Studies Of Protective Effects And Mechanisms Of17-methoxyl-7-hydroxyl-benzofuran Chalcone On Myocardial Ischemia Injury

Yulangsan(YLS), the root of one plant named Millettia pulchra Kurz var laxior (Dunn) Z.Wei, is a traditional Chinese herb used for the treatment of various diseases including hypertension, hyperlipidemia, hyperglycemia and senile dementia. Moreover, our studies have proven that YLS has a significant protective effect against ischemia reperfusion injury in rat hearts and brains in vitro and in vivo. Howerver, until now, the specific active component in YLS was unknown. We have identified eight flavone monomers. After preliminary screening, we determined that YLSC is bioactive. YLSC is a component of flavone whose chemical name is17-methoxyl-7-hydroxyl-benzofuran chalcone. Separation and extraction technology of YLSC has been awarded a national patent(patent number:200710034771.2).In recent studies, YLSC showed the effects of anti-blood coagulation, anti-apoptosis, inhibition of Ca2+overloading, and regulation of oxidation, in particular through its ability to scavenge free radicals. Our research is to observe the effects of YLSC on anti-ischemia and its mechanisms.Part Ⅰ. Protective effect of17-methoxyl-7-hydroxyl-benzofuran chalcone on myocardial ischemia induced by pituitrin in ratsAbstract Objective:To study the cardioprotective effect of17-methoxyl-7-hydroxyl-benzofuran chalcone (YLSC) in rats with myocardial injury induced by pituitrin. Method:60SD rats were randomly divided into six groups:normal group, vehicle group, model control group, positive control group and YLSC groups(2.5,5.0mg.kg-1). Pituitrin(0.75Ukg-1) was injected through sublingual vein to erect model of acute myocardium ischemia. The changes of ST segment on the II road electrocardiogram and contents of creatine kinase-MB(CK-MB), Troponin (cTnI), myoglobin(MyoG) in serum were observed and compared. Meanwhile, myocardium pathological structures were accessed with HE staining. Myocardial ischemia sizes were measured by Evans blue and TTC staining when the experiment was over. Result:Compared with the model control group, YLSC could significantly decrease the height of ST segment at15s,30s, lmin and5min in a dose-dependent manner(P<0.05or P<0.01). It also could reduce myocardial ischemia sizes and contents of CK-MB, cTnI, MyoG in serum (P<0.05or P<0.01). Furthermore,these damages induced by pituitrin including myocardium edema, hemorrhage and exudation of inflammatory cells were improved in YLSC treated groups. Conclusion:YLSC has obvious protective effect against pituitrin induced myocardial injury in rats.Part Ⅱ.17-methoxyl-7-hydroxyl-benzofuran chalcone participating in pharmacological preconditioning against cardiac ischemia reperfusion injury in isolated rat heartsAbstract Objective:To investigate whether17-methoxyl-7-hydroxyl-benzofuran chalcone (YLSC) participated in pharmacological preconditioning against cardiac ischemia reperfusion injury in isolated rat hearts. Methods:70SD rats were randomly divided into seven groups:normal group, ischemia reperfusion(I/R) group,vehicle group, ischema preconditioning(IPC) group, positive control group and YLSC groups (5.0mg·L-1,10.0mg·L-1). Isolated rat hearts were subjected to ischemia by stopping perfusion for30min followed by reperfusion for30min in Langendorff perfusion system. The coronary arterial flow(CF), heart rate(HR) and left ventricular developed pressure(LVSP) were recorded. The activity of creatine kinase (CK) in the coronary effluent fluid was measured. The infarction sizes were measured with TTC dying method. Contents of NO, NOS, SOD and MDA were measured when the experiment was over. Results:Compared with the I/R group, YLSC could significantly increase CF, HR and LVSP at5,10,20,30min after reperfusion in a dose-dependent manner(P<0.05or P<0.01).YLSC preconditioning could improve the recovery of cardiac function, decrease the activiy of CK, and minimize the size of myocardial infarction(P<0.05or P<0.01). It also could increase activity of NO, NOS and SOD, reduce content of MDA compared with the I/R group(P<0.05or P<0.01). Conclusion:YLSC has obvious