Activity Screening And Evaluation For The Lead Compounds Based On The Antioxidant Effects Of Cardiovascular Drugs

Cardiovascular disease is the leading killer that threatens people, including myocardial ischemia/reperfusion injury, hyperlipidemia and atherosclerosis. Several theories were hypothesized about the etiology of these disease, one of which is that reactive oxygen spices (ROS) induced cellular oxidative stress is considered to be initial factor of diseases. Many epidemiological studies suggested that the oxidative stress might be the common mechanism causing both ischemia/reperfusion injury and hyperlipidemia. Therefore, the objective of this investigation is to find the leading compounds for the cardiovascular protection based on antioxidative stress.The present study contains the following two parts. In the first part, six water soluble compounds extracted from Radix Salviae Miltiorrhixae, including salvianolic acid A, salvianolic acid B, tanshinol, rosmarinci acid, alkannic acid and protocatechualdehyde were screened in vitro based on their free radical scavenging activities. These findings suggested that salvianolic acid A (Sal A) exhibited a good scavenging effect. Thus we examined the protective effect of salvianolic acid A (Sal A) on myocardial ischemia/reperfusion injury. In the second part, seven novel structure compounds(WS070035, WS070117, WS070120, WS070121, WS070124, WS070135, and WS070143) synthesized by Institute of Material Medica & Chinese Medical Academy, had been screened in vitro based on their free radical scavenging activity as well. WS070117 was identified to possess a significant free radical scavenging activity. As a result, we selected WS070117 as the target chemical to examine its anti-lipid peroxidation effect.In the first part, we tested scavenging activities of Sal A for six free radicals (DPPH radical, superoxide anion, hydroxyl and peroxyl radical, peroxynitrite, and hydrogen peroxide) using chromatometry, fluorescence, and chemoluminescence methods based on a molecule-based ROS scavenging activities evaluation system. The results indicated that Sal A had intense scavenging effects on all of the six radicals. The intensity was:DPPH> peroxyl radical> hydroxyl radical> hydrogen peroxide>peroxynitrite> superoxide anion.On basis of the ROS scavenging assays, the effects of Sal A were tested against hypoxia/reoxygenation-induced cell damage to myocardial cells. The assay employed rat embryonic heart H9c2 cells as the model, and oxidative stress injury was induced by digital hypoxia/reoxygenation technique with hypoxia (1% O2) for 12 hours and then reoxygenation for 8 hours. Cells treated with a range of concentrations (0.001,0.01,0.1, 1 and 10μmol/L) of Sal A for 8 hours showed enhanced viability. Under the concentrations of 0.01,0.1 and 1μmol/L, Sal A inhibited hypoxia /reoxygenation-induced ROS increase and mitochondrion membrane potential (MMP,ΔΨm) decrease, implicating that the protective effects by Sal A can protect myocardial cells from injury of hypoxia/reoxygenation via anti-oxidation.A rat isolated heart ischemia/reperfusion injury model was established with stopping perfusion for 20 min and then recovering perfusion for 60 min to detect the protective effect of Sal A on isolated heart. It was demonstrated that coronary artery flow (CF), the peak of pressure development ((dP/dt)max), and the valley of pressure development ((dP/dt)min) were all decreased, whereas the lowered left ventricular end diastolic pressure (LVEDP) increased significantly, indicating that functions of coronary artery dilatation and left ventricular dilatation/constriction were both damaged, and single concentrations of 0.05,0.1,0.2,0.4,0.8,1.6 and 3.2 mg/ml of Sal A improved all above four indicators, respectively.Experimental data from three levels-molecule, cell and isolated organ indicated that Sal A protects against ischemia/reperfusion injury through anti-oxidative activity, which was consistent with results of in vivo experiments on rats and beagle dogs from other colleagues in our lab.In the second part, six compounds with novel structural synthesized by our institute were screening for their ROS scavenging activities. WS070117 was identified to have the most intense ROS scavenging activity, which could scavenge peroxyl radical, hydrogen peroxide, and DPPH, of which the intensity is:peroxyl radical> hydrogen peroxide> DPPH.On in vitro molecular level, all groups of HepG2 cells were pretreated with a range of concentrations (100,10,1,0.1 and 0.01μmol/L) of WS070117 for 30min and then stimulated with 10 ng/mL TNF-αfor 1 hour. The results showed that all concentrations of WS070117 contributed to a reduction on ROS level of HepG2 cells induced by TNF-a. Chemifluorescence assay suggested that WS070117 inhibited NADPH oxidase (NOX) activity.To establish a hyperlipidemic lipid peroxidation model with extremely high blood fat, apoE-/-mice were fed with high fat diet for 8 weeks, which were unable to be reversed by statins. The model mice were then administrated intragastrically with 5mg/kg WS070117 each day for 8 weeks to examine the anti-oxidative effect of WS070117 independent of hypolipidemic activity.Compared with the untreated mice, WS070117 exhibited anti-lipid peroxidation by decreasing malondialdehyde (MDA) level and raising superoxide Dismutase (SOD) activity in serum and liver, implying that WS070117 had protective effect against hyperlipidemic peroxyl injury.The findings proved that it was possible to screen liposoluble compounds with anti-oxidative activity on basis of in vitro ROS scavenging activities. Our screening results indicated that WS070117 could be a potential candidate for curing hyperlipidemia. In our lab, hypolipidemic effects of WS070117 have been confirmed in golden hamster and rat in vivo experiments, which are consistent with our previous findings.In summary, we used anti-oxidative activity as the breakthrough point, and identified the protective effects of water-soluble Sal A and liposoluble WS070117 through their ability of anti-ischemia and regulation of lipids based on experimental ischemia/reperfusion injury and lipid peroxidation models, respectively. The conclusion confirmed that oxidative injury is an important risk factor that caused myocardial ischemia and hyperlipidemia. Therefore, oxidative injury could be used as a target for drug screening, which could be an important avenue to search for novel drugs with myocardial protection.