Baolu Zhao

Preventive effect of Ginkgo biloba extract on apoptosis in rat cerebellar neuronal cells induced by hydroxyl radicals

The ability of oxidative stress to induce apoptosis, and the effect of Ginkgo biloba extract (EGb761) on this induction were studied in primary cultured rat cerebellar neuronal cells. Cells were exposed to hydroxyl radicals by treating them with 20-50 microM hydrogen peroxide (H2O2) and 100 microM ferrous sulfate. Hydroxyl radical treatment fragmented the DNA in a manner typical of apoptosis cells, producing a ladder pattern of 200 base pair increments on 1% agarose gel electrophoresis. Pretreatment of cells with 100 micrograms/ml EGb reduced hydroxyl radical induced cells apoptosis (determined by flow cytometry) and DNA fragmentation. The results indicate that hydroxyl radicals induce apoptosis in rat cerebellar neuronal cells and this induction can be prevented by EGb.

Sodium tanshinone IIA sulfonate mediates electron transfer reaction in rat heart mitochondria

In this paper, an electron transfer reaction mediated by sodium tanshinone IIA sulfonate (STS) was studied in rat heart mitochondria. It was found that STS could stimulate mitochondrial NADH oxidation dose-dependently and partly restore NADH oxidation in the presence of respiratory inhibitor (rotenone or antimycin A or KCN). It was likely that STS could accept electrons from complex I similar to ferricyanide and could be converted to its semiquinone form that could then reduce oxygen molecule. The data also showed that cytochrome c (Cyt c) could be reduced by STS in the presence of KCN, or STS could transfer the electron to oxygen directly. Free radicals were involved in the process. The results suggest that STS may protect ischemia-reperfusion injury through an electron transfer reaction in mitochondria against forming reactive oxygen radicals.

Different effects of the constituents of EGb761 on apoptosis in rat cerebellar granule cells induced by hydroxyl radicals.

The present study was conducted to evaluate the different effects of the constituents of EGb761(Ginkgo biloba Extract) on apoptosis in cerebellar granule cells induced by hydroxyl radicals. The total flavonoid component of EGb761, two pure EGb761 components (rutin and quercetin), and a mixture of flavonoids and terpenes protected cerebellar granule cells from oxidative damage and apoptosis induced by hydroxyl radicals. ESR(electron spin resonance) results showed that the IC50 of the flavonoids for scavenging hydroxyl radicals was almost the same as that of EGb761, even though flavonoids make up only 24% of EGb761, implying that other constituents of EGb761 besides flavonoids can scavenge hydroxyl radicals. Total terpenes of EGb761 did not protect against apoptosis. Flavonoids and terpenes did not show a synergistic effect in this regard. Terpenes did not scavenge hydroxyl radicals directly, which might be related to their “cage‐like” structures.

The antioxidant EPC-K1 attenuates NO-induced mitochondrial dysfunction, lipid peroxidation and apoptosis in cerebellar granule cells

In this study we investigated the effects of nitric oxide (NO) on cultured cerebellar granule cells. Exposure to NO donors, S-nitrosoglutathione (GSNO; 250 microM) or sodium nitroprusside (SNP; 500 microM), triggered apoptosis in immature cultures of cerebellar granule cells, which was characterized by chromatin condensation, nuclei fragmentation, and DNA laddering. Exposure of cerebellar granule cells to NO donors led to a decrease in the mitochondrial transmembrane potential and intracellular ATP content, which suggested that NO treatment caused mitochondrial dysfunction. NO treatment also induced oxidative stress in cerebellar granule cells as measured by thiobarbituric acid (TBA) assay. Pretreating cells with L-ascorbic acid 2-[3,4-dihydro-2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridecyl)-2H -1-benzopyran-6-yl-hydrogen phosphate] potassium salt (EPC-K1), a novel antioxidant, attenuated NO-induced mitochondrial dysfunction and oxidative stress to some extent, and prevented the cells from apoptosis. The results of the present investigation suggest that a superoxide/peroxynitrite-mediated oxidative stress may be an important pathway leading to NO-associated neuronal damage. Pretreating cells with the antioxidant EPC-K1 attenuated NO-induced neurotoxicity by scavenging superoxide/peroxynitrite and/or its breakdown products.

Scavenging effect of Chinonin on NO and oxygen free radicals and its protective effect on the myocardium from the injury of ischemia-reperfusion.

