Oxidative Damage In HepG2 Cells Research Articles

Protective Effect of 3,5-Dicaffeoylquinic Acid Isolated from Ligularia fischeri against Oxidative Damage in HepG2 Cells

Protective Effect of 3,5-Dicaffeoylquinic Acid Isolated from Ligularia fischeri against Oxidative Damage in HepG2 Cells

Isotetrandrine ameliorates tert-butyl hydroperoxide-induced oxidative stress through upregulation of heme oxygenase-1 expression.

1R, 1'S-isotetrandrine, a naturally occurring plant alkaloid found in Mahonia of Berberidaceae, possesses anti-inflammatory, antibacterial, and antiviral properties, but the antioxidative activity and mechanism action remain unclear. In this study, we demonstrated the antioxidative effect and mechanism of 1R, 1'S-isotetrandrine against tert-butyl hydroperoxide-induced oxidative damage in HepG2 cells. We found that 1R, 1'S-isotetrandrine suppressed cytotoxicity, reactive oxygen species generation, and glutathione depletion. Additionally, our study confirmed that 1R, 1'S-isotetrandrine significantly increased the antioxidant enzyme heme oxygenase-1 expression and nuclear translocation of factor-erythroid 2 p45-related factor 2 (Nrf2). Specifically, the nuclear translocation of Nrf2 induced by 1R, 1'S-isotetrandrine was associated with Nrf2 negative regulatory protein Keap1 inactivation and phosphorylation of both extracellular signal-regulated protein kinase and c-Jun NH2-terminal kinase. Preincubation with thiol-reducing agents reduced 1R, 1'S-isotetrandrine-induced heme oxygenase-1 expression, and treatment with either extracellular signal-regulated protein kinase or c-Jun NH2-terminal kinase inhibitors attenuated the levels of 1R, 1'S-isotetrandrine-induced Nrf2 activation and heme oxygenase-1 expression. Furthermore, the cytoprotective effect of 1R, 1'S-isotetrandrine was abolished by heme oxygenase-1, extracellular signal-regulated protein kinase, and c-Jun NH2-terminal kinase inhibitors. These results indicated that the 1R, 1'S-isotetrandrine ameliorated tert-butyl hydroperoxide-induced oxidative damage through upregulation of heme oxygenase-1 expression by the dissociation of Nrf2 from Nrf2-Keap1 complex via extracellular signal-regulated protein kinase and c-Jun NH2-terminal kinase activation and Keap1 inactivation.

Nanoceria Attenuated High Glucose-Induced Oxidative Damage in HepG2 Cells.

Hyperglycemia, a common metabolic disorder in diabetes, can lead to oxidative damage. The use of antioxidants can benefit the control and prevention of diabetes side effects. This study aims to evaluate the effect of nanoceria particles, as an antioxidant, on glucose induced cytotoxicity, reactive oxygen species (ROS), lipid peroxidation (LPO) and glutathione (GSH) content in a human hepatocellular liver carcinoma cell line (HepG2) cell line. In this experimental study, we divided HepG2 cells into these groups: i. Cells treated with 5 mM D-glucose (control), ii. Cells treated with 45 mM D- mannitol+5 mM D-glucose (osmotic control), iii. Cells treated with 50 mM D-glucose (high glucose), and iv. Cells treated with 50 mM D-glucose+nanoceria. Cell viability, ROS formation, LPO and GSH were measured and analyzed statistically. High glucose (50 mM) treatment caused significant cell death and increased oxidative stress markers in HepG2 cells. Interestingly, nanoceria at a concentration of 50 mM significantly decreased the high glucose-induced cytotoxicity, ROS formation and LPO. This concentration of nanoceria increased the GSH content in HepG2 cells (P<0.05). The antioxidant feature of nanoceria particles makes it an attractive candidate for attenuation of hyperglycemia oxidative damage in different organs.

