Nuclear factor erythroid 2-related factor 2 antioxidant response element pathways protect bovine mammary epithelial cells against H2O2-induced oxidative damage in vitro

The experiment was conducted to determine the role of nuclear factor (erythroid-derived 2)-like factor 2 (NFE2L2, formerly Nrf2) antioxidant response element (ARE) pathway in protecting bovine mammary epithelial cells (BMEC) against H₂O₂-induced oxidative stress injury. An NFE2L2 small interfering RNA (siRNA) interference or a pCMV6-XL5-NFE2L2 plasmid fragment was transfected to independently downregulate or upregulate expression of NFE2L2. Isolated BMEC in triplicate were exposed to H₂O₂ (600 μM) for 6 h to induce oxidative stress before transient transfection with scrambled siRNA, NFE2L2-siRNA, pCMV6-XL5, and pCMV6-XL5-NFE2L2. Cell proliferation, apoptosis and necrosis rates, antioxidant enzyme activities, reactive oxygen species (ROS) and malondialdehyde (MDA) production, protein and mRNA expression of NFE2L2 and downstream target genes, and fluorescence activity of ARE were measured. The results revealed that compared with the control, BMEC transfected with NFE2L2-siRNA3 had proliferation rates that were 9 or 65% lower without or with H₂O₂, respectively. These cells also had apoptosis and necrosis rates that were 27 and 3.5 times greater with H₂O₂ compared with the control group, respectively. In contrast, transfected pCMV6-XL5-NFE2L2 had proliferation rates that were 64.3% greater or 17% lower without or with H₂O₂ compared with the control group, respectively. Apoptosis rates were 1.8 times lower with H₂O₂ compared with the control. In addition, compared with the control, production of ROS and MDA and activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), and glutathione-S-transferase (GST) increased markedly in cells transfected with pCMV6-XL5-NFE2L2 and without H₂O₂. However, compared with the control, production of ROS and MDA and activity of CAT and GSH-Px increased markedly, whereas activities of SOD and GST decreased in cells transfected with pCMV6-XL5-NFE2L2 and incubated with H₂O₂. Compared with the control, cells transfected with NFE2L2-siRNA3 with or without H₂O₂ had lower production of ROS and MDA and activity of SOD, CAT, GSH-Px, and GST. Cells transfected with pCMV6-XL5-NFE2L2 with or without H₂O₂ had markedly higher protein and mRNA expression of NFE2L2, heme oxygenase-1 (HMOX-1), NADH quinone oxidoreductase 1, glutamate cysteine ligase catalytic subunit, and glutamyl cystine ligase modulatory subunit compared with the control incubations. Cells transfected with NFE2L2-siRNA3 without or with H₂O₂ had markedly lower protein and mRNA expression of NFE2L2, HMOX-1, NADH quinone oxidoreductase 1, glutamyl cystine ligase modulatory subunit, and glutamate-cysteine ligase catalytic subunit compared with the control incubations. In addition, expression of HMOX-1 was 5.3-fold greater with H₂O₂ compared with the control. Overall, results indicate that NFE2L2 plays an important role in the NFE2L2-ARE pathway via the control of HMOX-1. The relevant mechanisms in vivo merit further study.     

Effect of heat-shock protein B7 on oxidative stress in adipocytes from preruminant calves