The scavenging effect of Chinonin on NO and oxygen free radicals and its protective effect on myocardium from the ischemia-reperfusion injury was studied with electron spin resonance (ESR) and chemiluminescence techniques. Chinonin can effectively inhibit the oxidative activity of ONOO-, (the IC50 = 7 x 10 (-5) mmol/L) and scavenge oxygen free radicals generated from the reaction of xanthine and xanthine oxidase (the IC50 = 2/5 x 10(-4) mmol/l). It is difficult to find another antioxidant which can scavenge so effectively both ONOO- and oxygen free radicals simultaneously. In the system of ischemia-reperfusion injury of myocardium, Chinonin can, in parallel, scavenge the NO and oxygen free radicals generated from the ischemia-reperfused myocardium, and decrease the activities of lactate dehydrogenase (LDH) and creatine kinase (CK) in the coronary artery effluent of ischemia-reperfused heart and therefore protect the heart from ischemia-reperfusion injury. The protective effect of 0.1 mmol/l Chinonin is similar to that of 1500 U/ml SOD and catalase.

Detection of nitric oxide in tissue by spin trapping EPR spectroscopy and triacetylglycerol extraction

A modified method based on EPR spin trapping and triacetylglycerol extraction was used for tissue nitric oxide (NO) detection at room temperature. NO signal intensity was stable for about 1.5 h and the detection limit of this method was less than 200 pmol g−1 tissue. Using this method, we report evidence that NO production in vivo can be inhibited by adriamycin in mice livers.

STUDIES ON NITRIC OXIDE FREE RADICALS GENERATED FROM POLYMORPHONUCLEAR LEUKOCYTES (PMN) STIMULATED BY PHORBOL MYRISTATE ACETATE (PMA)

The interaction of NO and O−2free radicals generated from PMA (phorbol myristate acetate)‐stimulated PMN (polymorphonuclear leukocytes) was studied by a nitroxide spin trap, DMPO (5,5‐dimethyl‐1‐pyrroline‐1‐oxide). It was found that addition of L‐arginine to the system would significantly decrease the trapped O−2by DMPO and addition of NG‐monomethyl‐arginine (NGMA) would significantly increase the trapped O−2by DMPO. It was proved that the formation of ONOO−by the reaction of NO and O−2was the main reason for the decrease of trapped O−2in the experiment with xanthine/xanthine oxidase and irradiation of riboflavin systems. The yield of NO during this process was calculated. The generation dynamic of NO was studied by a luminol‐dependent chemiluminescence technique and it was found that after stimulation of PMN by PMA, there would be an immediate, significant chemi‐luminescence, which came mainly from the active oxygen free radicals generated by PMN. If L‐arginine was added to this system, the chemiluminescence would increase about 100‐fold, but NGMA inhibited the increase of the chemiluminescence. Ten minutes after addition of L‐arginine, this increase did not change, the chemiluminescence peak decreased gradually, but the half life increased. The ESR and chemiluminescence properties of NO and ONOO−synthesized were also studied in model systems.

Antioxidant effects of green tea polyphenols

The unprecedented interest in the antioxidant activity of green tea polyphenols (GTP) is due to the apparent health benefits of tea drinking and the experimental results with the polyphenols. The results suggest that the galloylated catechins show stronger antioxidant effect than that of nongalloylated catechins and the double bond in C ring also plays an important role in this effect. There are synergic effects between different catechins and the antioxidant effect of (+)-epimers is stronger than that of (−)-epimers. The active site to scavenge free radicals mainly locates in p-hydroxyl of phenol in gallic acid and hydroxyl of phenol in the pyrane also shows activity. The large π bond in chromane and benzene ring of catechin is the structure base for their antioxidant effects. The prevent effect of GTP against diseases and their redox regulation in cell signal pathway are very important to be studied in the future.

EPC‐K1 attenuates peroxynitrite‐induced apoptosis in cerebellar granule cells

Apoptosis induced by peroxynitrite in cultured cerebellar granule cells was confirmed morphologically by chromatin condensation and biochemically by DNA laddering. A 30 min exposure to peroxynitrite (10 μM) initiated oxidative stress, which caused the formation of thiobarbituric acid‐reactive substances (TBARS) and the alteration of cell membrane fluidity. Peroxynitrite treatment also caused ATP decrease and thus activated the apoptotic program. Pre‐treating cells with antioxidant EPC‐K1 (L‐ascorbic acid 2‐[3,4‐dihydro‐2,5,7,8‐tetramethyl‐2‐(4,8,12‐trimethyltridecyl)‐2H‐1‐benzopyran‐6‐yl‐hydrogen phosphate] potassium salt), a new water‐soluble derivative of vitamin C and vitamin E, attenuated oxidative injury and prevents cells from apoptosis. The results suggest that EPC‐K1 might be used as a potential therapeutic agent for diseases associated with NO/ONOO‐‐mediated neuronal injury.

Scavenging Effects of Chinese Herbs and Natural Health Products On Active Oxygen Radicals

Although the clinical efficacy of some Chinese herbs and natural products have been well known for a long time, investigation of their biological effects and reaction mechanisms is just at the very beginning. Many problems concerning the active ingrediests and mechanisms of the action remain to be solved. Exploitation in this field will definitely enrich our knowledge in free radical biology and benefit our health.