The Protective Effects Of Soybean Isoflavone On Cardiomyocytes And The Mechanism Research

Soybean isoflavone is a bioactive material existing naturally in the soywith similar structure to 17-estradiol. There is the effect of estrogen and wasnamed as phyto-estrogen(PE). Two main types of soy isoflavone are found inhuman body: genistein and daidzein,which are the main PE that could bedetected in human and animal body fluids. It is the most important bioactivecomponent in soybean. Isoflavone of soybeans is the micro substance insoybeans, which is the most medical valuable active composition in soybeans.Domestic and abroad research materials showed that SI had: the effect oflowering blood pressure and stimulating antihypoxia, sheeting bloodvessels,lowering chole-sterol, It also inhibited atherosclerotic occurrence anddevelopment, Soybean Isoflavone have the protective effects on myocardiumagainst ischemia reperfusion injury.It had positive effects on brain ishemia,lowered blood sugar,improved micro-circulation, showed anti-aging propertiesand imporved sex function.Soybean isoflavones play important roles in reduing risk of developingcardiolvascular disease, osteoporosis, cancer, Alzheimer's disease andclimacteric disease. More and more researches suggest that soy isoflavone hasmultiple modulative effects on cardiovascular system, which will hopefullybecome a favorable interventional medication for the primary rehabilitation ofcardiovascular diseases. It is suggested that SI can decrease the level ofplasmatriglyceride, and resist the rise of the peroxidative status inhyperlipidemic rats,and amelio-rate the tissue damage induced by oxidativestress.Large numbers of evidences indicate that isoflavones prevent and curecardiovascular diseases through influencing lipid metabolism, antioxidation oflow density lipoprotein and antilipid peroxidation, affecting vascular smoothmuscle cell and bloodvessel compliance, inducing antithrombotic effect, as wellas influencing the expression of atherosclerosis related gene.The pathology mechanism of the myocardial ischemia/reperfusion injury,MI/RI under the influence of hypoxia are free radical increase, intracellularcalcium overload and ATP synthesizing obstruction in the mitochondrion.These three factors may work separately or interact with each other to causecellular structure, function and metabolism obstruction, and eventual cell death.The most important factor is intracellular calcium overload in the pathologicaland physiological changes in both cell and the mitochondrion, the initial causeof ishemia inducing myocardial damage is hypoxia. and it was said that hypoxiacould cause apoptosis and necrosis of the endothelium and nerve cells.Likewise, hypoxia could cause apoptosis and necrosis in the cardiomyocyte too.Some literature said that apoptosis played an important.role in the mechanismof ishemic damage to the myocardium. Ishemic heart disease (IHD) is caused bycoronal blood circulation changes and induces the imbalance of myocardial supplyand demand. IHD includes myocardial ishemia which arises from functional andorganic alteration of the coronal arteries, which in turn, comes about due to acute andchronic diminished blood supply. Its most common pathogenisis is the straighteningand obstruction of the coronary arteries from atherosclerosis. Therefore, IHD is alsocalled Atherosclerotic heart disease. In our country, the mortality of cardio-vascularsystem diseases in urban areas has increased over America and Canada, havingbecome a susceptible country to cardio-vascular disease. It is obvious, at thistime,that everyone here needs to be educated about the dangers of IHD andtreated if suffering from it.Soybean Isoflavone have the protective effects on myocardium ischemiaand brain ischemia injury. Domestic and abroad research materials was taken invivo, but reported seldom on ion mechanism research of cardiac myocytes , noreport about the effect of SI on sodium current and KATP current so far. SIreduces the free radical from hypoxia,but it is not clear about the exactmechanism. We simulated the hypoxia damage model in vivo by H2O2 injury inthe cultured cells level and observed SI effects on cardiomyocytes apoptosisand on intracellular free calcium concentration of cultured ventricularmyocytes.So that to impove further the protective mechanism for the cardiacmyocytes and supply theorical basic for clinic.Ⅰ. Effects of SI on the action potential parameters of papillary muscles inguinea-pig.Intracellular microelectrode was used to record the action potential ofguinea pig papillary muscles. Compared with control group, SI 0.01μg/mlchanged the action potential parameters APD50 ,APD90 from(175.6±7.32)mS,(207.97±6.50)mS to(177.94±6.58)mS,(212.6±6.36)mS (p>0.05);APAwas reduced from (68.59±5.4)mV to (56.66±3.09)mV (p<0.05). SI 0.1μg/mlchanged the action potential parameters APD50 , APD90 , APA from(175.6±7.32)mS(207.97±6.50)mS,(68.59±5.4)mV to(146.43±18.91)mS,(181.47±6.10)mS,(33.19±10.11)mV(p<0.05);SI 1.0μg/ml changedthe action potential parameters APD50 ,APD90,APA from (175.6±7.32)mS, (207.97±6.50)mS,(68.59±5.4)mV to(141.77±2.39)mS,(174.9±5.87)mS,(30.18±2.39)mV(p<0.01);the above effect suggests: SI0.1μg/ml andSI1.0μg/ml could abbreviate the action potential duration and amplitude in adose-dependence manner. After wash out , APD90 was recovered to(190.07±7.10)mS. It was no difference after compared with control group(p>0.05). This result was consistent with current patch clamp techniquerecording the action potential experiment result in cultured ventricularmyocytes in neonatal rat.