Dairy cows with ketosis display excessive lipolysis in adipose tissue. Heat-shock protein B7 (HSPB7), a small heat-shock protein, plays important roles in mediating cytoprotective responses to oxidative stress in rodent adipose tissue. Accordingly, it is assumed that HSPB7 may also play important roles in the antioxidant response in adipose tissue of ketotic cows. Therefore, the aim of this study is to investigate (1) the redox state of adipose tissue in ketotic cows and (2) the role and mechanism of HSPB7 on the regulation of oxidative stress in adipocytes from preruminant calves. An in vivo study consisting of 15 healthy and 15 clinically ketotic cows was performed to harvest subcutaneous adipose tissue and blood samples. In addition, adipocytes isolated from calves were treated with different concentrations of H₂O₂ (0, 12.5, 25, 50, 100, or 200 μM) for 2 h, transfected with adenovirus-mediated overexpression of HSPB7 for 48 h, or transfected with small interfering RNA of HSPB7 for 48 h followed by exposure to H₂O₂ (200 μM) for 2 h. Serum concentrations of nonesterified fatty acids and β-hydroxybutyrate were greater in cows with clinical ketosis, whereas serum concentration of glucose was lower. Compared with healthy cows, the malondialdehyde content was greater but the activity of glutathione peroxidase and superoxide dismutase was lower in adipose tissue of clinically ketotic cows. The abundance of HSPB7 and nuclear factor, erythroid 2 like 2 (NFE2L2) was greater in adipose tissue of clinically ketotic cows. In vitro, H₂O₂ treatment induced the overproduction of reactive oxygen species and malondialdehyde, and inhibited the activity of antioxidant enzymes glutathione peroxidase and superoxide dismutase in adipocytes from preruminant calves. The low concentration of H₂O₂ (12.5, 25, and 50 μM) increased the abundance of HSPB7 and NFE2L2, but high concentrations of H₂O₂ (100 or 200 μM) reduced the abundance of HSPB7 and NFE2L2. The overexpression of HSPB7 improved the H₂O₂-induced oxidative stress in adipocytes via increasing the abundance of NFE2L2 and its downstream target genes heme oxygenase-1 (HMOX1) and NADH quinone oxidoreductase 1 (NQO1). Knockdown of HSPB7 markedly inhibited the expression of NFE2L2, HMOX1, and NQO1 and further exacerbated H₂O₂-induced oxidative stress. Overall, these results indicate that activation of the HSPB7-NFE2L2 pathway increases cellular antioxidant capacity, thereby alleviating oxidative stress in bovine adipocytes.     

Adenosine 5′-monophosphate-activated protein kinase ameliorates bovine adipocyte oxidative stress by inducing antioxidant responses and autophagy

Adipose tissue concentration of reactive oxygen species (ROS) increases in dairy cows with ketosis, suggesting that the tissue experiences oxidative stress. Autophagy, an adaptive response to cellular stress, has been shown to promote survival and plays a critical role in antioxidant responses. Dysregulation of adenosine 5′-monophosphate-activated protein kinase (AMPK) is closely related to antioxidant responses and autophagy of adipocytes in animal models of metabolic disorders, but its role in bovine adipose tissue during periods of stress is unknown. We hypothesized that AMPK may play important roles in the regulation of oxidative stress in adipose tissue of ketotic cows. Specific objectives were to evaluate autophagy status and AMPK activity in adipose tissue of ketotic cows, and their link with oxidative stress in isolated bovine adipocytes. Selection of 15 healthy and 15 clinically ketotic Holstein cows at 17 (±4) d postpartum was performed after a thorough veterinary evaluation for clinical symptoms and also based on serum β-hydroxybutyrate concentrations before collection of subcutaneous adipose tissue samples. Primary cultures of bovine adipocytes isolated from the harvested adipose tissue were stimulated with varying concentrations of H₂O₂ (0, 50, 100, 200, or 400 μM) for 2 h. In another experiment, adipocytes were cultured with the AMPK activator A769662 or adenovirus-containing small interfering RNA (ad-AMPKα-siRNA) for 3 or 48 h, respectively, followed by H₂O₂ exposure (200 μM) for 2 h. Compared with healthy cows, clinical ketosis led to increased abundance of AMPK and nuclear factor erythroid-derived 2-like 2 (NFE2L2), but lower abundance of Kelch-like ECH-associated protein 1 (KEAP1) in adipose tissue. Abundance of the key proautophagy proteins Beclin1, sequestosome 1 (SQSTM1), autophagy-related gene 7 (ATG7), ATG5, and ratio of microtubule-associated protein light chain 3 (LC3) II to LC3I were greater in adipose tissue of ketotic cows. In bovine adipocytes, treatment with H₂O₂ induced accumulation of ROS and malondialdehyde (MDA), whereas H₂O₂ stimulation inhibited activities of the antioxidant enzymes glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD). Addition of AMPK activator A769662 increased antioxidant response via activating NFE2L2 and its downstream targets heme oxygenase 1 (HMOX1), superoxide dismutase 1 (SOD1), catalase (CAT), and glutathione-S-transferase (GST) to improve H₂O₂-induced oxidative stress in adipocytes. Simultaneously, activation of AMPK increased abundance of Beclin1, SQSTM1, ATG7, ATG5, and ratio of LC3II to LC3I. In contrast, inhibition of AMPK downregulated abundance of NFE2L2, HMOX1, SOD1, CAT, Beclin1, SQSTM1, ATG7, ATG5, and ratio of LC3II to LC3I, and further aggravated H₂O₂-induced oxidative stress. Overall, these data indicate that activation of AMPK, as an adaptive mechanism for acute metabolic regulation of adipose tissue homeostasis, can induce antioxidant responses and autophagy, and further reduce oxidative stress in bovine adipocytes.     

