Tai-Wing Wu

Effect of sodium tanshinone IIA sulfonate in the rabbit myocardium and on human cardiomyocytes and vascular endothelial cells

Sodium tanshinone IIA sulfonate (STS) is a derivative of tanshinone IIA. The latter is a pharmacologically active component isolated from the rhizome of the Chinese herb Salvia miltiorrhiza. Liquid chromatographically pure STS was found to reduce myocardial infarct size by 53.14 +/- 22.79% relative to that in the saline control in a rabbit 1 hr-ischemia and 3 hr-reperfusion model. This effect was comparable to that of Trolox (a better characterized antioxidant serving as a reference cytoprotector), which salvaged the myocardium in the same infarct model by 62.13 +/- 18.91%. Also, like Trolox, STS did not inhibit oxygen uptake by xanthine oxidase (XO), a key enzyme in free radical generation. However, in contrast to Trolox, STS significantly prolonged the survival of cultured human saphenous vein endothelial cells but not human ventricular myocytes in vitro when these cells were separately exposed to XO-generated oxyradicals. Note that the endothelium is recognized to be a key site of oxidant generation and attack. Our findings in vitro and in vivo support the interpretation that STS is a cardioprotective substance, and that it may exert a beneficial effect on the clinically important vascular endothelium.

Unconjugated bilirubin inhibits the oxidation of human low density lipoprotein better than trolox

Oxidative modification of human low density lipoprotein (LDL) has been implicated in plaque formation in blood vessels leading to atherogenesis. Conversely, there is increasing evidence that prevention of LDL oxidation reduces the incidence of coronary artery disease. Here, we have compared the effect of unconjugated bilirubin (Bu) and Trolox (a vitamin E analogue) on the oxidation of LDL after treatment with Cu2+ under defined conditions. We observed that Bu, at or near the normal serum level (i.e. 17 microM) effectively inhibits oxidation of LDL, while it takes at least 500 microM Trolox to achieve a similar effect. This means that, on a per mole basis, Bu is > 20 times more effective than Trolox in preventing LDL oxidation. The oxidation of LDL was assessed by agarose gel electrophoresis. This was further corroborated by assaying the malondialdehyde formed upon reacting the presumptive peroxidation product(s) of LDL with thiobarbituric acid. Thus, we have directly verified that Bu and, less so, Trolox, can each prevent the oxidative damage of LDL in vitro. Our result supports the contention that Bu as an endogenous antioxidant can prevent LDL oxidation and hence reduce the risk of atherogenesis.

Morin: a wood pigment that protects three types of human cells in the cardiovascular system against oxyradical damage.

Morin is a yellowish pigment extractable from the wood of Chlorophora tinctoria. In the present study, we have determined that morin protects three types of human cells--ventricular myocytes, saphenous vein endothelial cells, and erythrocytes--against damage by oxyradicals generated in situ. In myocytes and endothelial cells, morin prolonged substantially and in a concentration-dependent manner the survival of cells exposed to either xanthine oxidase-generated oxyradicals or superoxide radicals produced with menadione. Morin protected erythrocytes from lytic attack by peroxyl radicals generated with 2,2-azo-bis (2-amidinopropane) dihydrochloride. In all three types of human cells, the protective effect of morin clearly excelled that displayed by Trolox (a vitamin E analog), ascorbate, or mannitol, which are water-soluble antioxidants of similar molecular size. Chemically, we verified that morin behaves as an antioxidant by diminishing markedly the amount of malondialdehyde (lipid peroxidation product) found in human cardiocytes despite their exposure to oxyradicals. In agreement with related reports, we also observed that morin is non-toxic in rats even when used at concentrations 2-3 orders of magnitude higher than those in our in vitro studies. Thus, morin acts as a broad-spectrum and non-toxic antioxidant.

Purpurogallin as an antioxidant protector of human erythrocytes against lysis by peroxyl radicals

Purpurogallin, a flavinol obtainable from oak nutgalls, prevented lysis of human erythrocytes exposed to an azo-initiator of peroxyl radicals [2,2-azo-bis (2-amidinopropane) dihydrochloride] at 37 degrees C. The percentage of this inhibition of erythrocyte lysis varied with the concentration of purpurogallin. In this system, the effect of purpurogallin surpassed those of lactosylphenyl-trolox, trolox and ascorbate, while mannitol, superoxide dismutase and/or catalase were without effect. While other concurrent mechanisms may be involved in purpurogallin action, it would appear that the amphipathic (both hydrophilic and lipophilic) nature of this flavinol and of trolox and lactosylphenyl-trolox, is an important factor. In fact, these three antioxidants, with purpurogallin as the best red cell protector here, excelled the protective effects of the other more hydrophilic antioxidants.

Demonstration of the myocardial salvage effect of lithospermic acid B isolated from the aqueous extract of Salvia miltiorrhiza

Lithospermic acid B has been isolated to > 95% purity by high performance liquid chromatography from the aqueous extract of the roots of Salvia miltiorrhiza. When infused at 5.5 mumoles/kg into the post-ischemic rabbit heart, it reduced by 62 +/- 10% (n = 8) the myocardial damage found in the saline control in a rabbit ischemia-reperfusion model.

