Wenjuan Xin

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.

Effects of EGb 761 on nitric oxide and oxygen free radicals, myocardial damage and arrhythmia in ischemia-reperfusion injury in vivo.

The cardioprotective effects of EGb 761 on the release of nitric oxide (NO), the concentration of serum thiobarbituric acid reaction substance (TBARS), the activity of creatine kinase (CK) and the incidence of ventricular arrhythmias were investigated in myocardial ischemia-reperfusion injury in vivo. Using sodium nitrite (NaNO2) as standard source of nitric oxide (NO), we compared the correlation coefficients of the three measuring methods used currently in the determination of NOFe2+(DETC)2 complex with that of the measuring method suggested in this study. The result showed that measuring the whole height of three splitting signals is the best linear correlation to the concentration of NO comparing with other methods in this system. Using this method, we observed the effects of EGb 761 on NOFe2+(DETC)2 complex in myocardial ischemia-reperfusion injury in vivo. The hearts of the Wistar rats were subjected to 30 min of ischemia and 10 min of reperfusion in vivo. Different doses of EGb 761 (25, 50, 100, 200 mg/kg i.p.), superoxide dismutase (SOD, 10(4) U/kg), l-arginine (50 mg/kg i.p.) and nitric oxide synthase (NOS) inhibitor NG-nitro-l-arginine (NNA, 50 mg/kg i.p.) were administered to the ischemia-reperfusion rats. EGb 761 under the dose of 100 mg/kg increased the signal intensity of NOFe2+(DETC)2 complex, while EGb 761 at 200 mg/kg showed an effect of decreasing the signal intensity of NOFe2+(DETC)2 complex. EGb 761 inhibited the formation of TBARS, the release of CK, and mitigated the incidence of ventricular arrhythmias in a dose dependent way. Both l-arginine and SOD increased the signal intensity of NOFe2+(DETC)2 complex and inhibited the formation of TBARS, the leakage of CK and the incidence of ventricular arrhythmia. NNA not only had no protective effects on myocardial injury, but also increased the incidence of reperfusion-induced arrhythmia. In conclusion, EGb 761 has cardiovascular protective effects by means of adjusting the level of NO and inhibiting oxygen free radicals induced lipid peroxidation in myocardial ischemia-reperfusion injury in vivo.

Tetramethylpyrazine scavenges superoxide anion and decreases nitric oxide production in human polymorphonuclear leukocytes

Tetramethylpyrazine is one of the active ingredients of the Chinese herb Ligusticum wallichii Franchat. By electron spin resonance spin trapping methods, effects of tetramethylpyrazine on superoxide anion and nitric oxide generated by human polymorphonuclear leukocytes were studied. During the respiratory burst of polymorphonuclear leukocytes induced by N-formylmethionyl-leucyl-phenylalanine, tetramethylpyrazine scavenges superoxide anion dose-dependently, and decreases the production of nitric oxide significantly, but shows no influence on oxygen consumption. These results suggest that the effective protection of tetramethylpyrazine against ischemic brain injury might be due to its scavenging of reactive oxygen species and regulation on nitric oxide production, and consequent prevention of peroxynitrite formation.

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.

Mechanisms of apoptosis in rat cerebellar granule cells induced by hydroxyl radicals and the effects of EGb761 and its constituents

In this study investigation is made on whether oxidative stress produced by treatment with hydroxyl radicals can induce apoptosis in rat cerebellar granule cells. The protective effects of Ginkgo biloba extract (EGb761) and its active constituents against apoptosis are also examined. The results show that hydroxyl radicals generated by the Fenton reaction induced apoptosis in cerebellar granule cells, which was associated with the decrease in the Bcl-2 mRNA level and the increase in the protein levels of the transcription factors Fos and Jun. Moreover, hydroxyl radicals induced time-dependent lipid peroxidation in cells and caused the changes in the sulfhydryl group binding sites on the membrane proteins. Hydroxyl radicals may induce apoptosis via different signaling pathways. EGb761 attenuated these changes and its different constituents showed different effects. The total flavonoid component of EGb761 and a mixture of flavonoids and terpenes protected cerebellar granule cells from oxidative damage and apoptosis induced by hydroxyl radicals. Total terpenes of EGb761 did not protect against apoptosis. Flavonoids and terpenes did not show a synergistic effect in this regard.

Effect of carotenoids on the respiratory burst of rat peritoneal macrophages.