Selenomethionine increases proliferation and reduces apoptosis in bovine mammary epithelial cells under oxidative stress

The decline in mammary epithelial cell number as lactation progresses may be due, in part, to oxidative stress. Selenium is an integral component of several antioxidant enzymes. The present study was conducted to examine the effect of oxidative stress and selenomethionine (SeMet) on morphology, viability, apoptosis, and proliferation of bovine mammary epithelial cells (BMEC) in primary culture. Cells were isolated from mammary glands of lactating dairy cows and grown for 3 d in a low-serum gel system containing lactogenic hormones and 0 or 100 μM H₂O₂ with 0, 10, 20, or 50 nM SeMet. Hydrogen peroxide stress increased intracellular H₂O₂ to 3 times control concentrations and induced a loss of cuboidal morphology, cell-cell contact, and viability of BMEC by 25%. Apoptotic cell number more than doubled during oxidative stress, but proliferating cell number was not affected. Supplementation with SeMet increased glutathione peroxidase activity 2-fold and restored intracellular H₂O₂ to control levels with a concomitant return of morphology and viability to normal. Apoptotic BMEC number was decreased 76% below control levels by SeMet and proliferating cell number was increased 4.2-fold. These findings suggest that SeMet modulated apoptosis and proliferation independently of a selenoprotein-mediated reduction of H₂O₂. In conclusion, SeMet supplementation protects BMEC from H₂O₂-induced apoptosis and increased proliferation and cell viability under conditions of oxidative stress.     

Curcumin protects mouse neuroblastoma Neuro-2A cells against hydrogen-peroxide-induced oxidative stress

Curcumin has been traditionally used in China and India for food and medicinal purposes. It has been shown to possess potent antioxidative activity both in vitro and in vivo. In the present study, the neuroprotective effects and the potential mechanisms of curcumin against H₂O₂-induced oxidative stress in mouse neuroblastoma Neuro-2A cells were investigated. Treatment with curcumin at 20 and 25 μg/mL for 1 h prior to H₂O₂ exposure significantly attenuated cell viability loss, reduced apoptosis, suppressed the elevation of intracellular reactive oxygen species (ROS) and calcium levels, and stabilised mitochondrial membrane potential. Furthermore, curcumin could block H₂O₂-mediated degradation of the protein IκBα and subsequent activation of nuclear factor κB, thus inhibiting the expression of its target gene cyclooxygenase 2. These results indicate that curcumin has potential protective effects against H₂O₂-induced oxidative stress in neuron cells, which might make curcumin a suitable therapeutic agent for prevention and treatment of neurodegenerative diseases associated with oxidative stress.     

Methionine supply alters mammary gland antioxidant gene networks via phosphorylation of nuclear factor erythroid 2-like 2 (NFE2L2) protein in dairy cows during the periparturient period

The periparturient period is the most critical period during the lactation cycle of dairy cows and is characterized by increased oxidative stress status. The objective of this experiment was to evaluate the effect of supplementing rumen-protected methionine on nuclear factor erythroid 2-like 2 (NFE2L2, formerly NRF2) protein and target gene expression in the mammary gland during the early postpartal period. Multiparous Holstein cows were used in a block design experiment with 30 cows per treatment. Treatments consisting of a basal control diet (control) or the basal diet plus rumen-protected methionine (methionine) were fed from d ?28 to 60 relative to parturition. Mammary tissue biopsies were harvested on d 21 postpartum from 5 cows per treatment. Compared with control, methionine increased dry matter intake, milk yield, and milk protein content. Among plasma parameters measured, methionine led to greater methionine and lower reactive oxygen metabolites. Compared with control, methionine supply resulted in greater mRNA abundance of the NFE2L2 target genes glutamate-cysteine ligase catalytic subunit (GCLC), glutamate-cysteine ligase modifier subunit (GCLM), glutathione reductase (GSR), glutathione peroxidase 1 (GPX1), malic enzyme 1 (ME1), ferrochelatase (FECH), ferritin heavy chain 1 (FTH1), and NAD(P) H quinone dehydrogenase 1 (NQO1) in the mammary tissue. In addition, methionine upregulated the mRNA abundance of NFE2L2, NFKB1, MAPK14 and downregulated KEAP1. The ratio of phosphorylated NFE2L2 to total NFE2L2 protein, and total heme oxygenase 1 (HMOX1) protein were markedly greater in response to methionine supply. In contrast, total protein abundance of Kelch-like ECH-associated protein 1 (KEAP1), which sequesters NFE2L2 in the cytosol and reduces its activity, was lower with methionine. Besides the consistent positive effect of methionine supply on systemic inflammation and oxidative stress status, the present data indicate a positive effect also on antioxidant mechanisms within the mammary gland, which are regulated, at least in part, via phosphorylation of NFE2L2 and its target genes. The exact mechanisms for these responses merit further study.     

