Sul-F对心肌损伤的保护作用及其机制研究
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摘要
芒柄花素(formononetin,7-羟基-4'-甲氧基异黄酮)为水溶性差、口服吸收差的异黄酮化合物,广泛分布于甘草、葛根、红车轴草等豆科植物中,具有解痉、降血脂、抗心律不齐、改善雌激素水平等作用;我们的体外研究发现其具有显著的心肌保护作用;芒柄花素磺酸钠(sodium formononetin-3'-sulfonate,Sul-F)是芒柄花素的磺化产物,在不影响药理作用的前提下大幅度提高了水溶性,为了快速起效,制成了无菌注射液;我们通过不同动物(大鼠、犬)、不同缺血模型(冠脉结扎致心肌缺血和缺血再灌注模型)、不同给药方法(单次静注、亚慢性连续静注),考察了Sul-F的心肌保护作用,并通过体内外心肌损伤模型,研究了Sul-F抗心肌缺血作用机制。
第一部分,Sul-F对心肌缺血损伤的保护作用
1.Sul-F对冠脉结扎致大鼠致急性心肌梗死的保护作用
结扎大鼠左侧冠脉制作急性心肌梗死模型,结扎5 min时尾静脉注射给予2.5~160 mg/kg的Sul-F,观察给药后4 h之内的心电图变化,给药6 h取心脏,TTC染色,比重法计算左心室心肌梗死百分比,ED50约为18.85 mg/kg。尾静脉注射给予10 mg/kg、20 mg/kg和40 mg/kg的Sul-F,能降低冠脉结扎大鼠30~240 min心电图J点的抬高,抑制血清酶LDH、AST和CK活性的升高,缩小心肌梗死范围。静脉给予20mg/kg的Sul-F可显著降低心肌梗死面积,而灌胃给予200和600mg/kg剂量的Sul-F无效。
2. Sul-F对冠脉结扎致大鼠心肌梗死的长期保护作用
结扎大鼠左侧冠脉制作急性心肌梗死模型,24 h后静脉给予20 mg/kg的Sul-F,每天给药1次,连续14天,Millar导管经颈总动脉导入左心室测定血流动力学指标,取心脏计算心脏系数。结果显示,Sul-F能显著升高±dp/dtmax、SP、DP和AP,对LVSP、LVEDP和HR未见明显影响。
3. Sul-F对冠脉结扎致犬急性心肌梗死的保护作用
结扎Beagle犬冠状动脉左前降支制作急性心肌梗死模型,结扎10 min时舌下静脉注射给予3 mg/kg、6 mg/kg和12 mg/kg的Sul-F,观察结扎4 h之内的血流动力学指标和EECG变化,并在结扎前、结扎2 h和结扎4 h分别取动脉血测定血氧,血流动力学测定完毕采血测定血清LDH、AST、CK活性,同时测定心肌梗死百分比。结果显示:6~12 mg/kg的Sul-F可抑制冠脉结扎犬的Σ-ST和N-ST升高、降低心肌梗死范围,抑制血清LDH、CK和AST活性升高。6~12 mg/kg的Sul-F在冠脉结扎后不同时间点抑制CO、SV、SW、CI和SI的降低,抑制SP、DP、AP、LVSP、±dp/dt_(max)的降低;6~12 mg/kg的Sul-F在冠脉结扎不同时间点抑制CBF、MBF和MOCI的降低,抑制MOUR的升高;12 mg/kg的Sul-F抑制TPVR的升高。3~12 mg/kg的Sul-F对冠状动脉结扎犬LVEDP、HR、MVO2无明显影响。
第二部分,Sul-F对心肌缺血再灌注损伤的保护作用
1. Sul-F对缺血再灌注致大鼠急性心肌梗死的保护作用;
制作大鼠心肌缺血再灌注心肌梗死模型,再灌5 min时尾静脉注射给予10 mg/kg、20 mg/kg和40 mg/kg的Sul-F,观察3 h之内的心电图变化,给药3 h后测定血流动力学,采血测定血清酶活,取心脏计算左心室心肌梗死百分比。结果显示,20~40 mg/kg的Sul-F能够降低缺血再灌注大鼠心电图J点的抬高和心肌梗死范围,升高+dp/dt_(max)、-dp/dt_(max)、LVSP、DP和AP,显著降低大鼠血清CK、LDH和AST活性。
2. Sul-F对缺血再灌注致大鼠心肌梗死的长期保护作用
制作大鼠心肌缺血再灌注心肌梗死模型,24 h后静注20 mg/kg的Sul-F,每天给药1次,连续14天,Millar导管测定血液动力学指标,取心脏,计算脏器系数,常规病理切片,行HE染色和Masson染色,观察病理组织学变化,计算心肌胶原容积分数(CVF)。结果显示,亚慢性给予Sul-F能抑制+dp/dt_(max)和-dp/dt_(max)的降低,可减轻缺血再灌注损伤引起的心肌纤维断裂缺损、心肌细胞坏死伴纤维化和炎性细胞浸润,降低CVF百分率。
第三部分,Sul-F抗心肌缺血和缺血再灌注损伤的作用机制研究
1. Sul-F对H9c2细胞氧化损伤的保护作用及机制研究
H9c2细胞贴壁24 h,加入含不同浓度的Sul-F培养24 h,加入100μM的H_2O_2,孵育1 h。采用细胞流式细胞仪,Annexin-V和PI双染心肌检测凋亡率,R123染色法检测细胞膜电位,Fluo-3染色法检测细胞内钙;ELISA法检测上清液Bcl-2和Bax表达。结果显示,31.2~62.5 nM的Sul-F可降低H_2O_2诱导的心肌细胞损伤;25 ~156 nM的Sul-F均能降低H_2O_2引起的细胞凋亡发生率,抑制H_2O_2诱导的细胞膜电位降低;62.5 nM Sul-F能抑制缺氧心肌细胞内Ca~(2+)浓度的升高;62.5~156nM的Sul-F可导致Bcl-2表达量和Bcl-2/Bax比值显著升高。