Ⅱ. Effects of SI on the calcium current of ventricular myocytes in culturedcardiomyocytes in neonatal rat.The whole cell patch clamp technique was used to record the calciumcurrent of ventricular myocytes, which was from holding potential -50mV ,stepping to +70 mV, Step order 10mV, the during time 400mS, frequency1Hz . The calcium current of 38 cultured cardiomyocytes in neonatal rat wererecorded. The calcium current of 15 observed normal cells and SI (0.01μg/ml)group at difference time was not reduced obviously. The calcium current ofseven observed cells in SI group(0.1μg/ml) was reduced from(0.253±0.06)nAto(0.225±0.05)nA(p<0.05).The calcium current of nine observed cells inSIgroup(1.0μg/ml)was reduced from(0.249±0.08)nA to(0.156±0.05)nA(p<0.01),the fall rate is 11.07% and 37.35%respectively. It proved that SIhave the block effect with a dose-dependence manner on the calcium current ofventricular myocytes .The result of experiment show the obvious block effect ofSI ( 0.1μg/ml and 1.0μg/ml) on the L-type calcium channel current.Ⅲ.Effects of SI on the sodium current of cultured ventricular myocytesThe whole cell patch clamp technique was used to record the sodiumcurrent of cultured ventricular myocytes, which was from holding potential-100 mV ,stepping to +30 mV, Step order 10mV, the during time 30mS,frequency 1Hz . The sodium current of 32 cultured cardiomyocytes in neonatalrat were recorded. The sodium current of 14 observed normal cells and SI(0.01μg/ml) group at difference time was not reduced obviously.The sodium current of six observed cells in SI group(0.1μg/ml) and sixobserved cells in SI group(1.0μg/ml) was reduced from(-7.85±0.48)nA,(-7.92±0.19)nA to(-6.72±0.46)nA(p<0.05),(-6.06±0.17)nA(p<0.01)respectively. The fall rate is 14.39% and 23.48% respectively. It proved that SIhave the block effect on the sodium current of ventricular myocytes, which isenhanced with the more dose.The result of experiment show the obvious block effect of SI ( 0.1μg/mland 1.0μg/ml) on the sodium current.Ⅳ . Effects of SI on the IKATP in ischemic guinea pig ventricular myocytes.In addition, this study isolated the cardiac myocyte of adult guinea pigsand simulated into hypoxia models observing the effects of SI on the cellularmemberane electricity flow of ATP-dependent K+channel with whole cellpatchclamp system. KATP channel current were recorded in the whole-cellconfiguration of the patch clamp technique by the isolated cell under conditionsof hypoxia. In our experiment, sodium and calcium current could be inhibitedwhen holding potential was -40mV.The whole cell patch clamp technique was used to record the potassiumcurrent of ventricular myocytes, which was from holding potential -100mV ,stepping to +100 mV Step order10mV,the during time300mS,After fiveminutes, the amplitude of potassium current was increased from (1.770±0.126)nA to,(1.832±0.415) nA and the I-V curve moved up(p>0.05) . Although SIincreased the KATP potassium current slightly ,there is no statistics.It proved thatit is not by the channel of sarcol KATP that the mechanism ofischemia/reperfusion injury take effect. It need to be researched further aboutwhether it have promotive effect on the open of the channel of mito KATP.Ⅴ. Measurement of intracellular free calcium concentration of culturedventricular myocytes by using fluo-3-AM dye.Supervise the dynamic change of intracellular free calcium concentration ofcultured ventricular myocytes under LSCM continuous scanning.It is the sameresult of experiment on calcium current partly. Experiments in vitro: simulatingthe hypoxia model on cultured cardiac myocytes in neonatal rats, observe theeffects on alteration of intracellular calcium with laser-confocal-microscopysystem.The result was that after application H2O2 0.3mmol·L-1, the intracellular freecalcium concentration was increased significantly. Compared with controlgroup, 0.3mmol·L-1H2O2 changed the intracellular free calcium averageconcentration from 743.15±34.78 to 1486.52±11.16(p<0.01);SI 0.1μg/ml ,1μg/ml ,10μg/ml changed the intracellular free calcium average concentrationfrom 1486.52±11.16 to 940.61±39.13,823.68±59.99,778.39±42.15 (p<0.01);The result was that after application SI, the intracellular free calciumconcentration with a dose-dependence manner was decreased significantly.This result was consistent with the above calcium current experiment result.Ⅵ . Detect cardiac myocytes apoptosis by flowcytometry(FCM).The result show that H2O2 with low concentration enhanced the percent ofcardiac myocytes apoptosis greatly, SI(0.1μg/ml,1μg/ml,10μg/ml)reduced thepercent of cardiac myocytes apoptosis caused by H2O2 obviously ,so that takethe protection effect on it .The above results impoved the SI protection effect on cardiac myocytes byion mechanism research, SI takes effect on APD50,APD90,APA throughinhibition of the L-type calcium channel current and the sodium current with adose-dependence manner. SI takes protection effect on myocardium throughtdecreasing intracellular calcium overload and the positive percent of cardiacmyocytes apoptosis caused by H2O2.