Glutamine pretreatment protects bovine mammary epithelial cells from inflammation and oxidative stress induced by γ-d-glutamyl-meso-diaminopimelic acid (iE-DAP)

Glutamine (GLN) has many types of biological activity in rats, including anti-inflammatory, antioxidative stress, and anti-apoptosis effects. However, little is known about the effects of GLN on bovine mammary epithelial cells (BMEC). γ-d-Glutamyl-meso-diaminopimelic acid (iE-DAP) is a cell wall peptidoglycan component of gram-negative bacteria that can be recognized by the intracellular receptor nucleotide-binding oligomerization domain-containing protein 1 (NOD1) and can cause bovine mastitis. The goal of the present study was to investigate whether GLN protects BMEC from iE-DAP–induced inflammation, oxidative stress, and apoptosis. We cultured BMEC in a GLN-free medium for 24 h and then separated them into 4 groups: cells treated with 1× PBS for 26 or 32 h (control); cells stimulated by 10 μg/mL iE-DAP for 2 or 8 h (2- or 8-h iE-DAP); cells pretreated with 8 or 4 mM GLN for 24 h followed by 2 or 8 h of 1× PBS treatment (8 or 4 mM GLN); and cells pretreated with 8 or 4 mM GLN for 24 h followed by 2 or 8 h of iE-DAP treatment (DG). In the 2-h iE-DAP group, when levels of inflammation peaked, iE-DAP treatment increased both the mRNA and protein expression of NOD1, inhibitor of nuclear factor-κB (NFKBIA, IκB), and nuclear factor-κB subunit p65 (RELA, NF-κB p65), as well as the mRNA expression of IL6 and IL8 and levels of IL-6 and tumor necrosis factor-α in cell culture supernatants. In contrast, 8 mM GLN pretreatment inhibited the mRNA and protein expression of inflammatory-related factors by suppressing the NOD1/NF-κB pathway. In the 8-h iE-DAP group, iE-DAP treatment decreased the mRNA and protein expression of extracellular regulated kinase (Erk, ERK) and nuclear factor erythroid 2–associated factor2 (NFE2L2, Nrf2), as well as the mRNA expression of superoxide dismutase 1 (SOD1), catalase (CAT), coenzyme II oxidoreductase 1 (NQO1), and heme oxygenase 1 (HMOX1, HO1). In addition, iE-DAP treatment increased the expression of malondialdehyde in BMEC when oxidative stress levels peaked. Interestingly, 4 mM GLN pretreatment induced the mRNA and protein expression of antioxidative stress–related factors and inhibited the expression of reactive oxygen species in BMEC by promoting the ERK/Nrf2 pathway. Moreover, GLN reduced apoptosis caused by inflammation and oxidative stress in BMEC. This is the first report showing that GLN protects against iE-DAP-induced inflammation and oxidative stress via the NOD1/NF-κB and ERK/Nrf2 pathways in BMEC.     