2. Sul-F对缺血再灌注大鼠心肌细胞凋亡的影响
制作大鼠心肌缺血再灌注心肌梗死模型,灌注5 min时尾静脉注射给予20 mg/kg的Sul-F,再灌注185min取心脏,常规病理切片,行TUNEL染色检测心肌细胞凋亡,免疫组化染色检测Bcl-2、Bax和p-Akt蛋白表达。结果显示:20 mg/kg的Sul-F能够抑制缺血再灌大鼠心肌细胞的凋亡,提高Bcl-2蛋白的表达和Bcl-2/Bax比值,增加p-Akt蛋白表达。
综上所述,Sul-F急性给药和亚慢性给药均可缩小心肌缺血或缺血再灌注损伤的心肌梗死面积,降低血清酶活性,改善血液动力学,增强心脏功能,减轻心肌病理改变;降低氧化损伤引起的细胞钙超载、抑制线粒体膜电位改变。其作用机制可能与激活PI3K/Akt信号转导通路,上调Bcl-2/Bax比值,抑制线粒体凋亡途径细胞凋亡有关。
Formononetin (7-hydroxy-4'-methoxy isoflavone), a kind of isoflavone with poor water solubility and poor oral absorption, widely distributes in the leguminous plants such as licorice, kudzu root, red grass, et al. Formononetin possessed spasm-relieving, blood-fat-reducing, anti-arrhythmic and estrogen-modulating activities. Sul-F (sodium formononetin-3'-sulfonate) was the sulfonated product of formononetin with good water solubility and no activity influenced. In order to make Sul-F produce rapid pharmacodynamic activity, it was prepared as sterile injection. By different animals (rat or dog), different models (induced by ligating the left anterior descending coronary artery (LAD)), and different administration methods (single-intravenous injection or continuous intravenous injection), anti-myocardial injury effects of Sul-F were investigated thoroughly, and the mechanisms were studied by in vitro and in vivo models of myocardial injury.
In the first part, protections of Sul-F on myocardial ischemic injury are displayed as follows:
1 Protection of Sul-F on acute myocardial infarcion induced by ligating LAD in rats
The acute myocardial infarcion model induced by myocardial ischemia was prepared by ligating LAD in rats. The rats were injected 2.5~160 mg/kg of Sul-F by tail vein at 5min after LAD ligation. The ECG changes were observed within 4h after administration. Taking heart in 6h and staining with TTC, the percentage of left ventricular myocardial infarction was counted by methods for specific gravity. The ED50 was about 18.85mg/kg. 10 mg/kg, 20 mg/kg and 40 mg/kg of Sul-F can reduce the raise of ECG J point after 30~240min LAD ligation in rats, inhibit the elevation of LDH, AST and CK enzyme activity in the serum and decrease myocardial infarction area. 20mg/kg of Sul-F by intravenous injection can reduce myocardial infarction area, but 200 and 600mg/kg of Sul-F can not do that by oral route.