Charge properties of peptides derived from casein affect their bioavailability and cytoprotection against H2O2-induced oxidative stress

The effects of charge properties of casein peptides on absorption stability, antioxidant activity, and cytoprotection were evaluated. Alcalase hydrolysates of casein were separated into 4 fractions by cation-exchange chromatography according to charge properties. After simulated digestion and Caco-2 cell transmembrane transport, we determined the total antioxidant capacity (Trolox equivalent antioxidative capacity and oxygen radical antioxidant activity) and nitrogen content of peptide fractions to estimate available antioxidant efficacy and bioavailability (BA) of peptides. Results showed that negatively charged peptide fractions had greater BA and antioxidant activities after digestion and absorption. The peptide permeates were used to test the cytoprotective effect against H2O2-induced oxidative damage in HepG-2 cells. All peptide permeates increased cell viability, elevated catalase activity, and decreased superoxide dismutase activity. However, negatively charged peptide fractions preserved cell viability to a greater degree. Therefore, the negatively charged peptides from casein may be potential antioxidants and could be used as ingredients in functional foods and dietary supplements.

Protective effects of the extracts of Barringtonia racemosa shoots against oxidative damage in HepG2 cells.

Barringtonia racemosa is a tropical plant with medicinal values. In this study, the ability of the water extracts of the leaf (BLE) and stem (BSE) from the shoots to protect HepG2 cells against oxidative damage was studied. Five major polyphenolic compounds consisting of gallic acid, ellagic acid, protocatechuic acid, quercetin and kaempferol were identified using HPLC-DAD and ESI-MS. Cell viability assay revealed that BLE and BSE were non-cytotoxic (cell viabilities >80%) at concentration less than 250 µg/ml and 500 µg/ml, respectively. BLE and BSE improved cellular antioxidant status measured by FRAP assay and protected HepG2 cells against H2O2-induced cytotoxicity. The extracts also inhibited lipid peroxidation in HepG2 cells as well as the production of reactive oxygen species. BLE and BSE could also suppress the activities of superoxide dismutase and catalase during oxidative stress. The shoots of B. racemosa can be an alternative bioactive ingredient in the prevention of oxidative damage.

Protective Mechanisms of Unsaponifiable Matter from Rice Bran Against Tert -Butyl Hydroperoxide-Induced Oxidative Damage in HepG2 Cells

Rice bran is rich source of phytochemicals, such as phytosterols, tocopherols, tocotrienols, policosanols, γ-oryzanol, triterpene alcohol and other minor compounds. In this study, we investigated the mechanisms underlying the protective effects of unsaponifiable matter from rice bran on tert-butyl hydroperoxide (TBHP)-induced oxidative stress in HepG2 cells. HepG2 cells were pretreated with different concentrations of USM (0–100 µg/mL) for 6 h and oxidative stress was induced by TBHP. The result indicated that pretreatment of HepG2 cells with USM significantly reduced TBHP-induced oxidative damage, as determined by cell cytotoxicity and intracellular reactive oxygen species generations. USM activated nuclear factor-erythroid 2-related factor 2 (Nrf2) nuclear translocation and subsequently enhanced the expression of phase II detoxifying enzymes including heme oxygenase-1, glutamate-cysteine ligase catalytic subunit, and NADPH: quinine oxidoreductase-1. These results suggest that USM-mediated up-regulation of phase II detoxifying enzymes via Nrf2-signaling pathways may provide a pivotal mechanism for its hepatoprotective action. Practical Applications Rice bran is a major byproduct of the rice milling process and is produced every year in an enormous amount in Korea. Rice bran is mainly used as animal feed, cooking oil, and cosmetics. It contains important bioactive compounds. This study provides biological evidence that supports the use of rice bran unsaponifiable matter (USM) in the prevention of oxidative damage. The assessment of the protective mechanism of rice bran USM against oxidative damage may give insight into its application as bioactive ingredients in the production and development of functional foods.

Protective effect of polypeptides from larva of housefly (Musca domestica) on hydrogen peroxide-induced oxidative damage in HepG2 cells

Housefly (Musca domestica) is an important medical insect and its larva is an ideal high protein food source. We isolated from housefly larvae the polypeptides hydrolyzed by neutral protease (PHNP), and investigated the protective effect of PHNP on hydrogen peroxide (H₂O₂)-induced oxidative damage in HepG2 cells. Cells exposed to H₂O₂ showed a marked decrease in proliferation and intracellular superoxide dismutase (SOD) activity, and a significant increase in reactive oxygen species (ROS) level and malondialdehyde (MDA) content. H₂O₂ also caused apoptosis and mitochondrial dysfunction including mitochondrial fragmentation and the loss of mitochondrial membrane potential. Pretreatment with PHNP at concentrations of 2.5, 5, 10 μg/mL blocked these H₂O₂-induced cellular events in a dose-dependent manner. The effect of PHNP at 10 μg/mL is equal to that of ascorbic acid at 10 μM. In summary, PHNP has a protective effect against H₂O₂-induced oxidative injury in cells due to its ability to decrease intracellular ROS and elevate antioxidant enzyme activities.

