Effects of a natural extract from Mangifera indica L,and its active compound,mangiferin, on energy state and lipid peroxidation of red blood cells

Following oxidative stress, modifications of several biologically important macromolecules have been demonstrated. In this study we investigated the effect of a natural extract from Mangifera indica L (Vimang), its main ingredient mangiferin and epigallocatechin gallate (EGCG) on energy metabolism, energy state and malondialdehyde (MDA) production in a red blood cell system. Analysis of MDA, high energy phosphates and ascorbate was carried out by high performance liquid chromatography (HPLC). Under the experimental conditions, concentrations of MDA and ATP catabolites were affected in a dose-dependent way by H₂O₂. Incubation with Vimang (0.1, 1, 10, 50 and 100 μg/mL), mangiferin (1, 10, 100 μg/mL) and EGCG (0.01, 0.1, 1, 10 μM) significantly enhances erythrocyte resistance to H₂O₂-induced reactive oxygen species production. In particular, we demonstrate the protective activity of these compounds on ATP, GTP and total nucleotides (NT) depletion after H₂O₂-induced damage and a reduction of NAD and ADP, which both increase because of the energy consumption following H₂O₂ addition. Energy charge potential, decreased in H₂O₂-treated erythrocytes, was also restored in a dose-dependent way by these substances. Their protective effects might be related to the strong free radical scavenging ability described for polyphenols.     

EGCG对H2O2诱导HLE细胞氧化损伤的保护作用

目的:探讨表没食子酸酯(EGCG)对体外培养的人晶状体上皮(human lens epithelial,HLE)细胞氧化损伤的保护作用及可能机制。方法:HLE细胞传代培养,分为阴性对照组:以正常培养液培养;氧化损伤组:100μmol·L~(-1)的H_2O_2作用12 h;EGCG低浓度组:10μmol·L~(-1)EGCG孵育24 h后,加入H_2O_2作用12 h;EGCG高浓度组:100μmol·L~(-1)EGCG孵育24 h后,加入H_2O_2作用12 h。MTT比色法检测细胞活力,流式细胞仪检测细胞凋亡率,Hochest33258染色观察凋亡细胞形态,比色法检测凋亡相关因子caspses-3及caspase-9的表达。结果:EGCG能明显抑制H_2O_2诱导的HLE细胞活力的下降,用不同浓度EGCG处理后,HLE细胞活性分别提高到51.00%±2.37%和63.67%±2.29%,与氧化损伤组(40.33%±2.86%)比较差异具有统计学意义(P0.05);经不同浓度EGCG处理后,HLE细胞凋亡率分别下降至33.33±3.12%和22.80±1.67%,与氧化损伤组(43.03±2.43%)比较差异具有统计学意义(P0.05);此外,EGCG还能明显减少H_2O_2所致HLE细胞内caspses-3及caspase-9的表达。结论:EGCG通过抑制caspses-3及caspase-9的表达有效抑制了H_2O_2对HLE细胞的损伤,从而为其用于治疗HLE细胞损伤提供可靠的实验依据。     

Epigallocatechin gallate preserves endothelial function by reducing the endogenous nitric oxide synthase inhibitor level