2 Long-term protection effects of Sul-F on myocardial infarction induced by LAD ligation in rats
The acute myocardial infarcion model induced by myocardial ischemia was prepared by ligating LAD in rats. At 24h after LAD ligation, 20mg/kg of Sul-F was intravenously injected once daily for 14 days. Millar catheter, which was inserted into left artrium by arteria carotis communis, can be used to measure blood hemodynamics parameters. The heart index was also calculated. The results showed Sul-F can raise±dp/dt_(max), SP, DP and AP and did not exhibit significant influences on LVSP, LVEDP and HR.
3 Protection of Sul-F on acute myocardial infarcion induced by ligating LAD in dogs
The acute myocardial infarcion model induced by myocardial ischemia was prepared by ligating LAD in dogs. At 10 min after ligation, 3 mg/kg, 6 mg/kg and 12mg/kg of Sul-F were sublingual intravenously administred. In the following 4h, ECG and hemodynamic changes were observed. Before ligation and at 2 h and 4 h after ligation, arterial blood oxygen was determined. After blood hemodynamics parameters were completely assayed, serum LDH, AST, CK activity, the percentage of myocardial infarction were investigated. The results showed, 6~12 mg/kg of Sul-F inhibited the decrease of CO, SV, SW, CI, SI CBF, MBF and MOCI, and the increase of MOUR at the different time points. 12 mg/kg of Sul-F inhibited the increase of TRVR. But no effects on LVEDP, HR and MVO2 were observed.
In the second part, protections of Sul-F on myocardial ischemic-reperfusion injury are displayed as follows:
1 Protection of Sul-F on myocardial infarcion induced by ischemic-reperfusion in rats
The myocardial infarcion model induced by myocardial ischemic-reperfusion was prepared by ligating LAD in rats. After LAD in rats was ligated for 30 min, reperfusion was begun. At 5 min after reperfusion, 10 mg/kg, 20 mg/kg and 40 mg/kg of the Sul-F were intravenously administred and ECG were continuously observed for 3h. After drug administration for 3h, enzyme activity in the serum and myocardial infarction range were determined. The results showed that, 20~40 mg/kg of Sul-F can inhibit the increase of ECG J point and decrease the myocardial infarction area in inchemic-reperfused rats, raise +dp/dt_(max), -dp/dt_(max), LVSP, DP and AP values, and decrease serum LDH, AST, CK activity.
2 Long-term protection effects of Sul-F on myocardial infarction induced by ischemia reperfusion in rats
The myocardial infarcion model induced by ischemic-reperfusion was prepared by ligating LAD in rats. After LAD in rats was ligated for 30 min, reperfusion was begun. The rats of the ECG J point elevation after 2h reperfusion were selected. After 24 h, 20mg/kg of Sul-F was intravenously injected, once daily for 14 days. Millar catheter was used to measure hemodynamics parameters. The heart index was calculated. The changes of pathohistology were observed by HE and Masson staining method. Myocardial CVF was calculated. The results showed that Sul-F can inhibit the reduction of +dp/dt_(max) and -dp/dt_(max). Sul-F can also prevent myocardial fiber breakage, necrosis, myocyte fibrosis and ruduce inflammatory cell infiltration caused by ischemia reperfusion and CVF ratio.
In the third part, anti-ischemic mechaniams of Sul-F are displayed as follows:
1 Protection and mechanisms of Sul-F on H9c2 cells injure induced by H_2O_2 simulated the oxidative stress.
H9c2 cells were allowed to adhere for 24 h. Different concentrations of Sul-F were added and the cells were cultured another 24 h. Then, 100μM of H_2O_2 were added and incubated for 1 h. By cell flow cytometry, Annexin-V and PI staining, R123 staining and Fluo-3 staining were used to determined apoptosis rate, membrane potential and intracellular calcium of cardiomyocytes, respectively. Bcl-2 and Bax expression in supernatant were assayed by ELISA kits. The results showed: 31.2~ 62.5 nM of Sul-F can provent myocardial cell injury induced by H_2O_2; 25~156 nM of Sul-F can reduce the incidence of cell apoptosis, inhibit H_2O_2-induced cell membrane potential decrease; 62.5 nM of Sul-F can inhibit the Ca~(2+) concentration increase in hypoxic myocardial cells; 62.5~156 nM of Sul-F can lead to the increase of Bcl-2 expression and Bcl-2/Bax ratio.
