Protective effects of hesperidin against oxidative stress of tert-butyl hydroperoxide in human hepatocytes

Food and Chemical Toxicology 48 (2010) 2980–2987

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Protective effects of hesperidin against oxidative stress of tert-butyl hydroperoxide in human hepatocytes Mingcang Chen a, Honggang Gu b, Yiyi Ye a, Bing Lin a, Lijuan Sun a, Weiping Deng a, Jingzhe Zhang b,**, Jianwen Liu a,* a b

State Key Laboratory of Bioreactor Engineering, Shanghai Key Laboratory of Chemical Biology, School of Pharmacy, East China University of Science and Technology, China Longhua Hospital, Shanghai University of Traditional Chinese Medicine, PR China

a r t i c l e

i n f o

Article history: Received 15 April 2010 Accepted 23 July 2010

Keywords: Hesperidin, Oxidative stress, tert-butyl hydroperoxide (t-BuOOH), heme oxygenase-1 (HO-1) Extracellular signal-regulated protein kinase 1/2 (ERK 1/2) Nuclear factor erythroid 2 related factor (Nrf2)

a b s t r a c t Increasing evidence regarding free radical generating agents and the inflammatory process suggest that accumulation of reactive oxygen species (ROS) could involve hepatotoxicity. Hesperidin, a naturally occurring flavonoid presents in fruits and vegetables, has been reported to exert a wide range of pharmacological effects that include antioxidant, anti-inflammatory, antihypercholesterolemic, and anticarcinogenic actions. However, the cytoprotection and mechanism of hesperidin to neutralize oxidative stress in human hepatic L02 cells remain unclear. In this work, we assessed the capability of hesperidin to prevent tert-butyl hydroperoxide (t-BuOOH)-induced cell damage by augmenting cellular antioxidant defense. Hesperidin significantly protected hepatocytes against t-BuOOH-induced cell cytotoxicity, such as mitochondrial membrane potential (MMP) deplete and lactate dehydrogenase (LDH) release. Hesperidin also remarkably prevented indicators of oxidative stress, such as the ROS and lipid peroxidation level in a dose-dependent manner. Western blot showed that hesperidin facilitated ERK/MAPK phosphorylation which appeared to be responsible for nuclear translocation of Nrf2, thereby inducing cytoprotective heme oxygenase-1 (HO-1) expression. Based on the results described above, it suggested that hesperidin has potential as a therapeutic agent in the treatment of oxidative stress-related hepatocytes injury and liver dysfunctions. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction Nutritional studies recommend the regular consumption of fruits and vegetables to favor a healthy quality of life. Epidemiological studies have shown that these foods may reduce the risk of death from coronary heart diseases and cancer (Di Stefani et al., 1999; Yochum et al., 1999). Citrus fruits and products are important sources of health-promoting constituents and are widely consumed around the world (Benavente Garcia et al., 1997). Flavonoids are one of the most important compounds present in genus Citrus. Recently, the health effects of citrus flavonoids have been attracting attention. These compounds have shown potential beneficial properties against several diseases, as they have shown biological activity on experiments with animals, cell lines, and in vitro assays associated with carcinogenic, cardiovascular, * Corresponding author. Address: State Key Laboratory of Bioreactor Engineering Shanghai Key Laboratory of Chemical Biology, School of Pharmacy, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, PR China. Tel./fax: +86 21 64252044. ** Corresponding author. Longhua Hospital, Shanghai University of Traditional Chinese Medicine, PR China. Tel.: +86 21 64385700x3815; fax: +86 21 64252044. E-mail addresses: [email protected] (J. Zhang), [email protected] (J. Liu). 0278-6915/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2010.07.037