Asymmetric dimethylarginine (ADMA), the endogenous nitric oxide synthase inhibitor, is thought to be a key factor contributing to endothelial dysfunction. Tea catechins can cause an endothelium-dependent vasorelaxation. The present study examined the effect of epigallocatechin gallate (EGCG), the major component of tea catechins, on endothelial dysfunction induced by native low density lipoprotein (LDL) in rats and oxidized LDL (ox-LDL) in cultured endothelial cells, and whether the protective effect of EGCG is related to reduction of ADMA level. A single injection of LDL (4 mg x kg(-1), i.v.) markedly reduced endothelium-dependent relaxation and the serum nitrite/nitrate (NO) level, and increased serum concentrations of ADMA, malondialdehyde (MDA), and tumor necrosis factor-alpha (TNF-alpha). EGCG (10 or 50 mg x kg(-1), i.p.) significantly attenuated the inhibition of vasodilator response to acetylcholine and the decreased serum nitrite/nitrate level, and reduced the elevated levels of ADMA, MDA, and TNF-alpha. Exposure of endothelial cells to ox-LDL (100 microg x mL(-1)) for 24 h markedly increased the medium levels of lactate dehydrogenase (LDH), ADMA, TNF-alpha, and MDA, and decreased the level of nitrite/nitrate in the medium and the activity of dimethylarginine dimethylaminohydrolase (DDAH) in the endothelial cells. EGCG (10 and 100 microg x mL(-1)) significantly decreased the levels of LDH, ADMA, TNF-alpha, and MDA, and increased the level of nitrite/nitrate and the activity of DDAH. These results suggest that EGCG protects endothelial dysfunction induced by native LDL in vivo or by ox-LDL in endothelial cells, and the protective effect of EGCG on the endothelium is related to decrease in ADMA level via increasing of DDAH activity.     

Vimang (Mangifera indica L. extract) induces permeability transition in isolated mitochondria, closely reproducing the effect of mangiferin, Vimang's main component

Mitochondrial permeability transition (MPT) is a Ca(2+)-dependent, cyclosporin A (CsA)-sensitive, non-selective inner membrane permeabilization process. It is often associated with apoptotic cell death, and is induced by a wide range of agents or conditions, usually involving reactive oxygen species (ROS). In this study, we demonstrated that Mangifera indica L. extract (Vimang), in the presence of 20 microM Ca(2+), induces MPT in isolated rat liver mitochondria, assessed as CsA-sensitive mitochondrial swelling, closely reproducing the same effect of mangiferin, the main component of the extract, as well as MPT-linked processes like oxidation of membrane protein thiols, mitochondrial membrane potential dissipation and Ca(2+) release from organelles. The flavonoid catechin, the second main component of Vimang, also induces MPT, although to a lesser extent; the minor, but still representative Vimang extract components, gallic and benzoic acids, show respectively, low and high MPT inducing abilities. Nevertheless, following exposure to H(2)O(2)/horseradish peroxidase, the visible spectra of these compounds does not present the same changes previously reported for mangiferin. It is concluded that Vimang-induced MPT closely reproduces mangiferin effects, and proposed that this xanthone is the main agent responsible for the extract's MPT inducing ability, by the action on mitochondrial membrane protein thiols of products arising as a consequence of the mangiferin's antioxidant activity. While this effect would oppose the beneficial effect of Vimang's antioxidant activity, it could nevertheless benefit cells exposed to over-production of ROS as occurring in cancer cells, in which triggering of MPT-mediated apoptosis may represent an important defense mechanism to their host.     

Protection of PC12 cells from hydrogen peroxide-induced injury by EPS2, an exopolysaccharide from a marine filamentous fungus Keissleriella sp. YS4108

A number of studies indicate that free radicals are involved in the neurodegeneration in Parkinson's and Alzheimer's diseases. EPS2, an exopolysaccharide with a mean molecular weight of 1.3 x 10(5) Da, was isolated by ion-exchange and sizing chromatography from the culture of Keissleriella sp. YS4108, a marine filamentous fungus. Compositionally, it is composed of galactose, glucose, rhamnose, mannose and glucuronic acid in an approximate proportion of 50:8:1:1:0.4. The protective effects of EPS2 on peroxide hydrogen (H2O2)-induced cell lesion, level of lipid peroxidation, antioxidant enzyme activities were investigated in the rat pheochromocytoma line PC12 cells. Following a 1-h exposure of the cells to H2O2, a significant reduction in cell survival and activities of glutathione peroxidase (GSH-Px) and catalase (CAT), as well as increased levels in malondialdehyde (MDA) production and lactate dehydrogenase (LDH) release were observed. However, preincubation of the cells with EPS2 prior to H2O2 exposure elevated the cell survival and GSH-Px and CAT activities, and decreased the level of MDA and LDH activity in a dose-dependent manner. In conclusion, EPS2 possesses pronounced protective effects against H2O2-induced cell toxicity. The finding is of a higher value in searching for new therapeutic agent for treating oxidative damage-derived neurodegenerative disorders.     