Lactobacillus plantarum AR501 Alleviates the Oxidative Stress of D‐Galactose‐Induced Aging Mice Liver by Upregulation of Nrf2‐Mediated Antioxidant Enzyme Expression

Lactic acid bacteria (LAB) have been used as ingredients of functional foods to promote health and prevent diseases because of their beneficial effects. This study aimed to investigate the antioxidative effects of LAB on the hepatotoxicity in D‐galactose‐induced aging mice. LAB were isolated from the traditional Chinese fermented foods and screened by the tolerance of hydrogen peroxide (H₂O₂). Male ICR (Institute of Cancer Research) mice were subcutaneously injected with D‐galactose for 5 weeks and then gastric gavage with LAB for 6 weeks. The results showed that Lactobacillus plantarum AR113 and AR501, and Pediococcu pentosaceus AR243 could tolerate up to 1.5 mM H₂O₂ in vitro, and they could live through simulated gastrointestinal tract (GIT) to colonizing the GIT of host. In vivo, oral administration of L. plantarum AR113 and AR501 improved the antioxidant status of D‐galactose‐induced oxidative stress mice such as alleviated liver damages and reduced abnormal activities of superoxide dismutase, glutathione peroxidase, and catalase to normal levels. In addition, L. plantarum AR501 markedly elevated the gene expression of nuclear factor erythroid‐2‐related factor 2 and upregulated the expressions of several antioxidant genes such as glutathione reductase, glutathione S‐transferase, glutamate‐cysteine ligase catalytic subunit, glutamate‐cysteine ligase modifier subunit, and NAD(P)H quinone oxidoreductase 1 in the liver of an aging mice. Therefore, L. plantarum AR501 could be a good candidate for producing antiaging functional foods.     

Catechin-7-O-β-d-glucopyranoside scavenges free radicals and protects human B lymphoma BJAB cells on H2O2-mediated oxidative stress

Catechin-7-O-β-d-glucopyranoside (CA-G) was previously isolated from red bean (the seed of Phaseolus calcaratus cv. Roxburgh). This study examined the ability of CA-G to scavenge reactive oxygen species generated by cell-free systems and to protect cells from oxidative stress caused by hydrogen peroxide (H₂O₂). The mechanism by which CA-G exerts its antioxidant and anti-apoptotic action on H₂O₂-exposed cells was also investigated. CA-G treatment prevented H₂O₂-mediated apoptosis and inhibited the formation of single stand breaks in DNA in H₂O₂-exposed BJAB cells. CA-G suppressed mitochondrial stress and caspase activation caused by H₂O₂. Mechanistic experiments revealed that the antioxidant mechanism of CA-G on H₂O₂-mediated oxidative damage was due to the direct scavenging of hydroxyl radicals and/or to the chelation of metal ions that were used to produce hydroxyl radicals from H₂O₂ via the Fenton reaction. Collectively, these findings suggest beneficial roles of CA-G or CA-G-rich red bean on the protection from oxidative damage.     

Collagen peptides from Acaudina molpadioides prevent CCl4-induced liver injury via Keap1/Nrf2-ARE,PI3K/AKT,and MAPKs pathways

Collagen peptide from Acaudina molpadioides (AMP) showed antioxidative activity in H₂O₂-induced RAW264.7 cells in our pervious study. In this study, it was observed that AMP could effectively improve the morphology and function of liver in CCl₄-induced mice. After 200 mg/kg AMP treatment, the content of MDA in liver decreased by 62.3%, and the level of antioxidant enzymes (SOD, GSH-Px, and CAT) increased by more than 65%. Western blot results disclosed that AMP (200 mg/kg) upregulated the Nrf2 level by 73.8% and downregulated Keap1 by 41.0% in CCl₄-induced mice liver. The levels of p-ERK, p-JNK, and p-p38 in 200 mg/kg AMP treatment groups decreased by 57.3%, 40.9%, and 40.6%, but the levels of p-PI3K and p-AKT increased by 162.6% and 60.3%, respectively. Furthermore, the trends of Nrf2, Keap1, p-ERK, p-JNK, p-p38, p-PI3K, and p-AKT levels in H₂O₂-induced RAW264.7 cells after AMP treatment were similar to the results in CCl₄-induced mice liver. These findings provided evidence that AMP exerted antioxidant activity via Keap1/Nrf2-ARE, PI3K/AKT, and MAPKs pathways in vivo and in vitro. Therefore, the collagen peptide from A. molpadioides might represent a novel functional food to prevent acute liver injury via attenuation of oxidative stress.     