inflammatory, allergic, and bleeding disorders (Galati et al., 1994; Ho et al., 1995; Koyuncu et al., 1999). As the most abundant flavonoids in Citrus, flavanones not only play an important physiological and ecological role but are also of commercial interest because of their multitude of applications in the food and pharmaceutical industries (Benavente Garcia et al., 1997; Del Río et al., 1998; Del Río et al., 2004; Marín et al., 2007). Reactive oxygen species are implicated in normal and pathological liver functions. In healthy organisms, ROS production is counterbalanced by antioxidant defense system to maintain an appropriate redox balance (Kruidenier and Verspaget, 2002). Organisms possess defense systems to escape the consequences of cell damage caused by ROS including enzymes such as catalase and superoxide dismutase and low molecular compounds, e.g., glutathione. Oxidative stress is the excessive exposure to ROS and results from the imbalance between prooxidants and antioxidants. And it is recognized to be a factor in many liver diseases, including ischemia/reperfusion injury, cholestasis, chronic hepatitis C, alcoholic and non-alcoholic fatty liver diseases (Comporti, 1985; Kaplowitz, 2000). Excessive ROS could directly lead to cell damage and tissue injury by targeting various biomacromolecules, such as protein, lipid, and DNA (Halliwell and Aruoma, 1991; Ozaki

M. Chen et al. / Food and Chemical Toxicology 48 (2010) 2980–2987

et al., 2000; Tsou and Yang, 1996). As the liver plays a crucial role in metabolic function, hepatic injury is concerned with the disorder of metabolic function (Bhandarkar and Khan, 2004). Therefore, studies which investigate scavenging free radicals as well as reducing oxidative stress, and protect liver from damage, have attracted much attention. Hesperidin, a naturally occurring flavonoid presents in fruits and vegetables (Justesen et al., 1998), has been reported to exert a wide range of pharmacological effects including antioxidant, anti-inflammatory, antiallergic and anticarcinogenic actions (Emin et al., 1994; Garg et al., 2001; Suarez et al., 1998; Tommasini et al., 2005). It is effectively used as a supplemental agent in the treatment protocols of complementary settings. Its deficiency has been linked to abnormal capillary leakiness as well as pain in the extremities causing aches, weakness and night leg cramps. Supplemental hesperidin also helps in reducing oedema or excess swelling in the legs due to fluid accumulation. Flavonoids such as hesperidin exhibit antioxidative properties by several different mechanisms, such as scavenging of free radicals, chelation of metal ions such as iron and copper which are of major importance for the initiation radical reactions, inhibition of enzymes responsible for free radical generation and facilitation endogenous antioxidative defense system (Benavente Garcia et al., 1997; Cai et al., 2006; Cotelle et al., 1996). In addition, epidemiological evidence strongly suggests protective effects of a diet rich in flavonoids such as fruits and vegetables with respect to the etiology of most cancers and also supports a role in coronary heart disease (Hollman et al., 1996). Hence, the flavonoids may protect biosystems against free radical attack probably involved in various cancers and coronary heart disease. However, whether hesperidin can alleviate human hepatocytes injury induced by t-BuOOH and the related mechanisms remains unclear. In the present work, we examined the protective potential of hesperidin against t-BuOOH-induced oxidative hepatocyte injury and the mechanisms underlying these protective effects in human hepatic L02 cells. Our results indicate that hesperidin could significantly protect hepatocytes against t-BuOOH-induced oxidative injury.

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2.4. Cell morphological assessment The morphology of cells was monitored under an inverted light microscope and stained with hematoxylin and eosin (H&E) according to standard protocols. 2.5. ROS production assay The generation of ROS was measured using oxidation-sensitive fluorescent probe HDCF-DA. L02 cell was treated with hesperidin (20, 40 and 80 lM) or 50 lM vitamin E for 24 h, and then incubated with t-BuOOH (150 lM) for a further 3 h. Then supernatants medium was removed and cells were washed twice with phosphate buffer saline (PBS) for analysis. Reactive oxygen species produced was determined according to our previous work (Ye et al., 2010). 2.6. Mitochondrial membrane potential (MMP) analysis The mitochondrial membrane potential was detected using rhodamine 123, a cationic fluorescent dye, described previously (Emaus et al., 1986). Briefly, after removing supernatants medium, cells were incubated with PBS containing 1.5 lM rhodamine 123 at 37 °C for 15 min. The intensities of fluorescence were detected by means of Synergy 2 multi-mode microplate reader, with an excitation wavelength of 485 nm and an emission wavelength of 530 nm. 2.7. Determination of LDH leakage L02 cell was treated with hesperidin (20, 40 and 80 lM) or 50 lM vitamin E for 24 h, and then incubated with t-BuOOH (150 lM) for a further 3 h. Then supernatant medium was collected and measured by LDH assay kit. The LDH activities were quantitated by reading optical densities at 420 nm by using spectrophotometer (KeDa Instrument Factory, WuXi, China). 2.8. Evaluation of MDA levels Cellular MDA levels were analyzed by MDA assay kit. L02 cell was treated with hesperidin (20, 40 and 80 lM) or 50 lM vitamin E for 24 h, and then incubated with t-BuOOH (150 lM) for a further 3 h. Then supernatants medium was removed and cells were washed twice for analysis. The levels of MDA were quantitated by reading optical densities at 532 nm using spectrophotometer. 2.9. Determination of HO-1 activity