Antioxidant properties and protective effects of a standardized extract of Hypericum perforatum on hydrogen peroxide-induced oxidative damage in PC12 cells

Free radical scavenging and antioxidant activities of a standardized extract of Hypericum perforatum (SHP) were examined for inhibition of lipid peroxidation, for hydroxyl radical scavenging activity and interaction with 1,1-diphenyl-2-picrylhydrazyl stable free radical (DPPH). Concentrations between 1 and 50 microg/ml of SHP effectively inhibited lipid peroxidation of rat brain cortex mitochondria induced by Fe2+/ascorbate or NADPH system. The results showed that SHP scavenged DPPH radical in a dose-dependent manner and also presented inhibitory effects on the activity of xanthine oxidase. In contrast, hydroxyl radical scavenging occurs at high doses. The protective effect of the standardized extract against H2O2-induced oxidative damage on the pheochromocytoma cell line PC 12 was investigated by measuring cell viability via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), lactate dehydrogenase (LDH) assays, caspase-3-enzyme activity and accumulation of reactive oxygen species [2',7'-dichlorofluorescin (DCF) assay]. Following 8-h cell exposure to H2O2 (300 microM), a marked reduction in cell survival was observed, which was significantly prevented by SHP (pre-incubated for 24 h) at 1-100 microg/ml. In a separate experiment, different concentrations of the standardized extract (0.1-100 microg/ml) also attenuated the increase in caspase-3 activity and suppressed the H2O2 -induced reactive oxygen species generation. Taken together, these results suggest that SHP shows relevant antioxidant activity both in vitro and in a cell system, by means of inhibiting free radical generation and lipid peroxidation.     

Modulation of tea and tea polyphenols on benzo(a)pyrene-induced DNA damage in Chang liver cells

The protective effects of three tea extracts (green tea, GTE; oolong tea, OTE; and black tea, BTE) and five tea polyphenols (epicatechin, EC; epicatechin gallate, ECG; epigallocatechin, EGC; epigallocatechin gallate, EGCG; and theaflavins, THFs) on benzo[a]pyrene (B[a]P)-induced DNA damage in Chang liver cells were evaluated using the comet assay. B[a]P-induced DNA damage in Chang liver cells was significantly (p < 0.05) inhibited by GTE and OTE at a concentration of 10 microg/ml and by BTE at 25 microg/ml. At a concentration of 100 microg/ml, the % tail DNA was reduced from 33% (B[a]P treated only) to 10, 9, 13%, by GTE, OTE and BTE, respectively. EC and ECG did not cause DNA damage in cells according to the results of the comet assay; however, EGC, EGCG and theaflavins caused DNA damage in cells at a concentration of 100 microM. The results indicated that EC and ECG had protective effects against B[a]P-induced DNA damage in cells at a concentration of 10-100 microM. Although EGC, EGCG and the theaflavins caused DNA damage at a high concentration, but they had protective effects against B[a]P-induced DNA damage in cells at a low concentration of 10-50 microM. The results also showed that the DNA damage in cells induced by EGC, EGCG, and the theaflavins was due to the generation of superoxide during incubation with cells at a higher concentration. Therefore, tea catechins and THFs play an important role in enabling tea extracts to inhibit DNA damage in Chang liver cells.     

