芦荟多糖对D-半乳糖致HepG2细胞氧化损伤的保护作用

目的:探讨芦荟多糖对HepG2细胞氧化损伤的保护作用,分析其体外抗氧化能力。方法:以HepG2细胞为研究对象,建立D-半乳糖诱导细胞氧化损伤模型,以不同浓度芦荟多糖进行预保护处理。采用细胞增殖与毒性检测试剂盒测定HepG2细胞存活率,生物化学法检测细胞超氧化物歧化酶、过氧化氢酶、谷胱甘肽过氧化物酶活力,酶联免疫吸附法测定细胞总抗氧化能力及丙二醛（malondialdehyde,MDA）含量,实时荧光定量PCR检测细胞Keap1、Nrf2、NQO1和HO-1基因表达水平。结果:与D-半乳糖模型组比较,25、50、100 μg/mL的芦荟多糖预保护处理能不同程度地提高HepG2细胞存活率;芦荟多糖能显著增强细胞中抗氧化酶的活性,降低细胞MDA的生成量（P<0.05）,且作用效果与芦荟多糖浓度成正比;细胞中Keap1基因表达显著降低,Nrf2、NQO1、HO-1 mRNA表达显著提高（P<0.05）。结论:芦荟多糖可以减轻HepG2细胞的氧化应激损伤,激活Nrf2表达并抑制其泛素化降解,提高下游NQO1、HO-1的转录水平,增强细胞抗氧化酶系活性并调控细胞氧化还原系统,以达到减轻细胞氧化损伤程度、提高细胞抗氧化应激损伤的效果。     

Pro-oxidative effects of melanoidin–copper complexes on isolated and cellular DNA

High molecular weight Maillard reaction products (melanoidins) are described to possess metal-chelating properties. Whereas in food systems, this ability contributes to antioxidant properties, the consequences on biological systems are not quite clear. The study was aimed to evaluate the implication of metal complexation by melanoidins on DNA damage. Melanoidins prepared with d-glucose and different l-amino acids under water-free reaction conditions were charged with cupric ions. The effect on isolated DNA was investigated by the PM2 assay and on cellular systems in the human colon carcinoma cell line HCT-116 by alkaline unwinding. Independent of the amino acid composition, pure melanoidins (MW >14 kDa) did not cause significant DNA damage. By charging melanoidins with Cu²⁺ ions, a considerable DNA strand breaking activity was detectable, which was again amplified in an oxidative milieu (addition of hydrogen peroxide). Since Cu²⁺ normally does not provoke the formation of reactive oxygen species (ROS) via Fenton-type reaction, the results obtained have to be attributed to reducing properties of melanoidins. Thus, in melanoidin–copper complexes redox cycling may take place leading to Cu⁺ which subsequently undergoes Fenton-type and Haber–Weiss reactions. As a consequence, ROS are formed, which may explain the generation of DNA strand breaks.     

Intracellular ROS scavenging and antioxidant enzyme regulating capacities of corn gluten meal-derived antioxidant peptides in HepG2 cells

The objective of this study was to investigate the intracellular reactive oxygen species (ROS) scavenging activities and antioxidant enzyme regulating capacities of corn gluten peptide fractions (CPFs) in HepG2 cells. A cellular antioxidant activity (CAA) assay was used to assess their antioxidant activities and revealed that both CPF1 (molecular weight < 1 kDa) and CPF2 (molecular weight between 1 and 3 kDa) exhibited high cellular antioxidant activities with EC₅₀ values of 2.85 ± 0.19 mg/mL and 5.05 ± 0.32 mg/mL, respectively. Both CPFs also exhibited cytoprotective effects and intracellular ROS scavenging activities in HepG2 cells subjected to oxidative stress by oxidation with H₂O₂. In addition, at concentrations of 2.50 mg/mL, the CPFs increased the activity levels of superoxide dismutase (SOD), catalase (CAT), and glutathione reductase (GR), as well as the total glutathione (GSH) levels in oxidized HepG2 cells (from 86.54% to 114.14% (CPF1) or 109.72% (CPF2) for SOD activity; from 71.91% to 107.64% (CPF1) or 106.50% (CPF2) for CAT activity; from 70.52% to 103.01% (CPF1) or 104.10% (CPF2) for GR activity; and from 81.39% to 114.00% (CPF1) or 108.82% (CPF2) for total GSH levels). These results suggested that both CPF1 and CPF2 exhibited positive effects on the activities of the intracellular antioxidant enzymes SOD, CAT and GR, as well as on the total GSH levels in HepG2 cells under conditions of oxidative stress. Furthermore, size exclusion gel chromatography and MALDI-TOF/TOF mass spectrometry revealed that the molecular weights of the antioxidant peptides in CPF1 were between 500 Da to 900 Da, and a novel antioxidant peptide consisting of GLLLPH (Gly-Leu-Leu-Leu-Pro-His) was identified in CPF1.     