2 Effects of Sul-F on cardiomyocyte apoptosis induced by ischemic-reperfusion in rats
The myocardial infarcion model induced by ischemic-reperfusion was prepared by ligating LAD in rats. After LAD was ligated for 30 min, reperfusion was begun. At 5 min after reperfusion, 20 mg/kg of Sul-F were intravenously administred. After reperfusion for 3h, the hearts were taken and routine pathological examination was carried out. The cardiomyocyte apoptosis were detected by TUNEL staining. Bcl-2, Bax and p-Akt protein expression were determined by immunohistochemistry method. The results showed: 20 mg/kg of Sul-F can inhibit myocardial apoptosis induced by ischemia-reperfusion, increase Bcl-2 protein expression, Bcl-2/Bax ratio and p-Akt protein expression.
In summary, acute or subchronic administration of Sul-F may potect myocardial injuries induced by ischemia or ischemia-reperfusion by reducing myocardial infarction area and serum enzyme activity, improving hemodynamics parameters, strenthening myocardial function and improving the crdiac pathology changes. Sul-F can reduce oxidative damage of cardiomyocyte induced by calcium overload, and inhibit the decrease of mitochondrial membrane potential. Possibly through PI3K/Akt signaling pathway, Sul-F can up-regulate Bcl-2/Bax ratio, inhibit mitochondrial apoptosis pathway and prevent cardiomyocytes from ischemic injury and ischemia-reperfusion injury.
第一部分,Sul-F对心肌缺血损伤的保护作用
1.Sul-F对冠脉结扎致大鼠致急性心肌梗死的保护作用
结扎大鼠左侧冠脉制作急性心肌梗死模型,结扎5 min时尾静脉注射给予2.5~160 mg/kg的Sul-F,观察给药后4 h之内的心电图变化,给药6 h取心脏,TTC染色,比重法计算左心室心肌梗死百分比,ED50约为18.85 mg/kg。尾静脉注射给予10 mg/kg、20 mg/kg和40 mg/kg的Sul-F,能降低冠脉结扎大鼠30~240 min心电图J点的抬高,抑制血清酶LDH、AST和CK活性的升高,缩小心肌梗死范围。静脉给予20mg/kg的Sul-F可显著降低心肌梗死面积,而灌胃给予200和600mg/kg剂量的Sul-F无效。
2. Sul-F对冠脉结扎致大鼠心肌梗死的长期保护作用
结扎大鼠左侧冠脉制作急性心肌梗死模型,24 h后静脉给予20 mg/kg的Sul-F,每天给药1次,连续14天,Millar导管经颈总动脉导入左心室测定血流动力学指标,取心脏计算心脏系数。结果显示,Sul-F能显著升高±dp/dtmax、SP、DP和AP,对LVSP、LVEDP和HR未见明显影响。
3. Sul-F对冠脉结扎致犬急性心肌梗死的保护作用
结扎Beagle犬冠状动脉左前降支制作急性心肌梗死模型,结扎10 min时舌下静脉注射给予3 mg/kg、6 mg/kg和12 mg/kg的Sul-F,观察结扎4 h之内的血流动力学指标和EECG变化,并在结扎前、结扎2 h和结扎4 h分别取动脉血测定血氧,血流动力学测定完毕采血测定血清LDH、AST、CK活性,同时测定心肌梗死百分比。结果显示:6~12 mg/kg的Sul-F可抑制冠脉结扎犬的Σ-ST和N-ST升高、降低心肌梗死范围,抑制血清LDH、CK和AST活性升高。6~12 mg/kg的Sul-F在冠脉结扎后不同时间点抑制CO、SV、SW、CI和SI的降低,抑制SP、DP、AP、LVSP、±dp/dt_(max)的降低;6~12 mg/kg的Sul-F在冠脉结扎不同时间点抑制CBF、MBF和MOCI的降低,抑制MOUR的升高;12 mg/kg的Sul-F抑制TPVR的升高。3~12 mg/kg的Sul-F对冠状动脉结扎犬LVEDP、HR、MVO2无明显影响。
第二部分,Sul-F对心肌缺血再灌注损伤的保护作用
1. Sul-F对缺血再灌注致大鼠急性心肌梗死的保护作用;
制作大鼠心肌缺血再灌注心肌梗死模型,再灌5 min时尾静脉注射给予10 mg/kg、20 mg/kg和40 mg/kg的Sul-F,观察3 h之内的心电图变化,给药3 h后测定血流动力学,采血测定血清酶活,取心脏计算左心室心肌梗死百分比。结果显示,20~40 mg/kg的Sul-F能够降低缺血再灌注大鼠心电图J点的抬高和心肌梗死范围,升高+dp/dt_(max)、-dp/dt_(max)、LVSP、DP和AP,显著降低大鼠血清CK、LDH和AST活性。
2. Sul-F对缺血再灌注致大鼠心肌梗死的长期保护作用