Extracellular adenosine triphosphate protects oxidative stress-induced increase of p21(WAF1/Cip1) and p27(Kip1) expression in primary cultured renal proximal tubule cells: role of PI3K and Akt signaling

Oxidative stress, the result of cellular production of reactive oxygen species (ROS), has been implicated in causing many renal diseases. Adenosine triphosphate (ATP) is an important extracellular signal in the regulation of many intracellular processes in normal tubular cells as well as in the pathogenesis of cell injury. This study investigated the effect of ATP on H(2)O(2)-induced increase of cyclin kinase inhibitors (CKI) expression and its related signal molecules in primary cultured renal proximal tubule cells (PTCs). H(2)O(2) inhibited DNA synthesis in a concentration- (>50 microM) and time-dependent manner (>2 h), as determined by thymidine and BrdU incorporation, and by increase in the p21(WAF/Cip1) and p27(Kip1) expression levels. In contrast, ATP increased the level of thymidine, BrdU incorporation (>10(-5) M), and decreased the p21(WAF/Cip1) and p27(Kip1) expression levels, suggesting that ATP has a protective effect against H(2)O(2)-induced oxidative damage. Suramin, reactive blue 2 (RB-2), MRS 2159, and MRS 2179 did block the reversing effect of ATP. In addition, AMP-CPP or 2-methylthio-ATP blocked H(2)O(2)-induced inhibition of DNA synthesis, suggesting all these P2 purinoceptors may be potentially involved. ATP-induced stimulation of DNA synthesis was blocked by phosphatidylinositol 3-kinase (PI3K) and Akt inhibitors. These results suggest the involvement of P2 purinoceptors-mediated PI3K/Akt signal pathway in the protective effect of ATP against H(2)O(2)-induced oxidative damage. Indeed, pre-treatment with PI3K or Akt inhibitors did not protect H(2)O(2)-induced lipid peroxide (LPO) production and inhibition of thymidine incorporation. In conclusion, ATP, in part, blocked H(2)O(2)-induced increase of p21(WAF1/Cip1) and p27(Kip1) expression through PI3K and Akt signal pathway in renal PTCs.     

Heat shock protein 70, heat shock protein 32, and vascular endothelial growth factor production and their effects on lipopolysaccharide-induced apoptosis in porcine aortic endothelial cells

Lipopolysaccharide (LPS) is a highly proactive molecule that causes in vivo a systemic inflammatory response syndrome and activates in vitro the inflammatory pathway in different cellular types, including endothelial cells (EC). Because the proinflammatory status could lead to EC injury and apoptosis, the expression of proinflammatory genes must be finely regulated through the induction of protective genes. This study aimed at determining whether an LPS exposure is effective in inducing apoptosis in primary cultures of porcine aortic endothelial cells and in stimulating heat shock protein (Hsp)70 and Hsp32 production as well as vascular endothelial growth factor (VEGF) secretion. Cells between third and eighth passage were exposed to 10 microg/mL LPS for 1, 7, 15, and 24 hours (time-course experiments) or to 1, 10, and 100 microg/mL LPS for 7 and 15 hours (dose-response experiments). Apoptosis was not affected by 1 microg/mL LPS but significantly increased in a dose-dependent manner with the highest LPS doses. Furthermore, apoptosis rate increased only till 15 hours of LPS exposure. LPS stimulated VEGF secretion in a dose-dependent manner; its effect became significant after 7 hours and reached a plateau after 15 hours. Both Hsp70 and Hsp32 expressions were induced by LPS in a dose-dependent manner after 7 hours. Subsequent studies were addressed to evaluate the protective role of Hsp32, Hsp70, and VEGF. Hemin, an Hsp32 inducer (5, 20, 50 microM), and recombinant VEGF (100 and 200 ng/mL), were added to the culture 2 hours before LPS (10 microg/mL for 24 hours); to induce Hsp70 expression, cells were heat shocked (42 degrees C for 1 hour) 15 hours before LPS (10 microg/mL for 24 hours). Hemin exposure upregulated Hsp32 expression in a dose-dependent manner and protected cells against LPS-induced apoptosis. Heat shock (HS) stimulated Hsp70 expression but failed to reduce LPS-induced apoptosis; VEGF addition did not protect cells against LPS-induced apoptosis at any dose tested. Nevertheless, when treatments were associated, a reduction of LPS-induced apoptosis was always observed; the reduction was maximal when all the treatments (HS + Hemin + VEGF) were associated. In conclusion, this study demonstrates that LPS is effective in evoking "the heat shock response" with an increase of nonspecific protective molecules (namely Hsp70 and Hsp32) and of VEGF, a specific EC growth factor. The protective role of Hsp32 was also demonstrated. Further investigations are required to clarify the synergic effect of Hsp32, Hsp70, and VEGF, thus elucidating the possible interaction between these molecules.     