Salvianolic acid B inhibits low‐density lipoprotein oxidation and neointimal hyperplasia in endothelium‐denuded hypercholesterolaemic rabbits

BACKGROUND: Atherosclerosis and restenosis are inflammatory responses involving free radicals and lipid peroxidation and may be prevented/cured by antioxidant‐mediated lipid peroxidation inhibition. Salvianolic acid (Sal B), a water‐soluble antioxidant obtained from a Chinese medicinal herb, is believed to have multiple preventive and therapeutic effects against human vascular diseases. In this study the in vitro and in vivo inhibitory effects of Sal B on oxidative stress were determined. RESULTS: In human aortic endothelial cells (HAECs), Sal B reduced oxidative stress, inhibited low‐density lipoprotein (LDL) oxidation and reduced oxidised LDL‐induced cytotoxicity. Sal B inhibited Cu²⁺‐induced LDL oxidation in vitro (with a potency 16.3 times that of probucol) and attenuated HAEC‐mediated LDL oxidation as well as reactive oxygen species (ROS) production. In cholesterol‐fed New Zealand White rabbits (with probucol as positive control), Sal B intake reduced Cu²⁺‐induced LDL oxidation, lipid deposition in the thoracic aorta, intimal thickness of the aortic arch and thoracic aorta and neointimal formation in the abdominal aorta. CONCLUSION: The data obtained in this study suggest that Sal B protects HAECs from oxidative injury‐mediated cell death via inhibition of ROS production. The antioxidant activity of Sal B may help explain its efficacy in the treatment of vascular diseases. Copyright © 2010 Society of Chemical Industry     

Effect of enzyme treatment with β-glucosidase on antioxidant capacity of mulberry (Morus alba L.) leaf extract

This study was carried out to investigate the effect of enzyme treatment with β-glucosidase on antioxidant capacity of the mulberry leaf extract (MLE) using oxygen radical absorbance capacity (ORAC) and cellular antioxidant capacity (CAC) assay. The MLE was prepared by autoclaving at 121°C for 15 min and treated with β-glucosidase for 9 hr. High pressure liquid chromatography (HPLC) analysis showed that only qercetin-3-β-d-glucose (QT-G) among quercetin (QT) glycosides of MLE, including QT-G, quercetin-3-O-glucose-6″-acetate (QT-GA), and rutin (RT), was converted into QT by 3 hr treatment with β-glucosidase. The in vitro peroxyl radical- and hydroxyl radical scavenging capacity significantly increased after the enzyme treatment using β-glucosidase for 6 and 9 hr, respectively. The metal chelating activity increased after the enzyme treatment using β-glucosidase for 3 hr. The intracellular antioxidant capacity of MLE to protect AAPH- and Cu²⁺-induced oxidative stress in HepG2 cells significantly increased after the enzyme treatment using β-glucosidase for 3 and 6 hr, respectively, indicating that QT may be released from QT-G by β-glucosidase and penetrate into cell membrane so that it can contribute to the intracellular antioxidant capacity of MLE.     

Effects of metal ions on growth, β-oxidation system,and thioesterase activity of Lactococcus lactis

The effects of divalent metal ions (Ca²⁺, Mg²⁺, Fe²⁺, and Cu²⁺) on the growth, β-oxidation system, and thioesterase activity of Lactococcus lactis were investigated. Different metal ions significantly influenced the growth of L. lactis: Ca²⁺ and Fe²⁺ accelerated growth, whereas Cu²⁺ inhibited growth. Furthermore, Mg²⁺ inhibited growth of L. lactis at a low concentration but stimulated growth of L. lactis at a high concentration. The divalent metal ions had significant effects on activity of the 4 key enzymes of the β-oxidation system (acyl-CoA dehydrogenase, enoyl-CoA hydratase, L-3-hydroxyacyl-CoA dehydrogenase, and thiolase) and thioesterase of L. lactis. The activity of acyl-CoA dehydrogenases increased markedly in the presence of Ca²⁺ and Mg²⁺, whereas it decreased with 1 mmol/L Fe²⁺ or 12 mmol/L Mg²⁺. All the metal ions could induce activity of enoyl-CoA hydratase. In addition, 12 mmol/L Mg²⁺ significantly stimulated activity of L-3-hydroxyacyl-CoA dehydrogenase, and all metal ions could induce activity of thiolase, although thiolase activity decreased significantly when 0.05 mmol/L Cu²⁺ was added into M17 broth. Inhibition of thioesterase activity by all 4 metal ions could be reversed by 2 mmol/L Ca²⁺. These results help us understand the effect of metal ions on the β-oxidation system and thioesterase activity of Lactococcus lactis.     

