Protective Effects Of Genistein On Myocardial Hypoxia Injury In Rats

Objectives: By acting on isoproterenol-induced rat model of acute myocardial ischemia and myocardial hypoxia model induced by cobalt dichloride in vitro, observed the protective effects of genistein on myocardial hypoxia injury and its mechanism, to provide experimental basis for the applications of genistein in cardiovascular disease.Methods:(1) Protective effect of genistein on isoproterenol-induced SD rats with acute ischemia impatient: 60 healthy SD rats with normal diet adaptive feeding one week. Then randomly divided into six groups of normal, model, positive drug and genistein(low, medium and high doses), to gavage once a day for 7 consecutive days dosing.Wherein the normal rats and modle rats were given the same amount of vehicle(DMSO: NaCl = 1: 9), positive drug group rats were given CSDP(85mg / kg), and genistein group rats were given different doses of genistein(7.5, 15, 30 mg / kg). Day 8, at 1 h after gavage, rats in each experimental group(except the normal group) were multi-point injected subcutaneously of isoproterenol(2 mg ? kg-1 ? day-1), repeated continuously for 3 days, to establish model of SD rats with acute myocardial ischemia and hypoxia injury. After the last injection of isoproterenol, simultaneously recorded the Ⅱ-lead ECG in rats with BL-420 F biological functional experimental system, and analysis the changes in T-wave amplitude, position of J point and heart rate. Then blood in rats’ apical, and serum was prepared, to detect the activity of lactate dehydrogenase(LDH), creatine kinase(CK), malondialdehyde(MDA) and superoxide dismutase(SOD), by UV-visible spectrophotometer according to the instructions of kits. Finally, took myocardial tissue that the tip of 1/3 heart and thoracic aorta,with 10% formalin-fixed, paraffin-embedded, sliced, HE staining to observe the changes of pathological in the electron microscope.(2) Protective effect of genistein on cultured rat myocardial cells with hypoxia injury induced by cobalt chloride: First, H9c2 cardiomyocytes treated at different times(0, 6, 12, 24, 48 h) with different concentrations of cobalt chloride(0, 200, 400, 600, 800, 1000 μM). MTT assay was used to detect cell activity, and Western-Blot method to detect the expression of hypoxia inducible factor(HIF-1α) and apoptosis-related proteins(Bax, Bcl-2,Caspase-3). In small cell damage but high expression of HIF-1α as a main reference index to choose the best treatment concentration and optimal treatment time of cobalt chloride.Then, give H9c2 cardiomyocytes with different concentrations of genistein(0, 50, 100, 150, 200 μM) pre-incubation at different times(0, 0.5, 4, 8 h), then with the above optimal treatment concentration and optimal treatment time of cobalt chloride to stimulate H9c2 cardiomyocytes, and detecting cell activity by MTT, as well as Western-Blot method to detect the expression of HIF-1α, Notch1, Bax,Bcl-2 and Caspase-3 protein.Results:(1) Genistein could improve the cardiac injury which induced by isoproterenol in rats with acute myocardial ischemia, such as elevate T-wave amplitude, move up the J point position, reduce heart rate. It also can inhibit the isoproterenol-induced oxidative stress damage, reduce the serum levels of MDA, LDH, CK, and increase SOD activity. In addition, it can reduce cardiac index, and improve the severity of myocardial tissue in rats with myocardial ischemia induced by isoproterenol.(2) 400μmol / L of cobalt chloride acts on H9c2 cardiomyocytes 24 h, can build the best model of hypoxia, when cell damage small but high expression of HIF-1α, so the conditions for subsequent damage.(3) Pretreatment with genistein can protect the H9c2 cardiomyocytes hypoxia induced by cobalt chloride, to reduce the protein expression of HIF-1α and increase the expression of Notch1. And that may also interfere H9c2 cardiomyocytes apoptosis induced by cobalt chloride, to downregulate the expression of Bax and Caspase-3 protein, upregulate the expression of Bcl-2, increase the ratio of Bcl-2 / Bax.Conclusions:(1) Genistein can protection the isoproterenol-induced myocardial ischemia and hypoxia injury in SD rats by improving cardiac function and inhibiting oxidative stress damage.(2) Genistein may intervene H9c2 myocardial hypoxia induced by cobalt dichloride, which may be associated with downregulate the expression of HIF-1α protein and upregulate the expression of Notch1 protein.