Characterization of reactive oxygen species induced effects on human spermatozoa movement and energy metabolism

Reactive oxygen species (ROS) inhibit sperm movement and have been implicated in male infertility. In this study, we determined the effects of specific ROS produced by activated leukocytes on human spermatozoa and investigated their metabolic site of action. We used chemiluminescence and electron paramagnetic resonance (EPR) to characterize the ROS generated by both blood and seminal leukocytes. We also determined the effects of these ROS on sperm energy metabolism using biochemical analyses and flow cytometry. Both blood and seminal leukocytes produced the same characteristic ROS which were determined to be hydrogen peroxide (H2O2) and superoxide radicals (O2*-). EPR using the spin trapping technique indicated that superoxide radical-dependent hydroxyl radicals (HO.) were also generated. ROS generated by PMA-stimulated blood leukocytes (2-5 x 10(6)/ml) caused inhibition of sperm movement in 2 h (p < .01). Using the hypoxanthine/ xanthine oxidase (0.5 U/ml) system to generate ROS, we determined that spermatozoa ATP levels, after ROS treatment, were reduced approximately eight-fold in 30 min (0.10 x 10(10) moles/10(6) sperm cells) compared to control (0.84 X 10(-10) moles/10(6) sperm cells) (p < .01). Sperm ATP reduction paralleled the inhibition of sperm forward progression. Neither superoxide dismutase (100 U/ml) nor dimethyl sulfoxide (100 mM) reversed these effects; however, protection was observed with catalase (4 X 10(3) U/ml). Flow cytometric analyses of sperm treated with various doses of H2O2 (0.3 mM-20.0 mM) showed a dose-dependent decrease in sperm mitochondrial membrane potential (MMP); however, at low concentrations of H2O2, sperm MMP was not significantly inhibited. Also, sperm MMP uncoupling with CCClP had no effect on either sperm ATP levels or forward progression. These results indicate that H2O2 is the toxic ROS produced by activated leukocytes causing the inhibition of both sperm movement and ATP production. O2*- and HO. do not play a significant role in these processes. Low concentrations of H2O2 causing complete inhibition of sperm movement and ATP levels inhibit sperm energy metabolism at a site independent of mitochondrial oxidative phosphorylation.     

A polysaccharide isolated from Cordyceps sinensis,a traditional Chinese medicine,protects PC12 cells against hydrogen peroxide-induced injury

Cordyceps sinensis, a well-known traditional Chinese medicine, possesses activities in anti-tumour, anti-oxidation and stimulating the immune system; however, the identity of active component(s) is not determined. By using anti-oxidation activity-guided fractionation, a polysaccharide of molecular weight approximately 210 kDa was isolated from cultured Cordyceps mycelia by ion-exchange and sizing chromatography. The isolated polysaccharide, having strong anti-oxidation activity, contains glucose, mannose and galactose in a ratio of 1 : 0.6 : 0.75. The pre-treatment of isolated polysaccharide on the cultured rat pheochromocytoma PC12 cells shows strong protective effect against hydrogen peroxide (H(2)O(2))-induced insult. Treatment of the cells with the isolated polysaccharide at 100 microg/ml prior to H(2)O(2) exposure significantly elevated the survival of PC12 cells in culture by over 60%. In parallel, the H(2)O(2)-induced production of malondialdehyde in cultured cells was markedly reduced by the polysaccharide treatment. Moreover, the pre-treatment of the isolated polysaccharide significantly attenuated the changes of glutathione peroxidase and superoxide dismutase activities in H(2)O(2)-treated cells in a dose-dependent manner. This is the first report in identifying a polysaccharide from Cordyceps, which protects against the free radical-induced neuronal cell toxicity.     