黄酮对AAPH诱导的HepG2细胞氧化应激的作用

研究部分黄酮对AAPH诱导的HepG2细胞氧化应激的作用。建立AAPH诱导HepG2细胞氧化应激的模型,比较对照组和实验组细胞内的氧化自由基ROS的含量。结果表明,用不同浓度的黄酮处理HepG2细胞1h后,经过相同浓度的氧化剂作用后,细胞内自由基ROS的含量相对空白对照组均发生变化。这些选择性的黄酮对于AAPH诱导的HepG2细胞氧化应激具有促进或者抑制作用,其作用机制可能清除HepG2细胞内ROS以及与细胞内氧化酶和抗氧化酶系统的作用有关。     

Protective effect of whey protein hydrolysates on H2O2-induced PC12 cells oxidative stress via a mitochondria-mediated pathway

Whey protein hydrolysates (WPHs) were prepared with pepsin and trypsin. A PC12 cell model was built to observe the protective effect of WPHs against H₂O₂-induced oxidative stress. The results indicated that WPHs reduced apoptosis by 14% and increased antioxidant enzyme activities. Flow cytometry was used to assess the accumulation of reactive oxygen species (ROS), Ca²⁺ levels and the mitochondrial membrane potential (MMP). The results showed that WPHs suppressed ROS elevation and Ca²⁺ levels and stabilised MMP by 16%. The anti-apoptosis/pro-apoptosis proteins Bcl-2/Bax and poly (ADP-ribose) polymerase (PARP) were investigated by Western-blot analysis, which indicated that WPHs increased the expression of Bcl-2 while inhibiting the expression of Bax and the degradation of PARP. WPHs also blocked Caspase-3 activation by 62%. The results demonstrate that WPHs can significantly protect PC12 cells against oxidative stress via a mitochondria-mediated pathway. These findings indicate the potential benefits of WPHs as valuable food antioxidative additives.     

鱼鳔胶原肽抗氧化稳定性研究

以DPPH自由基、OH自由基清除能力和还原力为评价指标,探究pH、温度、食品配料、金属离子、防腐剂以及体外模拟胃、肠道消化对鱼鳔胶原肽抗氧化稳定性的影响。结果表明:鱼鳔胶原肽在酸性环境中抗氧化活性保持得较好,在碱性条件下丧失较快;鱼鳔胶原肽具有较强的耐热性,高温不会降低其抗氧化活性;蔗糖、葡萄糖以及防腐剂(苯甲酸钠和山梨酸钾)对鱼鳔胶原肽的活性影响不明显,但添加高浓度的食盐会降低鱼鳔胶原肽的抗氧化活性;金属离子Zn~(2+)、Cu~(2+)对鱼鳔胶原肽抗氧化活性的影响较大,Ca~(2+)的影响较小,K~+、Mg~(2+)无显著影响;模拟胃肠道消化对鱼鳔胶原肽抗氧化活性无显著影响。     

Caseinophosphopeptides exert partial and site-specific cytoprotection against H2O2-induced oxidative stress in Caco-2 cells

Caseinophosphopeptides can sequester prooxidant metals and scavenge free radicals, and may thus be used as functional food ingredients. The total antioxidant capacity (TEAC and ORAC) of two pools of caseinophosphopeptides (1–3 mg/ml), obtained from casein subjected to simulated gastrointestinal digestion (at two different pH values) and selective precipitation, was evaluated to determine dose–response activity. Pool B (which showed the highest antioxidant capacity due to the presence of more antioxidant amino acids) was used to test its cytoprotective effect against H₂O₂-induced oxidative stress in Caco-2 cells. Caseinophosphopeptides protected the cells against oxidative damage by preserving cell viability, increasing GSH content, inducing catalase enzyme activity, diminishing lipid peroxidation and maintaining a correct cell cycle progression. However, they failed to exert protection at a mitochondrial level (ROS and mitochondrial membrane potential), implying a partial and site-specific effect. Thus, their mechanism of action is not only related to free radical scavenging activity, but also to metal chelation and the modulation of intracellular signaling cascades.     