2. Materials and methods

Cellular HO-1 activity was detected described previously (Suttner et al., 1999) with little modification. Briefly, 20 lL aliquots of cell suspension (2  106 cells) were reacted with 20 lL hemin (150 lM) and 20 lL NADPH (4.5 mM) in subdued lighting at 37 °C for 15 min. Blanks were cell sonicates reacted with hemin only. The reaction was stopped with dry ice (78 °C).

2.1. Chemicals

2.10. Western blot assay

Hesperidin (purity P99%, HPLC grade) was purchased from SHANXI SCIPHAR BIOTECHNOLOGY (Xi’an, Shanxi, China). RPMI 1640 medium and fetal bovine serum were obtained from Gibco (Grand Island, New York, USA). Lactate dehydrogenase (LDH) assay kit and malondialdehyde (MDA) assay kit were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China). Antibodies against Nrf2, extracellular signal-regulated protein kinase 1/2(ERK1/2), phosphor ERK1/2, p38, phosphor p38, c-Jun N-terminal kinase (JNK), phosphor JNK, b-actin and AP-labeled goat anti-rabbit immunoglobulin were purchased from Bipec Biopharma (Cambridge, MA, USA). BCIP/NBT color development substrate was obtained from Promega Biotech (Madison, WI, USA). ERK1/2 inhibitor, PD98059, p38 inhibitor, SB203580 and JNK inhibitor, SP600125 were obtained from Calbiochem (San Diego, CA, USA). The other chemicals were purchased from Sigma–Aldrich (Saint Louis, MO, USA) unless otherwise indicated.

Cells were seeded in 10 cm dishes and incubated with indicated concentrations of hesperidin for the tested times. Total protein extracts were isolated according to our previous work (Xu et al., 2008). The extraction and isolation of nuclear fraction were performed according to previous work (Levites et al., 2002). Protein concentrations were determined using the BCA assay (Pierce, Rockford, IL, USA). The western blot assay was performed according to our previous work (Xu et al., 2008) with little modification. Briefly, equal amounts of proteins (40 lg) were separated by SDS–PAGE and transferred to a nitrocellulose membrane. Membranes were blocked with 2% bovine serum albumin (BSA) and then incubated with appropriate primary antibodies overnight at 4 °C. Expression of protein was detected by staining with NBT and BCIP. b-Actin and Lamin B were used as loading controls for total/cytoplasm fraction extracts and nuclear fraction extracts, respectively. 2.11. Real-time quantitative PCR

2.2. Cell culture The normal human hepatic cell strain, L02, was purchased from Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). L02 cells were maintained in RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum, 100 U/mL penicillin, and 100 lg/mL streptomycin at 37 °C in a 5% CO2humidified environment. Cells were plated onto appropriate multiwells plates or dishes and experiments were performed when cells reached 60% confluence.

2.3. Cell viability assay Cell viability was determined by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) tetrazolium dye assay according to our previous work (Xu et al., 2008).