EGCG attenuates AMPA-induced intracellular calcium increase in hippocampal neurons

We have investigated the protective effect of (-)-epigallocatechin gallate (EGCG) on alpha-amino-3-hydroxy-5-methyl-4-isoxazolo propionate (AMPA)-induced toxicity in cultured rat hippocampal neurons. Treatment of 24 h AMPA (10 microM) reduced the neuronal viability in both survival neuron counting and MTT reduction assay compared with control, with increase in cellular concentrations of hydrogen peroxide and malondialdehyde. These responses to AMPA were significantly blocked by co-treatments with EGCG (10 microM), which effect was very similar to the protective ability of a known antioxidant catalase (2000 U/ml). AMPA (50 microM) elicited the increase in intracellular calcium concentration ([Ca(2+)]i) on which EGCG significantly attenuated both peak amplitude and sustained nature of that [Ca(2+)]i increase in a dose-dependent manner. These data suggest that EGCG has a neuroprotective effect against AMPA through inhibition of AMPA-induced [Ca(2+)]i increase and consequent attenuation of reactive oxygen species production and lipid peroxidation as an antioxidant and a radical scavenger.     

Protective effects of Paeonia lactiflora pall on hydrogen peroxide-induced apoptosis in PC12 cells

This study was conducted to examine the antioxidative and neuroprotective effects of Paeonia lactiflora pall (PLE). Total phenolic content of PLE was 89.65 mg of gallic acid equivalent per gram of PLE. IC(50) values for reducing power, hydrogen peroxide scavenging activity, and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity were 297.57, 3.33, and 32.74 microg, respectively. The protective effect of PLE against H(2)O(2)-induced oxidative damage to PC12 cells was investigated by an 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) reduction assay and lactate dehydrogenase (LDH) release assay. After 2 h of cell exposure to 0.5 mM H(2)O(2), a marked reduction in cell survival was observed. However, this reduction was significantly prevented by 10-100 microg/ml of PLE. H(2)O(2) also induced severe apoptosis of the PC12 cells, which was indicated by a flow cytometric analysis. Interestingly, the H(2)O(2)-stressed PC12 cells that had been incubated with PLE had greatly suppressed apoptosis. The results suggest that PLE could be a candidate for a new antioxidant against neuronal diseases.     

Effect of hydrogen peroxide on neuron sensitivity to acetylcholine

The dose-dependent effect of hydrogen peroxide on snail neuromembrane chemosensitivity was studied by means of standard voltage-clamp method. Short-term exposure (7 min) of neurons to H(2)O(2) (10(-11)-10(-4) M) caused dose-dependent depression of Acetylcholine (Ach)-induced ionic currents in the membrane. The H(2)O(2)-induced depression of Ach-sensitivity of membrane was more pronounced in K(+)-free solution than in normal physiological solution and it disappeared in cold medium (5 degrees C). The H(2)O(2) (10(-11)-10(-4) M) decreased membrane electrical conductivity and cell volume. The dose-dependent decrease in Ach-sensitivity of the snail neuromembrane by H(2)O(2) may be due to a decrease in the number of functionally active membrane receptors caused by a decrease in membrane active surface. H(2)O(2)-induced decrease in Ach-sensitivity has a metabolic but Na(+)-K(+) pump independent character, the nature of which is the subject for current investigation.     