Rosmarinic acid attenuated SIN-1-induced cytotoxicity in HepG2 cells through the HO-1 induction and radical scavenging activity

Rosmarinic acid (RA), a food-derived polyphenolic compound, has been reported to possess anti-oxidant, antiallergic, and anti-inflammatory activities. In this study, we investigated whether RA has cytoprotective activity in SIN-1 induced HepG2 cells and how to achieve it. The results show that RA attenuates SIN-1 induced cytotoxicity in HepG2 cells. RA significantly reduces the intracellular reactive oxygen species (ROS) generated from SIN-1 and induced heme oxygenase-1 (HO-1) expression. RA-induced HO-1 expression was attenuated by a pretreatment with specific inhibitors for JNK, ERK, and phosphoinositide 3-kinase (PI3K). The addition of N-acetyl-cysteine (NAC) also reduced HO-1 induction in RA-induced cells. Furthermore, RA induced the translocation of NF-E2-related factor 2 (Nrf2). Taken together, we conclude that RA attenuates cytotoxicity in SIN-1-induced HepG2 cells by direct radical scavenging activity and HO-1 induction through a ROS/PI3K/MAPKs/Nrf2 pathway.     

Inhibitory effect of the ethyl acetate fraction from astringent persimmon on H2O2-induced oxidative stress in HepG2 cells

Persimmon cv. Sangjudungsi (Diospyros kaki Thunb.) is a major astringent cultivar in Korea. A phenolic extract of cv. Sangjudungsi was obtained using acetone with homogenization, then sequentially fractionated into n-hexane, chloroform, ethyl acetate, n-butanol, and water fractions. These 5 fractions were used to evaluate levels of total phenolics and total flavonoids, and the antioxidant capacities, and to investigate whether the fractions protected human hepatoma HepG2 cells from deleterious oxidative stress. The ethyl acetate fraction had the highest levels of total phenolics and total flavonoids, and the greatest antioxidant capacity. Under oxidative stress caused by H₂O₂, the ethyl acetate fraction at non-toxic concentrations significantly (p<0.05) restored the viability of HepG2 cells in a dose-dependent manner, compared with a control. The ethyl acetate fraction also alleviated intracellular oxidative stress, possibly due to effective antioxidant activities in cells. Astringent persimmons are a good source of antioxidants for reduction of oxidative stress.     

Ranking the efficacies of selected red wine phenolic anti-oxidants using reversed-phase HPLC

Several studies have assessed total anti-oxidant activity of wine or individual components in isolation using chemical-based assays. In this study, a quantitative approach was developed to assess the relative anti-oxidant efficacies of selected red wine phenolics via peak reduction, using reversed-phase HPLC. Both intact red wine and phenolic standard solutions were challenged with five oxidant model systems as follows: (1) hydrogen peroxide (H₂O₂); redox-active metal ions (2) Fe³⁺ and (3) Cu²⁺; and the Fenton reagents (4) H₂O₂ + Fe²⁺; and (5) H₂O₂ + Cu⁺. Treatment with oxidants (1–3) resulted in loss of 47–60% of phenolic standards, which increased to 66–89% for treatment with the Fenton systems, with quercetin exhibiting the optimal anti-oxidant activity. For intact red wine, treatment with oxidants (1–3) led to all phenolic compounds being oxidised (27–77% loss), with caffeic acid and quercetin as the most effective anti-oxidants. For both Fenton systems (4–5), activities in red wine were considerably enhanced for caffeic acid and quercetin, which exhibited the highest anti-oxidant efficacies with 100% peak reduction, while p-coumaric acid and gallic acid were less effective anti-oxidants with peak reductions of 60–68%. The ranking, facilitated by this new quantitative approach, allows comparison of the individual efficacies of the anti-oxidants in a complex matrix.     

Application of beech sawdust for removal of heavy metals from water: biosorption and desorption studies

In this work the efficiency of applying non-modified beech sawdust for the removal of Cu²⁺ and Cr³⁺ heavy metal ions from water solutions was examined. Parameters taken into consideration in the analysis of environment conditions were influence of sorbent concentration, initial concentration of metal ions, temperature effect, presence of additional substances in solution (NaCl and MgCl₂ inorganic salts and anionic and cationic surface-active compounds). Results of kinetic experiments were described by two models: pseudo-first-order and pseudo-second-order; equilibrium results were approximated with five non-linear isotherm models. Maximum sorption capacity at a temperature of 20 °C and pH 5.0 was 30.22 mg g^?1 for Cu²⁺ ions and 41.86 mg g^?1 for Cr³⁺ ions. The positive value of the thermodynamic parameter ΔH° indicates the endothermic nature of the process. Application of 0.1 M HCl as the eluent enables effective desorption of precious metals and reuse of sorbent for purifying water solutions of Cu²⁺ and Cr³⁺ ions.     