Total RNA was isolated by Total RNA (Mini) kit (Watson Biotechnologies, Shanghai, China). RNA was quantitated by optical density measurement at 260 and 280 nm using a spectrophotometer (all RNA samples had an A260/A280 ratio >1.8) and integrity was confirmed by running RNA on a 1.2% agarose gel. Primers were obtained from Shanghai Sangon Biological Engineering Technology & Services (Shanghai, China) and their sequences were: 50 -GGTGATAGAAGAGGCCAAGACTGC-30 (sense) and 50 -TGTAAGGACCCATCGGAGAAGC-30 (anti-sense) for HO-1, 50 -GGTCGGAGTCAACGGATTTG-30 (sense) and 50 -ATGAGCCCCAGCCTTCTCCAT-30 (anti-sense) for GAPDH. Equal quantities (2.0 lg) of total RNA were converted to first-strand cDNA using Revert Aid First Stand cDNA Synthesis kit (Fermentas Life Sciencs, Lithuania, EU) for RT-PCR. Real-time quantitative PCR and melt-curve analyses were performed with the SYBR Green Realtime PCR kit (TOYOBO, Osaka, Japan) and an iCycler machine (Bio-Rad, Hercules, CA, USA). Amplification was comprised of 40 cycles (95 °C for 30 s, 56 °C for 30 s, 72 °C for 30 s for HO-1 and 95 °C for 30 s, 54 °C for 30 s, and

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M. Chen et al. / Food and Chemical Toxicology 48 (2010) 2980–2987 72 °C for 30 s for GAPDH). Relative quantities of expression of the genes of interest in different samples were calculated by the comparative Ct (threshold cycle) value method (Mandal et al., 2006).

2.12. Statistical analysis All results shown represent as means ± standard deviations from triplicate experiments performed in a parallel manner unless otherwise indicated. Significance was tested among and between groups using the one-way analysis of variance (ANOVA) followed by Dunnett’s posthoc test.

3. Results 3.1. Hesperidin attenuated t-BuOOH-induced cell injury

Fig. 1. Effects of t-BuOOH and hesperidin on the proliferation of L02 cells. L02 cells were treated with different concentrations of t-BuOOH for 3 h. After the medium was removed, cells were incubated with RPMI 1640 medium for a further 24 h and prepared for measurement by MTT analysis. Meanwhile the cells were treated with different concentrations of hesperidin for 24 h and then measured by MTT analysis.*p < 0.05 and **p < 0.01 represent significant differences compared with control group.

In order to assess effects of t-BuOOH on cell viability, we used the MTT tetrazolium dye assay. As shown in Fig. 1, t-BuOOH significantly inhibited L02 cell proliferation. However, hesperidin had no noticeable effect on the viability of L02 cells when its concentration less than 100 lM. To assess the ability of hesperidin to protect against t-BuOOHinduced cell death, L02 cell was treated with hesperidin (20, 40

Fig. 2. Hesperidin attenuated t-BuOOH-induced cell injury. (A) L02 cell was treated with hesperidin (20, 40 and 80 lM) or 50 lM vitamin E for 24 h, and then incubated with t-BuOOH (150 lM) for a further 3 h. Cell viability was measured with MTT. *p < 0.05 and **p < 0.01 represent significant differences compared with control group. #p < 0.05 and ##p<0.01 represent significant differences compared with t-BuOOH model group. (B) Morphologic changes of cells were observed under an inverted light microscope (250  H&E). Cell was treated with 80 lM hesperidin or 50 lM vitamin E for 24 h, and then incubated with 150 lM t-BuOOH for a further 3 h. Cells were stained with H&E as described in material and method section. (a) Control group; (b) the t-BuOOH group; (c) Hesperidin group; (d) Vitamin E group.

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M. Chen et al. / Food and Chemical Toxicology 48 (2010) 2980–2987 Table 1 Comparison of oxidative stress indices after hesperidin treatment in t-BuOOH-induced L02 cells. LDH (% of control) 20 lM Hesperidin + t-BuOOH 40 lM Hesperidin + t-BuOOH 80 lM Hesperidin + t-BuOOH 50 lM vitamin E + t-BuOOH t-BuOOH group 80 lM Hesperidin group Control group

*

127.62 ± 4.24 109.95 ± 5.96*** 104.97 ± 2.89** 103.87 ± 2.10** 133.15 ± 4.33* 98.89 ± 2.97** 100 ± 3.70

MMP (% of control) ***

81.94 ± 4.82 92.70 ± 4.31** 94.15 ± 6.60** 97.21 ± 6.31** 62.64 ± 9.66* 101.33 ± 14.38** 100 ± 8.14

ROS (% of control) *

189.34 ± 33.24 150.60 ± 27.11*** 108.15 ± 22.37** 112.52 ± 30.60** 209.02 ± 34.74* 102.32 ± 26.03** 100 ± 18.09