Iptakalim protects PC12 cell against H2O2-induced oxidative injury via opening mitochondrial ATP-sensitive potassium channel

The final common pathway in the demise of dopaminergic neurons in Parkinson's disease may involve oxidative stress and excitotoxicity. In this study, we examined the neuroprotective effects of a novel ATP-sensitive potassium channel (K(ATP)) opener, iptakalim (IPT), against H(2)O(2)-induced cytotoxicity in rat dopaminergic PC12 cells. Pretreatment with IPT could attenuate increased extracellular glutamate levels and inhibit calcium influxing induced by H(2)O(2). Moreover, IPT regulated the expressions of bcl-2 and bax which were responsible for inhibiting apoptosis in PC12 cells. These protective effects of IPT were abolished by selective mitoK(ATP) channel blocker 5-hydroxydecanoate. Therefore, IPT can protect PC12 cells against H(2)O(2)-induced oxidative injury via activating mitoK(ATP) channel.     

A water extract of Curcuma longa L. (Zingiberaceae) rescues PC12 cell death caused by pyrogallol or hypoxia/reoxygenation and attenuates hydrogen peroxide induced injury in PC12 cells

A number of studies indicate that free radicals are involved in the neurodegeneration in Alzheimer's disease (AD). The role of superoxide anion (O2*-) in neuronal cell injury induced by reactive oxygen species (ROS) was examined in PC12 cells using pyrogallol (1,2,3-benzenetrior), a donor to release O2*-. Pyrogallol induced PC12 cell death at concentrations, which evidently increased intracellular O2*-, as assessed by O2*- sensitive fluorescent precursor hydroethidine (HEt). A water extract of Curcuma longa L. (Zingiberaceae) (CLE), having O2*- scavenging activity rescued PC12 cells from pyrogallol-induced cell death. Hypoxia/reoxygenation injury of PC12 cells was also blocked by CLE. The present study was also conducted to examine the effect of CLE on H2O2 -induced toxicity in rat pheochromocytoma line PC12 by measuring cell lesion, level of lipid peroxidation and antioxidant enzyme activities. Following a 30 min exposure of the cells to H2O2 (150 microM), a marked decrease in cell survival, activities of glutathione peroxidase and catalase as well as increased production of malondialdehyde (MDA) were found. Pretreatment of the cells with CLE (0.5-10 microg/ml) prior to H2O2 exposure significantly elevated the cell survival, antioxidant enzyme activities and decreased the level of MDA. The above-mentioned neuroprotective effects are also observed with tacrine (THA, 1 microM), suggesting that the neuroprotective effects of cholinesterase inhibitor might partly contribute to the clinical efficacy in AD treatment. Further understanding of the underlying mechanism of the protective effects of these radical scavengers reducing intracellular O2*- on neuronal cell death may lead to development of new therapeutic treatments for hypoxic/ischemic brain injury.     

Hepatoprotective and antioxidant effects of Hibiscus sabdariffa extract against carbon tetrachloride-induced hepatocyte damage in Cyprinus carpio

The present study aims to evaluate the hepatoprotective and antioxidant effects of Hibiscus sabdariffa extract on the carbon tetrachloride (CCl(4))-induced hepatocyte damage in fish and provide evidence as to whether it can be potentially used as a medicine for liver diseases in aquaculture. H. sabdariffa extract (100, 200, and 400?μg/mL) was added to the carp primary hepatocyte culture before (pre-treatment), after (post-treatment), and both before and after (pre- and post-treatment) the incubation of the hepatocytes with CCl(4). CCl(4) at 8?mM in the culture medium produced significantly elevated levels of lactate dehydrogenase (LDH), glutamate oxalate transaminase (GOT), glutamate pyruvate transaminase (GPT), and malondialdehyde (MDA) and significantly reduced levels of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px). Pre-treatment and pre- and post-treatment of the hepatocytes with H. sabdariffa extract significantly reduced the elevated levels of LDH, GOT, GPT, and MDA and increased the reduced activities of SOD and GSH-Px in a dose-dependent manner; post-treatment did not show any protective effect. The results suggest that H. sabdariffa extract can be potentially used for preventing rather than curing liver diseases in fish.     

