Studies On Protective Effects And Mechanisms Of Sul-f On Myocardial Injury

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 ratsThe acute myocardial infarcion model induced by myocardial ischemia was prepared by ligating LAD in rats. The rats were injected 2.5¹60 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 30²40min 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 ratsThe 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 dogsThe 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, 6¹2 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 ratsThe 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, 20⁴0 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 ratsThe 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 H₂O₂ 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 H₂O₂ 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 H₂O₂; 25¹56 nM of Sul-F can reduce the incidence of cell apoptosis, inhibit H₂O₂-induced cell membrane potential decrease; 62.5 nM of Sul-F can inhibit the Ca²⁺ concentration increase in hypoxic myocardial cells; 62.5¹56 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 ratsThe 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.