MDA (nmol/mg prot) 3.10 ± 0.53* 2.20 ± 0.39** 1.69 ± 0.46** 1.78 ± 0.29** 3.75 ± 0.56* 1.15 ± 0.44** 1.05 ± 0.24

Values are mean ± SD of three separate experiments. p < 0.05 represent significant differences compared with control group. ** p < 0.05 represent significant differences compared with t-BuOOH model group. *

and 80 lM) or 50 lM vitamin E (Vit E) for 24 h, and then incubated with t-BuOOH (150 lM) for a further 3 h. Cell viability, MMP decrease and lactate LDH leakage were assayed. As shown in Fig. 2A, hesperidin exerted a dose response effect on the protection against t-BuOOH-induced cell death. In addition, hesperidin dose dependently prevented t-BuOOH incuced LDH release and MMP decrease in Table 1. We also observed the cell morphology phenomena using H&E staining. As shown in Fig. 2B, the untreated cells displayed normal, healthy shape demonstrated by the clear skeletons. On the contrary, cells distorted severely and grew slowly in the t-BuOOH model group. Furthermore, cell microvillus disappeared in t-BuOOH group. Excitedly, after treated with hesperidin, L02 cells exhibited considerable improvement in shape as compared to the t-BuOOH group.

while hesperidin noticeably and dose dependently attenuated this increase. MDA is a secondary oxidation product of lipids and serves as a good marker for lipid peroxidation progress. As shown in Table 1, t-BuOOH increased MDA production in L02 cells. However, hesperidin significantly prevented this unfavorable condition.

3.2. Effects of hesperidin on the indicators of oxidative stress

Most of the genes encoding antioxidant enzymes and phase II detoxifying have an antioxidant response element (ARE) sequence in their promoter region. Nrf2 is an important transcription factor that regulates ARE-driven gene expression. To examine whether hesperidin activated Nrf2 nuclear translocation, cells were treated with indicated concentrations of hesperidin for the indicated times

To examine the protective effects of hesperidin on t-BuOOH-induced oxidative stress, we tested the formation of ROS and MDA. As shown in Table 1, t-BuOOH significant increased the intracellular ROS levels (more than tow-fold) as compared to control group,

3.3. Hesperidin upregulated HO-1 gene expression We studied the ability of hesperidin to upregulate HO-1 in L02 cells. As shown in Fig. 3, hesperidin upregulated HO-1 expression at mRNA levels (Fig. 3B), protein levels (Fig. 3C and D) and HO-1 enzyme activity levels (Fig. 3A) in a dose-dependent manner. 3.4. Hesperidin facilitated Nrf2 nuclear translocation

Fig. 3. Effects of hesperidin on HO-1 expression. Cells were treated with or without hesperidin (20, 40 and 80 lM) for 24 h, and then prepare for measurements. (A) Effects of hesperidin on HO-1 enzyme activity. HO-1 activity was carried out as described in materials and methods section. (B) Effects of hesperidin on HO-1 mRNA expression. HO-1 mRNA expression was analyzed by RT-Q-PCR. (C) Effects of hesperidin on HO-1 protein expression. HO-1 protein expression was analyzed by western blot. Data shown are representative of twice independent experiments. (D) Scanning densitometry was used for semi-quantitative analysis in compared to control group levels. *p < 0.05 represent significant differences compared with control group.

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Fig. 4. Hesperidin facilitated Nrf2 nuclear translocation. (A) Effects of hesperidin on Nrf2 nuclear translocation. Cells were treated with different concentrations of hesperidin (20, 40 and 80 lM) for 24 h for western blot. (B) Scanning densitometry was used for semi-quantitative analysis in compared to control group levels. *p < 0.05 represent significant differences compared with control group.