protective effect against ischemia reperfusion injury in the isolated rat hearts, the mechanism may involve in stimulation of NO release and inhibition of lipid peroxidation.Part Ⅲ. Protective effects of17-methoxyl-7-hydroxyl-benzofuran chalcone on rats with myocardial ischemia reperfusion injuryAbstract Purpose:To study the cardioprotective effect of7-methoxyl-7-hydroxyl-benzofuran chalcone (YLSC) on rats with myocardial ischemia reperfusion injury. Methods:60SD rats were randomly divided into six groups: Sham group, Model group, Vehicle group, Dilthiazem(Dil) group, YLSCL group(2.5mg/kg) and YLSCH group(5.0mg/kg). Myocardial ischemia reperfusion injury model was established through occluding left anterior descending branch of coronary artery for30min and removing ligation later to reperfuse for1h. The changes of hemodynamic, including HR, MAP, LVEDP and±dp/dtmax were recorded. Contects of SOD, MDA, Na+-K+-ATPase and Ca2+-ATPase were detected when the experiment was over. Myocardium apoptosis rate was measured by TUNEL.Expressions of apoptosis signal regulating kinase1(ASK1),cytochrome c (cyt c), Bcl-2and Bax protein were tested by western blot.Meanwhile, Bcl-2/Bax ratio was calculated. Results: Compared with the Model group, YLSC could decrease HR, MAP and±dp/dtmax, increase LVEDP, inhibit a increase of MDA, and increase the activity of SOD, Na+-K+-ATPase and Ca2+-ATPase in a dose-dependent manner (P<0.05or P<0.01).Compared with that in the Model group, apoptosis rate was significantly decreased in YLSC groups(P<0.01).Expression levels of ASK1, cyt c and Bax protein were lower in YLSC groups than that in Model group (P<0.05or P<0.01), while the level of Bcl-2protein and Bcl-2/Bax ratio were higher in YLSC groups than that in Model group(P<0.01), respectively. Conclusion: YLSC plays a protective role in rats with myocardial ischemia reperfusion injury. The mechanism may involve in anti-apoptosis, inhibting effect on lipid peroxidation, protecting activity of Na+-K+-ATPase and Ca2+-ATPase, decreasing ASK1, cyt c, Bax protein expression, increasing Bax protein expression, and augmenting Bcl-2/Bax ratio in myocardium of the rat.Part Ⅳ. The protective effects of YLSC on cultured cardiomyocytes in anoxia/reoxygenation injuryAbstract Objective:To study protective effects and mechanisms of17-methoxyl-7-hydroxyl-benzofuran chalcone(YLSC) on damage induced by anoxia/reoxygenation(A/R) in myocardial cell. Methods:Primary cultured neonate rat myocardial cells were pretreated with2h anoxia following4h reoxygenation. They were randomly assigned to six groups:control group, A/R group, vehicle group, Diltiazem (Dil) group(30μmol·L-1), YLSL group(75μg·mL-1), YLSH group(150μg·mL-1). At the end of experiment, myocardial cells beating rate was calculated, intracellular Ca2+([Ca2+];) and mitochondrial membrane potential (ΔΨm) were determined by flow cytometric analysis. Besides, Na+-K+-ATPase and Ca2+-Mg2+-ATPase, Alphac subunit of T-type Ca2+channels(Cav1.2), Sodium-calcium exchanger (NCX) and SR calcium ATPase-2a(SERCA2a) expressions were also evaluated in each group. Results: Compared with the A/R group, the beating rate,[Ca2+]; and ΔΨm of YLSC groups was significantly increased(P<0.05or P<0.01). Compared with the A/R group, Cav1.2and NCX mRNA and protein expressions were significantly decreased(P<0.05or P<0.01), but SERCA2a mRNA and protein expression were not changed. Compared with the A/R group, treatment with YLSC significantly decreased apoptosis(P<0.01). Furthermore, YLSC inhibited cyt c release from the mitochondria and prevented caspase9/3activation(P<0.05. or P<0.01). Conclusion:YLSC exerts significantly cardioprotective effects against A/R in cultured myocardial cells. The protection of calcium overload and anti-apoptosis may be involved in the mechanism of YLSC protection.In summary, the present results suggest that YLSC is effective in anti-ischemia. As a possible mechanism, YLSC can scavenge free radicals, reduce the contents of myocardium kinase, regulate hemodynamic, ruduce myocardial contractility, inhibit apoptosis, inhibit calcium overload, and inhibit the expressions of calcium overload-related gene.