制作大鼠心肌缺血再灌注心肌梗死模型,24 h后静注20 mg/kg的Sul-F,每天给药1次,连续14天,Millar导管测定血液动力学指标,取心脏,计算脏器系数,常规病理切片,行HE染色和Masson染色,观察病理组织学变化,计算心肌胶原容积分数(CVF)。结果显示,亚慢性给予Sul-F能抑制+dp/dt_(max)和-dp/dt_(max)的降低,可减轻缺血再灌注损伤引起的心肌纤维断裂缺损、心肌细胞坏死伴纤维化和炎性细胞浸润,降低CVF百分率。
第三部分,Sul-F抗心肌缺血和缺血再灌注损伤的作用机制研究
1. Sul-F对H9c2细胞氧化损伤的保护作用及机制研究
H9c2细胞贴壁24 h,加入含不同浓度的Sul-F培养24 h,加入100μM的H_2O_2,孵育1 h。采用细胞流式细胞仪,Annexin-V和PI双染心肌检测凋亡率,R123染色法检测细胞膜电位,Fluo-3染色法检测细胞内钙;ELISA法检测上清液Bcl-2和Bax表达。结果显示,31.2~62.5 nM的Sul-F可降低H_2O_2诱导的心肌细胞损伤;25 ~156 nM的Sul-F均能降低H_2O_2引起的细胞凋亡发生率,抑制H_2O_2诱导的细胞膜电位降低;62.5 nM Sul-F能抑制缺氧心肌细胞内Ca~(2+)浓度的升高;62.5~156nM的Sul-F可导致Bcl-2表达量和Bcl-2/Bax比值显著升高。
2. Sul-F对缺血再灌注大鼠心肌细胞凋亡的影响
制作大鼠心肌缺血再灌注心肌梗死模型,灌注5 min时尾静脉注射给予20 mg/kg的Sul-F,再灌注185min取心脏,常规病理切片,行TUNEL染色检测心肌细胞凋亡,免疫组化染色检测Bcl-2、Bax和p-Akt蛋白表达。结果显示:20 mg/kg的Sul-F能够抑制缺血再灌大鼠心肌细胞的凋亡,提高Bcl-2蛋白的表达和Bcl-2/Bax比值,增加p-Akt蛋白表达。
综上所述,Sul-F急性给药和亚慢性给药均可缩小心肌缺血或缺血再灌注损伤的心肌梗死面积,降低血清酶活性,改善血液动力学,增强心脏功能,减轻心肌病理改变;降低氧化损伤引起的细胞钙超载、抑制线粒体膜电位改变。其作用机制可能与激活PI3K/Akt信号转导通路,上调Bcl-2/Bax比值,抑制线粒体凋亡途径细胞凋亡有关。
Formononetin (7-hydroxy-4'-methoxy isoflavone), a kind of isoflavone with poor water solubility and poor oral absorption, widely distributes in the leguminous plants such as licorice, kudzu root, red grass, et al. Formononetin possessed spasm-relieving, blood-fat-reducing, anti-arrhythmic and estrogen-modulating activities. Sul-F (sodium formononetin-3'-sulfonate) was the sulfonated product of formononetin with good water solubility and no activity influenced. In order to make Sul-F produce rapid pharmacodynamic activity, it was prepared as sterile injection. By different animals (rat or dog), different models (induced by ligating the left anterior descending coronary artery (LAD)), and different administration methods (single-intravenous injection or continuous intravenous injection), anti-myocardial injury effects of Sul-F were investigated thoroughly, and the mechanisms were studied by in vitro and in vivo models of myocardial injury.
In the first part, protections of Sul-F on myocardial ischemic injury are displayed as follows:
1 Protection of Sul-F on acute myocardial infarcion induced by ligating LAD in rats
The acute myocardial infarcion model induced by myocardial ischemia was prepared by ligating LAD in rats. The rats were injected 2.5~160 mg/kg of Sul-F by tail vein at 5min after LAD ligation. The ECG changes were observed within 4h after administration. Taking heart in 6h and staining with TTC, the percentage of left ventricular myocardial infarction was counted by methods for specific gravity. The ED50 was about 18.85mg/kg. 10 mg/kg, 20 mg/kg and 40 mg/kg of Sul-F can reduce the raise of ECG J point after 30~240min LAD ligation in rats, inhibit the elevation of LDH, AST and CK enzyme activity in the serum and decrease myocardial infarction area. 20mg/kg of Sul-F by intravenous injection can reduce myocardial infarction area, but 200 and 600mg/kg of Sul-F can not do that by oral route.