Modulation of the antioxidant/pro-oxidant balance,cytotoxicity and antiviral actions of grape seed extracts

Grape seed extracts (GSEs) were investigated in yeast cells harbouring defects in their antioxidant system (regarding the cellular growth and growth recovery from H₂O₂ insult). GSEs antioxidant activity was detected in wild-type and mutant strains Δcta1, Δgsh1 and Δoye2glr1, while pro-oxidant activity in Δsod1 cells was seen. Assessment of proliferation of prostate cancer PC3 and HBV-replicating HepG2 2.2.15 cells treated with GSEs has shown higher cytotoxicity of red grape seed extract (RW) than white grape seed extract (WW) subjective to dose and period of administration. No antiviral effect was detected by measuring the secreted virion particles in HepG2 2.2.15 cells treated with GSEs. The GSEs play a dual antioxidant/pro-oxidant role in vivo according with the cellular antioxidant system deficiencies and exhibit cytotoxic properties in PC3 and HepG2 2.2.15 cell lines, but no antiviral action against HBV.     

Neferine induces reactive oxygen species mediated intrinsic pathway of apoptosis in HepG2 cells

Evidence has accumulated concerning the medicinal application of Nelumbo nucifera in the treatment of various diseases. Neferine, an alkaloid from N. nucifera was found to exert cytotoxicity on liver cancer cells HepG2 in a dose-dependent manner. We evaluated its anticancer potential by studying its effect on mitochondrial membrane potential, intracellular calcium levels [Ca²⁺]_i, cell membrane integrity, apoptotic body formation and DNA fragmentation in cultured HepG2 cells. The reactive oxygen species level has been increased upon neferine treatment with concomitant decrease in reduced glutathione. Our data further indicate reduction of ΔψM and increased [Ca²⁺]_i during apoptosis induction by neferine with increased expression of apoptotic proteins such as Bax, Bad, cleaved forms of caspase 3, caspase 9 and PARP, with the downregulation of anti-apoptotic protein Bcl2 in HepG2 cells. Moreover, the expressions of tumour suppressor proteins p53 and PTEN were upregulated along with the downregulation of P-Akt. In addition, expression levels of TNF-α, p38 and ERK1/2 MAP kinases were increased upon neferine treatment. These results imply that mitochondrial-mediated ROS generation induced by neferine leads to caspase-dependent apoptosis in HepG2 cells.     

Chemical characterization and chemo-protective activity of cranberry phenolic powders in a model cell culture. Response of the antioxidant defenses and regulation of signaling pathways

Oxidative stress and reactive oxygen species (ROS)-mediated cell damage are implicated in various chronic pathologies. Emerging studies show that polyphenols may act by increasing endogenous antioxidant defense potential. Cranberry has one of the highest polyphenol content among commonly consumed fruits. In this study, the hepato-protective activity of a cranberry juice (CJ) and cranberry extract (CE) powders against oxidative stress was screened using HepG2 cells, looking at ROS production, intracellular non-enzymatic and enzymatic antioxidant defenses by reduced glutathione concentration (GSH), glutathione peroxidase (GPx) and glutathione reductase (GR) activity and lipid peroxidation biomarker malondialdehyde (MDA). Involvement of major protein kinase signaling pathways was also evaluated. Both powders in basal conditions did not affect cell viability but decreased ROS production and increased GPx activity, conditions that may place the cells in favorable conditions against oxidative stress. Powder pre-treatment of HepG2 cells for 20 h significantly reduced cell damage induced by 400 μM tert-butylhydroperoxide (t-BOOH) for 2 h. Both powders (5–50 μg/ml) reduced t-BOOH-induced increase of MDA by 20% (CJ) and 25% (CE), and significantly reduced over-activated GPx and GR. CE, with a significantly higher amount of polyphenols than CJ, prevented a reduction in GSH and significantly reduced ROS production. CJ reversed the t-BOOH-induced increase in phospho-c-Jun N-terminal kinase. This study demonstrates that cranberry polyphenols may help protect liver cells against oxidative insult by modulating GSH concentration, ROS and MDA generation, antioxidant enzyme activity and cell signaling pathways.