Protective effect of 70% ethanolic extract of Lindera obtusiloba Blume on tert-butyl hydroperoxide-induced oxidative hepatotoxicity in rats

Lindera obtusiloba Blume, a native plant of East Asia, has traditionally been used as a folk medicine for liver disease. We studied the in vitro antioxidant and in vivo hepatoprotective activities of a 70% ethanolic extract of L. obtusiloba (LOE) containing 62.9% quercitrin and 22.0% afzelin. LOE prevented tert-butyl hydroperoxide (t-BHP)-induced oxidative damage in HepG2 cells. Along with its high antioxidant potency in vitro, our animal study confirmed that pretreatment with LOE (500 or 2000mg/kg) for 7days prior to a single dose of t-BHP (i.p.: 0.5mmol/kg) significantly lowered the serum levels of alanine and aspartate aminotransferases. In addition, glutathione levels were increased in the liver, and lipid peroxidation levels were decreased in a dose-dependent manner. The histopathological examinations of rat livers showed that LOE significantly reduced the incidence of liver lesions induced by t-BHP. Therefore, we concluded that LOE has merit as a potent candidate to protect the liver against oxidative damage.

Hepatoprotective and cytoprotective properties of Hyptis suaveolens against oxidative stress–induced damage by CCl4 and H2O2

ObjectiveTo investigate capacity of Hyptis suaveolens (H. suaveolens) methanol extract as an antioxidant to protect against carbon tetrachloride (CCl4)-induced oxidative stress, hepatotoxicity in Albino Wistar rats and cytoprotective effect of hydrogen peroxide (H2O2) induced cell death in HepG2 cell line. MethodsTwo different doses of methanol extract of H. suaveolens were evaluated for the hepatoprotective activity against carbon tetrachloride (CCl4) induced hepatotoxicity in rats. Animals in Group I: served as control, group II: H. suaveolens (100 mL/kg b.w), group III: H. suaveolens (50 mL/kg b.w) + CCl4 (1 mg/kg), group IV: H. suaveolens (100 mL/kg b.w) + CCl4 (1 mL/kg) and group V: CCl4 (1 mL/kg). Histopathologic changes of liver were also evaluated. Cytotoxicity was also determined by 3, (4,5-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide (MTT) assay. ResultsOral sigle dose treatment of CCl4 produced a marked elevation in the serum levels of aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP) and Lactate dehydrogenase (LDH). Histopathological analysis of the liver of CCl4-induced rats revealed marked liver cell necrosis with inflammatory collections that were conformed to increase in the levels of SOD, GSH, GST, GR and LPO. Treatment with H2O2 significantly induced death of HepG2 cell. Pretreatment with H. suaveolens methanol extract inhibited or attenuated H2O2 induced cytotoxicity. ConclusionsThis study shows that H. suaveolens methanol extract can be proposed to protect the liver against CCl4-induced oxidative damage in rats and protect the cells against H2O2-induced oxidative damage in HepG2 cells. The hepatoprotective and cytoprotective effects might be correlated with its antioxidant and free radical scavenger effects.

Protective effects of oligomeric and polymeric procyanidin fractions from defatted grape seeds on tert-butyl hydroperoxide-induced oxidative damage in HepG2 cells

Oligomeric and polymeric procyanidins have been reported to possess different antioxidant capacities. However, the intracellular antioxidant mechanisms of oligomeric and polymeric procyanidins are still poorly understood. In this study, we evaluated the cytoprotective effects of the oligomeric procyanidin fraction (OPF) and the polymeric procyanidin fraction (PPF) from grape seeds against the oxidative damage induced by tert-butyl hydroperoxide (TBHP) in HepG2 cells. The levels of cellular reactive oxygen species (ROS), cellular lipid peroxidation, glutathione (GSH), and antioxidant enzyme activities were measured as biomarkers of cellular oxidative status. HepG2 cells were treated with different concentrations of procyanidin samples (0–20μg/ml) for 6h prior to treatment with TBHP for 3h. The incubation of HepG2 cells with TBHP led to an approximately 60% decrease in cell viability. However, pretreatment of the cells with the samples, at 5–20μg/ml, rescued cell viability in a dose-dependent manner. Cellular generation of ROS, formation of malondialdehyde (MDA), and depletion of GSH were reduced by OPF and PPF. Moreover, TBHP treatment increased the activities of glutathione peroxidase (GPx), catalase (CAT), and superoxide dismutase (SOD). However, a 6h pretreatment with each of the samples at 20μg/ml significantly decreased the activities of these enzymes. These results clearly showed that treatment with OPF and PPF protected against oxidative damage by modulating ROS production, GSH levels, MDA generation, and antioxidant enzyme activities in HepG2 cells.