2 Long-term protection effects of Sul-F on myocardial infarction induced by LAD ligation in rats
The acute myocardial infarcion model induced by myocardial ischemia was prepared by ligating LAD in rats. At 24h after LAD ligation, 20mg/kg of Sul-F was intravenously injected once daily for 14 days. Millar catheter, which was inserted into left artrium by arteria carotis communis, can be used to measure blood hemodynamics parameters. The heart index was also calculated. The results showed Sul-F can raise±dp/dt_(max), SP, DP and AP and did not exhibit significant influences on LVSP, LVEDP and HR.
3 Protection of Sul-F on acute myocardial infarcion induced by ligating LAD in dogs
The acute myocardial infarcion model induced by myocardial ischemia was prepared by ligating LAD in dogs. At 10 min after ligation, 3 mg/kg, 6 mg/kg and 12mg/kg of Sul-F were sublingual intravenously administred. In the following 4h, ECG and hemodynamic changes were observed. Before ligation and at 2 h and 4 h after ligation, arterial blood oxygen was determined. After blood hemodynamics parameters were completely assayed, serum LDH, AST, CK activity, the percentage of myocardial infarction were investigated. The results showed, 6~12 mg/kg of Sul-F inhibited the decrease of CO, SV, SW, CI, SI CBF, MBF and MOCI, and the increase of MOUR at the different time points. 12 mg/kg of Sul-F inhibited the increase of TRVR. But no effects on LVEDP, HR and MVO2 were observed.
In the second part, protections of Sul-F on myocardial ischemic-reperfusion injury are displayed as follows:
1 Protection of Sul-F on myocardial infarcion induced by ischemic-reperfusion in rats
The myocardial infarcion model induced by myocardial ischemic-reperfusion was prepared by ligating LAD in rats. After LAD in rats was ligated for 30 min, reperfusion was begun. At 5 min after reperfusion, 10 mg/kg, 20 mg/kg and 40 mg/kg of the Sul-F were intravenously administred and ECG were continuously observed for 3h. After drug administration for 3h, enzyme activity in the serum and myocardial infarction range were determined. The results showed that, 20~40 mg/kg of Sul-F can inhibit the increase of ECG J point and decrease the myocardial infarction area in inchemic-reperfused rats, raise +dp/dt_(max), -dp/dt_(max), LVSP, DP and AP values, and decrease serum LDH, AST, CK activity.
2 Long-term protection effects of Sul-F on myocardial infarction induced by ischemia reperfusion in rats
The myocardial infarcion model induced by ischemic-reperfusion was prepared by ligating LAD in rats. After LAD in rats was ligated for 30 min, reperfusion was begun. The rats of the ECG J point elevation after 2h reperfusion were selected. After 24 h, 20mg/kg of Sul-F was intravenously injected, once daily for 14 days. Millar catheter was used to measure hemodynamics parameters. The heart index was calculated. The changes of pathohistology were observed by HE and Masson staining method. Myocardial CVF was calculated. The results showed that Sul-F can inhibit the reduction of +dp/dt_(max) and -dp/dt_(max). Sul-F can also prevent myocardial fiber breakage, necrosis, myocyte fibrosis and ruduce inflammatory cell infiltration caused by ischemia reperfusion and CVF ratio.
In the third part, anti-ischemic mechaniams of Sul-F are displayed as follows:
1 Protection and mechanisms of Sul-F on H9c2 cells injure induced by H_2O_2 simulated the oxidative stress.
H9c2 cells were allowed to adhere for 24 h. Different concentrations of Sul-F were added and the cells were cultured another 24 h. Then, 100μM of H_2O_2 were added and incubated for 1 h. By cell flow cytometry, Annexin-V and PI staining, R123 staining and Fluo-3 staining were used to determined apoptosis rate, membrane potential and intracellular calcium of cardiomyocytes, respectively. Bcl-2 and Bax expression in supernatant were assayed by ELISA kits. The results showed: 31.2~ 62.5 nM of Sul-F can provent myocardial cell injury induced by H_2O_2; 25~156 nM of Sul-F can reduce the incidence of cell apoptosis, inhibit H_2O_2-induced cell membrane potential decrease; 62.5 nM of Sul-F can inhibit the Ca~(2+) concentration increase in hypoxic myocardial cells; 62.5~156 nM of Sul-F can lead to the increase of Bcl-2 expression and Bcl-2/Bax ratio.