The effect of four carotenoids (beta-carotene, lutein, bixin and canthaxanthin) on the respiratory burst of rat peritoneal macrophages was investigated. The results obtained showed that carotenoids suppressed the luminol-dependent chemiluminescence generated from PMA-stimulated macrophages at the beginning and after 2 min of the stimulation. Canthaxanthin and bixin had higher suppressive activity than beta-carotene and lutein. The changes in absorption spectra of carotenoids showed that the absorption by carotenoids was diminished during the stimulation of macrophages by PMA and their absorption peaks were either further diminished or blue-shifted after addition of L-arginine to the system, indicating that the carotenoids were consumed and converted to new compounds during the two processes. By using cell-free systems, it was found that carotenoids could scavenge superoxide anion generated by xanthine/xanthine oxidase system. Their ability to scavenge superoxide anion decreased in the order of canthaxanthin > bixin > lutein > beta-carotene. Canthaxanthin also showed the scavenging effect on superoxide anion generated from irradiation of riboflavin. The hydroxyl radical scavenging activity of carotenoids was investigated in the reaction system of Fe2+ and H2O2. There was little difference among their activities. The reaction between carotenoids and nitric oxide led to the decreasing absorption between 400 and 540 nm and the concomitant appearance of the new absorption peaks between 330 and 395 nm. Bleaching of beta-carotene, bixin and canthaxanthin by peroxynitrite resulted in the increasing absorption between 290 and 365 nm and the diminishing absorption between 400 and 500 nm. But the increasing absorption between 280 and 490 nm was observed in bleaching of lutein by peroxynitrite. Carotenoids inhibited thiobarbituric acid-reactive substance (TBARS) formation in AAPH-induced lipid peroxidation of PC liposomes in air. The results suggest that the suppressive effect of carotenoids on the respiratory burst of macrophages may be just a way by which carotenoids in vivo protect host cells and tissues from harmful effects of oxygen metabolites overproduced by macrophages and enhance the generation of specific immune responses.

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 ESeroS-GS inhibits NO production and prevents oxidative stress in astrocytes.

Within the central nervous system uncontrolled production of large amounts of nitric oxide (NO) by activated glial cells might be the common pathogenesis of several neurodegenerative disorders, including Alzheimers disease and Parkinsons disease. In the present investigation, we measured the effect of a novel antioxidant gamma-L-glutamyl-S-[2-[[[3,4-dihydro-2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridecyl)-2H-1-benzopyran-6-yl]oxy]carbonyl]-3-[[2-(1H-indol-3-yl)ethyl]amino]-3-oxopropyl]-L-cysteinyl-glycine sodium salt (ESeroS-GS) on NO production in cultured rat astrocytes. Upon stimulation with 1 microg/mL lipopolysaccharide plus 100 U/mL interferon-gamma which induced the expression of inducible nitric oxide synthase, cultured astrocytes generated large amounts of NO as measured by nitrite assay and ESR technique. The endogenous NO caused oxidative damage in astrocytes, which was confirmed by the accumulation of both cytosolic and extracellular peroxides, the decrease in the cellular glutathione level, and the formation of thiobarbituric acid reactive substrates. Production of endogenous NO resulted in cell death finally. Pretreatment with the novel antioxidant ESeroS-GS effectively decreased the expression of iNOS gene, inhibited the formation of endogenous NO, and prevented NO-induced oxidative damage and cell death in astrocytes. The results suggest that ESeroS-GS might be used as a potential agent for the prevention and therapy of diseases associated with the overproduction of NO by activated astrocytes.

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.

ANTIOXIDANT PROPERTIES OF EPC-K1 : A STUDY ON MECHANISMS

Scavenging effects of L-ascorbic acid 2-[3,4-dihydro-2,5,7,8- tetramethyl-2-(4,8,12-trimethytridecyl)-2H-1-benzopyran- 6-yl-hydrogen phosphate] potassium salt (EPC-K1) on hydroxyl radicals, alkyl radicals and lipid radicals were studied with ESR spin trapping techniques. The inhibition effects of EPC-K1 on lipid peroxidation were assessed by TBA assay. The kinetics of EPC-K1 reacting with hydroxyl radicals and linoleic acid radicals were studied by pulse radiolysis. The active site of EPC-K1 and the structure-antioxidative activity relationships were discussed. The superoxide radicals scavenging capacity of the brain homogenate of EPC-K1-treated rats was measured. The results revealed that in comparison with Trolox and vitamin C, EPC-K1 showed better overall antioxidative capacity in vitro and in vivo. EPC-K1 was a moderate scavenger on hydroxyl radicals and alkyl radicals, a potent scavenger on lipid radicals, and an effective inhibitor on lipid peroxidation. EPC-K1 could react with hydroxyl radicals with a rate constant of 7.1 x 10(8) dm3 mol-1 s-1 and react with linoleic acid radicals with a rate constant of 2.8 x 10(6) dm3 mol-1 s-1. The active site of EPC-K1 was the enolic hydroxyl group. After administration of EPC-K1, the ability of rat brain to scavenge superoxide radicals was significantly increased. The potent scavenging effects of EPC-K1 on both hydrophilic and hydrophobic radicals were relevant with its molecular structure, which consisted of both hydrophilic and hydrophobic groups.