Anticancer effect of tert-butyl-2(4,5-dihydrogen-4,4,5,5-tetramethyl-3-O-1H-imidazole-3-cationic-1-oxyl-2)-pyrrolidine-1-carboxylic ester on human hepatoma HepG2 cell line

Tert-butyl-2(4,5-dihydrogen-4,4,5,5-tetramethyl-3-O-1H-imidazole-3-cationic-1-oxyl-2)-pyrrolidine-1-carboxylic ester (L-NNP) is a stable nitroxyl nitroxide radical, which have displayed cytotoxicity on human breast cancer MCF-7 and MDA-MB-231 cell lines. In the present study, we investigated the selective cytotoxicity of L-NNP on isogenetic human hepatoma HepG2 and normal L-02 cell lines. Cell growth inhibition, intracellular reactive oxygen species production, the mitochondrial membrane potential loss, malondialdehyde generation and glutathione levels were analyzed. The expression of Bax, Bcl-2 and NF-κBp65 proteins was also examined. The anticancer activity was evaluated in a HepG2 cell xenograft nude mice model. The results showed that 10, 20, 40μg/ml L-NNP exposure for 48h caused 52%, 82% and 91% cell growth inhibition of HepG2 cells, compared with 5%, 10% and 15% that of L-02 cells (p<0.01). Concentrations of 10, 20, 40μg/ml L-NNP induced cell death by increasing the generation of intracellular reactive oxygen species and MDA, by depolarizing the mitochondrial membrane potential, and by decreasing intracellular GSH levels in HepG2 cells. Western blot assay showed that Bax, Bcl-2 and NF-κBp65 might be implicated in L-NNP-induced selective HepG2 cell death. L-NNP was also found to inhibit HepG2 hepatoma growth and extend the life span of nude mice model (p<0.01). The pretreatment and co-treatment of 10mM N-acetyl-cysteine alleviated L-NNP exposure induced intracellular reactive oxygen species increase and cell growth inhibition demonstrated that L-NNP exhibited neoplasm-selective cytotoxicity and pro-apoptotic activities via reactive oxygen species mediated oxidative damage in HepG2 cells. It might be promising for developing a new class of anticancer agent for liver cancer.

Thioredoxin-1 contributes to protection against DON-induced oxidative damage in HepG2 cells

Leucocytes are susceptible to the toxic effects of deoxynivalenol (DON), which is a trichothecene mycotoxin produced by a number of fungi including Fusarium species. One mechanism of action is mediated by reactive oxygen species (ROS). The liver is an important target for toxicity caused by foreign compounds including mycotoxins. On the other hand, little is known about the influence of the redox state on hepatocytes treated with DON. The present study investigated the effect of DON on the cytosolic redox state and antioxidative system in the human hepatoma cell line HepG2. The cell viability of human monocyte cell line THP-1 or leukemia cell line KU812 treated with 2.5 and 5μmol/l DON were significantly reduced. However, HepG2 cells showed no toxic effects under the same conditions and did not exhibit an increased oxidative state. Further experiments showed that thioredoxin-1 (Trx-1) protein levels but not glutathione increased in the cells treated with 10μmol/l DON. In addition, the enhancement of Trx-1 content was repressed by antioxidants. These results suggest that DON-induced accumulation of Trx-1 in HepG2 cells plays one of the key roles in protection against cytotoxicity caused by DON and that the mechanism may be mediated by the antioxidant properties of Trx-1.