2 Effects of Sul-F on cardiomyocyte apoptosis induced by ischemic-reperfusion in rats
The myocardial infarcion model induced by ischemic-reperfusion was prepared by ligating LAD in rats. After LAD was ligated for 30 min, reperfusion was begun. At 5 min after reperfusion, 20 mg/kg of Sul-F were intravenously administred. After reperfusion for 3h, the hearts were taken and routine pathological examination was carried out. The cardiomyocyte apoptosis were detected by TUNEL staining. Bcl-2, Bax and p-Akt protein expression were determined by immunohistochemistry method. The results showed: 20 mg/kg of Sul-F can inhibit myocardial apoptosis induced by ischemia-reperfusion, increase Bcl-2 protein expression, Bcl-2/Bax ratio and p-Akt protein expression.
In summary, acute or subchronic administration of Sul-F may potect myocardial injuries induced by ischemia or ischemia-reperfusion by reducing myocardial infarction area and serum enzyme activity, improving hemodynamics parameters, strenthening myocardial function and improving the crdiac pathology changes. Sul-F can reduce oxidative damage of cardiomyocyte induced by calcium overload, and inhibit the decrease of mitochondrial membrane potential. Possibly through PI3K/Akt signaling pathway, Sul-F can up-regulate Bcl-2/Bax ratio, inhibit mitochondrial apoptosis pathway and prevent cardiomyocytes from ischemic injury and ischemia-reperfusion injury.
引文
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[2] Lloyd-Jones D, Adams RJ, Brown TM, et al. Heart disease and stroke statistics--2010 update: a report from the American Heart Association. Circulation, 2010, 121 (7):e46.
[3] Murray C, Lopez A. Alternative projections of morta Dereklity and disability by cause 1990-2020:Global Burden of Disease Study.Lancet, 1997, 349:1498-1504.
[4]吴锡桂.我国人群冠心病流行现况与趋势.中国慢性病预防与控制, 2003,11(4):190-191.
[5]石晶,姚裕家,李炜如.缺氧缺血与线粒体DNA损伤.国外医学儿科分册,2002,29(1):26-28.
[6] Hess ML, Manson NH. Molecular oxygen: friend and foe. The role of the oxygen free radical system in the calcium paradox, the oxygen paradox and ischemia/reperfusion injury. J Mol Cell Cardiol, 1984, 16 (11):969-85.
[7] Bueno OF, De Windt LJ, Tymitz KM, et al. The MEK1-ERK1/2 signaling pathway promotes compensated cardiac hypertrophy in transgenic mice. EMBO J, 2000, 19 (23):6341-50.
[8]颜红兵,马长生,霍勇等译.美国不稳定型心绞痛和非ST段抬高心肌梗死治疗指南(2007修订版).中国环境科学出版社.
[9] Kajstura J, Cheng W, Reiss K, et al. Apoptotic and necrotic myocyte cell deaths are independent contributing variables of infarct size in rats. Lab Invest, 1996, 74 (1):86-107.
[10] Yellon DM, Hausenloy DJ. Myocardial reperfusion injury. N Engl J Med, 2007, 357 (11):1121-35.
[11] Tsao PS, Aoki N, Lefer DJ, et al. Time course of endothelial dysfunction and myocardial injury during myocardial ischemia and reperfusion in the cat. Circulation, 1990, 82 (4):1402-12.
[12] Gheorghiade M, Sopko G, De Luca L, et al. Navigating the crossroads of coronary artery disease and heart failure. Circulation, 2006, 114 (11):1202-13.
[13]幸奠霞,薛存宽,黄畦,等.刺芒柄花素对大鼠血脂及肝脏雌激素受体的作用.中国医院药学杂志, 2009, 29(8): 1558-1561.
[14] Ha H, Lee HY, Lee JH, et al. Formononetin prevents ovariectomy-induced bone loss in rats. Arch Pharm Res, 2010, 33 (4):625-32.
[15] Mu H, Bai YH, Wang ST, et al. Research on antioxidant effects and estrogenic effect of formononetin from Trifolium pratense (red clover). Phytomedicine, 2009, 16 (4):314-9.
