in each group: sham operation (SHAM) group, myocardial ischemia reperfusion injury (MIRI) group, PLF low dose (PLFL) group, PLF high (PLFH) dose group,positive drug (DIL)group. Different doses of drug were administered into duodenum 30 min before ligation of the left anterior descending coronary artery (LAD) for 60 min and followed with reperfusion for 180 min to establish MIRI model. Determine the following indexes: 1. MPA-cardiac function acquisition and analytical system (MPA-CFS) was used to record HR, LVSP, Lvedp,+dp/dtmax, -dp/dtmax, IT, ET and IT/ET. 2. Myocardial ischemic tissues were taken to make histological section and electron microscopic section S.A. for observing the changes of histopathology and ultrastructure of myocardium under optical microscope and transmission electron microscope respectively, and taking the pictures with camera simultaneously. 3.The blood was collected via the femoral vein, centrifuged, and the upper serum was taken for detecting activities of AST, CK, CK-MB, LDH, LDH1, SOD, GSH-Px and TNOS, and content of MDA and NEFA in each Beagle dog. The myocardial ischemic tissues were taken for determining the activities of AST, CK, CK-MB, LDH, LDH1, SOD, GSH-Px, TNOS and ATPase, and content of MDA and NEFA. 4. The myocardial ischemic tissues were taken for determining Bcl-2 and Bax protein expressions by immunohistochemical method.Results: 1. Compared with MIRI group, at 180min after reperfusion, HR, LVSP, +dp/dtmax and ET in PLFH group increased significantly (P<0.01 or P<0.05), while the Lvedp, -dp/dtmax, IT, IT/ET decreased significantly (P<0.01 or P<0.05). 2. Myocardial fibers were in disorder condition under optical microscope in MIRI group, in which cellular edema, break or necrosis, infiltration of inflammatory cells, or obvious leakage of red blood cells could be observed. The above injuries could be significantly alleviated in PLFL and PLFH group. The obvious damages of myocardial fibers, mitochondria and intercalated disks could be observed in MIRI group under electronic microscope, but these damages were attenuated significantly in PLFL and PLFH group. 3. Compared with MIRI group, persimmon leaf flavonoid could inhibited the increasing of myocardial enzyme activity and the content of MDA in the serum significantly (P<0.01), and inhibited the decreasing in the activities of SOD, GSH-Px and TNOS in the serum significantly (P<0.01 or P<0.05). Persimmon leaf flavonoid also could decrease the content of MDA and NEFA in the myocardial ischemic tissues significantly (P<0.01 or P<0.05), and increase the myocardial enzyme activity and the activities of SOD, GSH-Px and ATPase in the myocardial ischemic tissues(P<0.01 or P<0.05). 4. Compared with the MIRI group, the expression of Bc1-2 protein increased significantly in PLFL and PLFH group (P> 0.05), the expresion of NF-κBp65 protein,Bax protein and the ratio of Bax protein to Bc1-2 decreased significantly (P <0.05),Conclusion: Persimmon leaf extracts have the protective effects on myocardial ischemical reperfusion injury in dogs. The mechanism may be related to anti-oxidation, protect the activity of ATPase, decrease the content of NEFA, increase the activity of TNOS, improve hemodynamics of heart and inhibit the cardiocyte apoptosis.
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