Protective effects of bamboo (Sasa borealis) leaf extracts against oxidative stress induced by t‐BHP in HepG2 cells

Bamboo (Sasa borealis) has been widely utilized as a medicinal plant in Korea. In this study, hepato‐protective effects of bamboo leaf extracts against oxidative damage induced by tert‐butyl hydroperoxide (t‐BHP) were evaluated using HepG2 cells. Two crude extracts and five fractions of the 70% ethanol (EtOH) extract of the bamboo leaves were prepared by extracting using water or 70% ethanol and by fractionating sequentially. The two crude extracts and the five fractions were non toxic up to 150 ¥ì g/mL concentration, as determined by the 3‐(4,5)‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐tetrazolium bromide (MTT) assay. Decreased cell viability (%), increased leakage of lactate dehydrogenase (LDH), increased activities of superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione reductase (GR), and raised concentration of malonedialdehyde (MDA) and decreased reduced glutathione (GSH) by t‐BHP were restored dose‐dependently by adding the crude extract of water or 70% EtOH or each fraction of the 70% EtOH extract. Among the two crude extract and five fractions, chloroform, ethyl acetate, and n‐butanol fractions showed the highest protective potential against oxidative damage. The results indicate that bamboo leaf extracts protect against oxidative damage in HepG2 cells.

Potential in vitro antioxidant and protective effects of Soymida febrifuga on ethanol induced oxidative damage in HepG2 cells

There is increasing evidence that oxidative stress is implicated in pathogenesis of various diseases, including alcoholic liver injury. In the present study, we investigated the comparative protective effects of leaf, bark, root and root bark extracts of Soymida febrifuga (Roxb.) A. Juss. ( Meliaceae) against ethanol induced oxidative damage in HepG2 cells. Comparatively, methanolic and aqueous extracts of bark and leaf significantly attenuated the cytotoxicity of the ethanol, as determined by cytotoxicity, lipid peroxidation, lactate dehydrogenase, alanine aminotransferases and asparatate aminotransferases, than the root and root bark extracts. Ethanol induces liver toxicity through free radical generation so initially in vitro antioxidant activity of the extracts was evaluated. Methanolic and aqueous extracts of bark and leaf have shown higher total phenolic content, reducing power, metal chelating, superoxide, hydroxyl radical, hydrogen peroxide and nitric oxide (murine macrophage cells) scavenging activity than the root and root bark extracts.

Antioxidant flavone glycosides from the leaves ofSasa borealis

Sasa borealis (Poaceae) is a perennial medicinal plant which is a major source of bamboo leaves in Korea. The n-BuOH extract of S. borealis leaves exhibited significant antioxidant activity against the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and a cytoprotective effect against oxidative damage in HepG2 cells. Bioactivity-guided fractionation by column chromatography led to the isolation of two antioxidative flavonoid C-glycoside derivatives, isoorientin (2) and isoorientin 2"-O-alpha-L-rhamnoside (4) along with tricin 7-O-beta-D-glucopyranoside (1) and apigenin 6-C-beta-D-xylopyranosyl-8-C-beta-D-glucopyranoside (3). Their structures were identified on the basis of chemical and spectroscopic methods. The radical scavenging activity and cytoprotective effect against oxidative damage of all the isolated compounds were also evaluated. Isoorientin (2) and isoorientin 2-O-alpha-L-rhamnoside (4) showed potent free radical scavenging activity with IC50 values of 9.5 and 34.5 microM, respectively, and strong cytoprotective effects against t-BOOH-induced oxidative damage in HepG2 cells, at very low concentrations of 1.1 microM isoorientin and 0.8 microM isoorientin 2-O-alpha-L-rhamnoside. This is the first report of the isolation and antioxidant activity of compounds 2 and 4 from S. borealis.

Increased expression of uncoupling protein 2 in HepG2 cells attenuates oxidative damage and apoptosis

Oxidative damage plays a major part in the pathogenesis of liver disease. Uncoupling proteins (UCPs) may be able to limit the generation of reactive oxygen species (ROS) and be cytoprotective. We investigated the effect of up-regulation of UCP2 in a hepatoblastoma cell line exposed to menadione or hypoxia/re-oxygenation. Lipid and protein oxidation was increased in HepG2 cells exposed to ROS but this increase was significantly lower in cells over-expressing UCP2 under identical conditions. LDH release increased 2.5-fold in response to hypoxia/re-oxygenation in control HepG2 cells with no significant increase in UCP2 transfected cells. Hypoxia/re-oxygenation resulted in a reduction in liver-specific protein secretion that was attenuated in transfected cells and UCP2 over-expression also resulted in a 66% reduction in apoptosis compared with non-transfected controls. These data suggest that UCP2 can limit oxidative damage in HepG2 cells in response to oxidative stress resulting in improved cell function and resistance to apoptosis.