[16] Park J, Kim SH, Cho D, et al. Formononetin, a phyto-oestrogen, and its metabolites up-regulate interleukin-4 production in activated T cells via increased AP-1 DNA binding activity. Immunology, 2005, 116 (1):71-81.
[17] Wu JH, Li Q, Wu MY, et al. Formononetin, an isoflavone, relaxes rat isolated aorta through endothelium-dependent and endothelium-independent pathways. J Nutr Biochem, 2010, 21 (7):613-20.
[18] Yu DH, Bao YM, An LJ, et al. Protection of PC12 cells against superoxide-induced damage by isoflavonoids from Astragalus mongholicus. Biomed Environ Sci, 2009, 22 (1):50-4.
[19] Chen HQ, Wang XJ, Jin ZY, et al. Protective effect of isoflavones from Trifolium pratense on dopaminergic neurons. Neurosci Res, 2008, 62 (2):123-30.
[20]王秋亚,孟庆华,张尊听,等.芒柄花素磺化物的合成、溶解性能及降脂保肝活性.药学学报, 2009, 44 (4): 386-389.
[21]张荣庆,韩正康.异黄酮植物雌激素对小鼠的免疫功能的影响.南京农业大学学报, 1993, 16(2):64-68.
[22]户田静男.异黄酮对活性氧引起脂质过氧化的抑制作用.国外医学中医中药分册, 1999年, 21(3):58.
[23] Huh JE, Kwon NH, Baek YH, et al. Formononetin promotes early fracture healing through stimulating angiogenesis by up-regulating VEGFR-2/Flk-1 in a rat fracture model. Int Immunopharmacol, 2009, 9 (12):1357-65.
[24]叶向荣.益气活血化痰方对大鼠缺血心肌保护作用的机理研究.中国中西医结合杂志, 1995, 6(15):17-18.
[25]徐叔云.药理实验方法学.第二版.北京:人民卫生出版社, 1991: 855-856.
[26]顾菊康,邓开伯.临床心功能学.合肥:安徽科学技术出版社,1992:32-33.
[27]胡梦娟,周双俊.人体解剖学.北京:北京医科大学,中国协和医科大学联合出版社,1995:140.
[28] Pell S, Fayerweather WE. Trends in the incidence of myocardial infarction and in associated mortality and morbidity in a large employed population, 1957-1983. N Engl J Med, 1985, 312 (16):1005-11.
[29] Rackley CE, Russell RO, Jr., Mantle JA, et al. Modern approach to the patient with acute myocardial infarction. Curr Probl Cardiol, 1977, 1 (10):1-47.
[30] Bolli R, Becker L, Gross G, et al. Myocardial protection at a crossroads: the need for translation into clinical therapy. Circulation research, 2004, 95 (2):125.
[31]张松,沈祥春,徐立,等.注射用灯盏花素对麻醉犬急性心肌缺血的影响.中药药理与临床,2004,20(2):13.
[32]吴襄,林坤伟.生理学大纲.北京:高等教育出版社,1992:110-114.
[33]程晓莉,邱彦,张黎,等.丹参总酚酸与三七总试配伍对犬心肌氧摄取率的影响及组方分析.中国药物应用与监测, 2004,3:43-45.
[34] Chimenti S, Carlo E, Masson S, et al. Myocardial infarction: animal models. Methods in molecular medicine, 2004, 98:217-26.
[35] Kastl S, Kotschenreuther U, Hille B, et al. Simplification of rat intubation on inclined metal plate. Adv Physiol Educ, 2004, 28 (1-4):29-32.
[36] Galinanes M, Hearse DJ. Species differences in susceptibility to ischemic injury and responsiveness to myocardial protection. Cardioscience, 1990, 1 (2):127-43.
[37] Hearse DJ. Species variation in the coronary collateral circulation during regional myocardial ischaemia: a critical determinant of the rate of evolution and extent of myocardial infarction. Cardiovasc Res, 2000, 45 (1):213-9.
[38] Tang XL, Sato H, Tiwari S, et al. Cardioprotection by postconditioning in conscious rats is limited to coronary occlusions< 45 min. American Journal of Physiology-Heart and Circulatory Physiology, 2006, 291 (5):H2308.
[39] Goldhaber JI, Weiss JN. Oxygen free radicals and cardiac reperfusion abnormalities. Hypertension, 1992, 20 (1):118-27.
[40]张庆柱.分子药理学.北京:高等教育出版社,2006,186-203.
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