Cytoprotective effect of white tea against H2O2-induced oxidative stress in vitro

The protective effect of water extracts of white tea (WEWT) on oxidative stress in vitro is investigated. WEWT, like water extracts of green tea (WEGT) and water extracts of Pu-erh tea (WEPT), demonstrates a marked inhibition of the oxidation of liposome, albumin and LDLmodel systems. WEWT protects against H₂O₂-induced cytotoxicity, in a dose-dependent manner. The inhibition of ROS generation and MDA formation by WEWT in H₂O₂-induced Clone 9 cells parallels the effects on cell viability. Moreover, GSH and antioxidant enzymes may play an important role in the protective effect that is associated with H₂O₂-induced oxidative stress. The HPLC-DAD and HPLC–MS/MS analysis, shows that sixteen bioactive compounds are present in WEWT, which may partially account for its protective effect against oxidative insult. These results suggest that the mechanism of the protective actions of WEWT is related to its antioxidant potential and the maintenance of the normal redox status of the cell.     

Cytoprotective effect of fucoxanthin isolated from brown algae <Emphasis Type="Italic">Sargassum siliquastrum</Emphasis> against H<Subscript>2</Subscript>O<Subscript>2</Subscript>-induced cell damage

In this study, the cytoprotective effect of fucoxanthin, which was isolated from Sargassum siliquastrum, against oxidative stress induced DNA damage was investigated. Fucoxanthin, a kind of carotenoid, was pretreated to the medium and the protective effect was evaluated via 2′,7′-dichlorodihydrofluorescein diacetate, 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide, and comet assays. Intracellular reactive oxygen species were over produced by addition of hydrogen peroxide (H₂O₂), but the production was significantly reduced by the treatment with fucoxanthin. The fucoxanthin strongly enhanced cell viability against H₂O₂ induced oxidative damage and the inhibitory effect of cell damage was a dose-dependent manner. Furthermore, a protective effect against oxidative stress-induced cell apoptosis was also demonstrated via nuclear staining with Hoechst dye. These results clearly indicate that fucoxanthin isolated from S. siliquastrum possesses prominent antioxidant activity against H₂O₂-mediated cell damage and which might be a potential therapeutic agent for treating or preventing several diseases implicated with oxidative stress.     

Cytoprotective effects of lotus (Nelumbo nucifera Gaertner) seed extracts on oxidative damaged mouse embryonic fibroblast cell

This study was conducted to evaluate the cytoprotective activity of lotus (Nelumbo nucifera Gaertner) seed extract (LSE) on mouse embryonic fibroblast (MEF) cells. The DPPH free radical scavenging activities of LSE increased in a concentration dependent manner. The cells, damaged by oxidative stress, decreased their viability following increasing concentration of H₂O₂, but the cotreatment of ethyl acetate fraction of LSE and H₂O₂ resulted in an increase in cell growth, by about 25%, compared to the cells treated with H₂O₂. The ethyl acetate fraction of LSE inhibited the cytotoxicity induced by H₂O₂ in a concentration dependent manner. The treatment of the n-butanol fraction of LSE on MEF cell was also examined by analyzing the DNA content and apoptotic rate, using flow cytometry. The oxidative damage to the cells, measured by apoptotic and necrotic cell accumulation, was similar with the addition of the ethyl acetate fraction of LSE to H₂O₂. These results suggest that LSE inhibited the cytotoxicity which is induced by H₂O₂, and has a protective effect on MEF cell against oxidative stress.     

Cytoprotective activity of lotus (Nelumbo nucifera Gaertner) leaf extracts on the mouse embryonic fibroblast cell

This study was conducted to evaluate the cytoprotective activity of lotus (Nelumbo nucifera Gaertner) leaf extract (LLE) on mouse embryonic fibroblast (MEF) cells. The 2-diphenyl-1-picrylhydrazyl hydrate (DPPH) free radical scavenging activities of LLE increased in a concentration dependent manner. The cells, damaged by oxidative stress, decreased their viability following increasing concentration of H₂O₂, but the co-treatment of n-butanol fraction of LLE and H₂O₂ resulted in an increase in cell growth, by about 25%, compared to the cells treated with H₂O₂. The n-butanol fraction of LLE inhibited the cytotoxicity induced by H₂O₂ in a concentration dependent manner. The oxidative damage to the cells, measured by apoptotic and necrotic cell accumulation, was similar with the addition of the n-butanol fraction of LLE to H₂O₂. Taken together, these results suggest that LLE inhibited the cytotoxicity which is induced by H₂O₂, and has a protective effect on MEF cell against oxidative stress.