Water-soluble antioxidant specificity against free radical injury using cultured human ventricular myocytes and fibroblasts and saphenous vein endothelial cells ☆

To better understand the protective effect of water-soluble antioxidants against free radical injury to the reperfused ischemic myocardium, we studied the antioxidant effectiveness of superoxide dismutase (SOD), catalase, ascorbic acid, and Trolox, a water-soluble analogue of alpha-tocopherol, in protecting cultured adult human ventricular myocytes and fibroblasts and saphenous vein endothelial cells from hypoxanthine-xanthine oxidase generated free radicals. The cells were cultured at oxygen tension to 150 and 40 mmHg. Passage P2 to P4 cells were injured by a hypoxanthine-xanthine oxidase free radical generation system. The time when all the cells became shriveled divided by the cell count expressed in terms of 100,000 cells was used to compare cellular susceptibilities to free radical injury and the relative effectiveness of the antioxidants. Fibroblasts were more resistant to free radical injury than myocytes which were more resistant than endothelial cells, when all three cell types were cultured at the same oxygen tension. Trolox and ascorbic acid were effective antioxidants for myocytes while SOD and catalase were ineffective. SOD and catalase were more effective than ascorbic acid as antioxidants for endothelial cells and fibroblasts, while Trolox was ineffective. In summary, we have shown that each cultured cell type has a different susceptibility to free radical damage and that antioxidants are not effective for all cell types.

Comparative Cytoprotection of Cultured Corneal Endothelial Cells by Water-Soluble Antioxidants Against Free-Radical Damage

We reported previously that purpurogallin (PPG) markedly protects the cultured rabbit corneal endothelial cells (RCEC) against oxyradical damage generated with hypo-xanthine (HX) and xanthine oxidase (XO)(1). In this study, we further compared the cytoprotective activities of PPG versus Trolox (TX, α-tocopherol, a water-soluble analogue of vitamin E) and ascorbate (Asc) in confluent cultured RCEC with phase contrast microscopy and confirmed by transmission electron microscopy. PPG prolonged survival of the oxyradical damaged cells longer than those without PPG present (18.6 ± 1.4 min at 1.0 mM and 11.2 ± 1.0 min at 0.25 mM respectively vs. 7.3 ± 0.8 min in control). At levels equimolar to PPG, TX, and Asc were less effective in delaying cell necrosis caused by HX and XO (p < 0.01). When exposed to superoxide radicals generated by menadione, RCEC necrosed at 29.8 ± 1.5 min compared to PPG 47.2 ± 1.0 min at 1.0 mM and 38.9 ± 1.0 min at 0.25 mM. This was significantly different from TX and Asc at corresponding concentrations (p < 0.01). PPG scavenges not only HX-XO-generated oxyradicals, but also nonenzymatically produced superoxide radicals, more actively than two well known antioxidants—TX and Asc.

Radiochemical quantitation of conjugated dienes during ischemia and reperfusion in the rat liver

Conjugated dienes (CD) are putative chemical imprints of oxyradical damage. Employing a highly selective assay for dienes based on their condensation with 14C-tetracyanoethylene (14C-TCNE), and a 14C-TCNE reagent with 60 times higher specific radioactivity than that used by Waller and Recknagel (1978), we have described the profile of phospholipid CD during global ischemia and reperfusion of the rat liver. During 70 min of ischemia, the hepatic CD appeared to increase moderately, but not statistically significantly, relative to that in sham-operated controls (with mean CD = 0.0397 +/- 0.0040 nmoles CD/nmole phosphate, for n = 7). In subsequent reperfusion, CD increased 2.6-3.3 fold higher than in sham controls during the first 10-15 min, declining thereafter to ischemia-like levels by 30 min of reflow. Our data demonstrate that oxyradicals are generated mostly during reperfusion of the post-ischemic rat liver, and that the refined TCNE method can quantitate tissue CD sensitively and relatively specifically.

The opposing effects of an inhibitor of nitric oxide synthesis and of a donor of nitric oxide in rabbits undergoing myocardial ischemia reperfusion

We observed that N-nitro-L-arginine (NOLA), a nitric oxide biosynthesis inhibitor, exacerbated necrosis in the rabbit heart during ischemia-reperfusion while 3-morpholino-sydnonimine-hydrochloride (SIN-1) (a nitric oxide donor) reduced myocardial damage in the same model. In rabbits undergoing 1-h ligation of the anterior ventricular coronary artery, a single bolus injection of NOLA (30 mg/kg) or continuous infusion of SIN-1 (3 mg/kg) were introduced into the post-ischemic heart immediately before 4-h reperfusion. Against negligible necrosis in 6 sham-operated control animals, and 33.8 (SD 13.5)% necrosis in the area at risk for the saline control group (n = 8), the NOLA-treated group (n = 8) had a necrosis of 44.3 (SD 8.6)% whereas the SIN-1-treated group (n = 10) showed a necrosis of 16.8 (SD 4.9)% (both with p < 0.05 vs saline control group). The pressure-rate index increased in the NOLA-treated group but decreased in the SIN-1-treated group. These data support the contention that a nitric oxide donor is an effective cardioprotector during ischemia-reperfusion in vivo.

Purpurogallin Inhibits DNA Synthesis of Murine Fibrosarcoma L-929 and Human U-87 MG Glioblastoma Cells in vitro

We demonstrated, for the first time, that the flavonoid purpurogallin (PPG) at 0.2-0.5 mM inhibits DNA synthesis of murine fibrosarcoma L-929 and human U-87 MG glioblastoma cells in vitro. In the human U-87 MG glioblastoma cell experiments, we found that when cells were incubated with PPG at 0.5 mM for 0.5 and 24 h, about 25 and 50% inhibition of DNA compared with control were observed respectively. In contrast, 0.5 mM Trolox (a more polar analogue of vitamin E) did not inhibit DNA synthesis in both cell lines. These data indicate that PPG inhibits the synthesis of DNA in two distinct tumour cell lines.