Effects of epigallocatechin gallate and quercetin on oxidative damage to cellular DNA

Phenolic phytochemicals are thought to promote optimal health, partly via their antioxidant effects in protecting cellular components against free radicals. The aims of this study were to assess the free radical-scavenging activities of several common phenolic phytochemicals, and then, the effects of the most potent phenolic phytochemicals on oxidative damage to DNA in cultured cells. Epigallocatechin gallate (EGCG) scavenged the stable free radical, alpha,alpha-diphenyl-beta-picrylhydrazyl (DPPH), most effectively, while quercetin was about half as effective. Genistein, daidzein, hesperetin, and naringenin did not scavenge DPPH appreciably. Jurkat T-lymphocytes that were pre-incubated with relatively low concentrations of either EGCG or quercetin were less susceptible to DNA damage induced by either a reactive oxygen species or a reactive nitrogen species, as evaluated by the comet assay. More specifically, control cells had a comet score of only 17+/-5, indicating minimal DNA damage. Cells challenged with 25 microM hydrogen peroxide (H(2)O(2)) or 100 microM 3-morpholinosydnonimine (SIN-1, a peroxynitrite generator) had comet scores of 188+/-6 and 125+/-12, respectively, indicating extensive DNA damage. The H(2)O(2)-induced DNA damage was inhibited with 10 microM of either EGCG (comet score: 113+/-23) or quercetin (comet score: 82+/-7). Similarly, the SIN-1-mediated DNA damage was inhibited with 10 microM of either EGCG (comet score: 79+/-13) or quercetin (comet score: 72+/-17). In contrast, noticeable DNA damage was induced in Jurkat T-lymphocytes by incubating with 10-fold higher concentrations (i.e., 100 microM) of either EGCG (comet score: 56+/-17) or quercetin (comet score: 64+/-13) by themselves. Collectively, these data suggest that low concentrations of EGCG and quercetin scavenged free radicals, thereby inhibiting oxidative damage to cellular DNA. But, high concentrations of either EGCG or quercetin alone induced cellular DNA damage.     

胶原蛋白肽经由Nrf2信号通路对H_2O_2诱导的肝细胞氧化损伤的保护作用(英文)

目的:氧化应激在肝脏疾病中扮演着重要的角色。胶原蛋白肽是天然的抗氧化剂,其在动物实验中已经被证实有抑制氧化应激的作用。最新研究证实胶原蛋白肽将有可能被应用在肝脏疾病的预防中,但是很少有研究报道其分子作用机制。因此本研究在胶原蛋白肽是对H2O2诱导的正常人的肝细胞系HL7702氧化损伤有保护作用的基础上,并探索其分子作用机制。方法:实验设空白对照组,H2O2模型组,胶原蛋白肽低、中、高剂量组(10,100,200μg/ml)。胶原蛋白肽各组加入相应浓度的药物预处理12 h后,与模型组一起加入300μM H2O2的H2O2共同培养12 h,空白对照组正常培养。细胞毒性是由CCK8和乳酸脱氢酶(LDH)的释放检测。抗氧化试剂盒检测细胞内活性氧的水平,超氧化物歧化酶(SOD)、过氧化氢酶(CAT)活性和丙二醛(MDA)含量的变化。Western blot检测细胞内Nrf2蛋白的表达水平。结果:胶原蛋白肽对H2O2诱导的正常人的肝细胞系HL7702氧化损伤有保护作用。胶原蛋白肽能够及时清除细胞内的活性氧,增加Nrf2的蛋白表达水平,提高超氧化物歧化酶(SOD)、过氧化氢酶(CAT)的活性,减轻脂质过氧化反应,从而保护正常人的肝细胞系HL7702。结论:总之,胶原蛋白肽通过增加Nrf2的蛋白表达水平,提高抗氧化活性,对H2O2诱导损伤的肝细胞发挥保护作用。本研究为胶原蛋白肽的分子作用机制提供了新的证据,将有助于预防氧化应激所致的肝损伤。