and prepared for western blot analysis. Hesperidin did not significantly change cytoplasm Nrf2 levels, but remarkably increased Nrf2 accumulation in the nucleus (Fig. 4). 3.5. Hesperidin activated ERK/MAPK phosphorylation To further elucidate the upstream signaling pathway involved in hesperidin-mediated HO-1 upregulation and Nrf2 nuclear translocation activation, we additionally determined the functional significance of ERK/MAPK, p38/MAPK and JNK/MAPK pathways. Using western blot analysis, we found that hesperidin markedly activated phosphorylation of ERK1/2 in Fig. 5. No visibly changes in the expression of phosphorylation of JNK1/2 and p38 were detected. 3.6. Hesperidin regulated HO-1 and Nrf2 translocation via ERK/MAPK We additional investigated the role of MAPK signals in regulation of HO-1 and Nrf2 translocation. As shown in Fig. 6A and B, HO-1 gene expression induced by hesperidin was dependent on ERK/MAPK activations, as an inhibitor of the ERK/MAPK pathway remarkably abolished the enhancement role of HO-1 induced by hesperidin. In addition, Nrf2 translocation was also dependent on ERK/MAPK activations in Fig. 6C and D. 4. Discussion Recently, there has been a global trend toward the use of natural antioxidant phytochemicals found in natural resources, such as fruits, vegetables and herbs. Hesperidin, a naturally occurring flavonoid presents in fruits and vegetables, has been reported to exert a wide range of pharmacological effects including antioxidant, antihypercholesterolemic, anti-inflammatory and anticarcinogenic actions (Emin et al., 1994; Garg et al., 2001; Suarez et al., 1998; Tommasini et al., 2005). Many research studies have focused on the potential use of hesperidin as free radical scavengers and inhibitors of lipid peroxidation to prevent oxidative damage (Cai et al., 2006; Cotelle et al., 1996). In this study, we demonstrated that hesperidin facilitated ERK/MAPK phosphorylation, activated Nrf2 nuclear translocation, then upregulated HO-1 expression and thus prevented t-BuOOH-induced hepatocytes oxidative injury. In the present study, t-BuOOH dose dependently decreased the viability of L02 cells in the model system used. t-BuOOH at 150 lM showed about 50% inhibition of cell viability. t-BuOOH also affected cell membrane system integrity which displayed at MMP decrease and LDH release. Interestingly, pretreatment with hesperidin effectively increased cell survival and decreased LHD release

Fig. 5. Effects of hesperidin on the phosphorylation of MAPKs. Cells were treated with or without indicated concentrations of hesperidin (20, 40 and 80 lM) for 24 h and then cell extracts were prepared for western blot. (A) Effect of hesperidin on the phosphorylation of MAPKs. (B) Scanning densitometry was used for semi-quantitative analysis in compared to control group levels. Data shown are representative of twice independent experiments.*p < 0.05 represent significant differences compared with control group.

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Fig. 6. Effects of MAPKs on HO-1 expression and Nrf2 translocation. Cells were treated with or without 10 lM MAPKs inhibitors for 30 min, then incubated with 80 lM hesperidin for a further 24 h, and then prepared for western blot. (A) Effects of MAPKs inhibitors on hesperidin-medicated HO-1 upregulation. (B) Scanning densitometry was used for semi-quantitative analysis in compared to control group levels. (C) Effects of MAPKs inhibitors on hesperidin-medicated Nrf2 nuclear translocation. (D) Scanning densitometry was used for semi-quantitative analysis in compared to control group levels. Column 1: hesperidin + PD98059 group; Column 2: hesperidin + SP600125 group; Column 3: hesperidin + SB203580 group; Column 4: hesperidin group; Column 5: control group. Data shown are representative of twice independent experiments. *p < 0.05 represent significant differences compared with control group.

and MMP degrade. These indicated that hesperidin, in the range of concentrations tested, may protect L02 cells against t-BuOOH-induced cytotoxicity associated with its effects on the suppression of LHD release and MMP degrade. Oxidative stress refers to the mismatched redox equilibrium between the production of free radicals and the ability of cells to defend against them. There is a growing body of evidence implicating oxidative stress as a major cause of cellular injury in a variety of human diseases including liver cirrhosis and fibrosis (Loguercio and Federico, 2003). Several studies have indicated that the protective effects of flavonoids are derived from their free radical-scavenging activities, whereas the antioxidant properties are insufficient to explain their protective mechanism (Horvathova et al., 2003). In present study, t-BuOOH significantly enhanced the ROS generation in L02 cells. The increase in ROS generation may be instrumental in inducing cell injury, and consequently, may decrease the cell viability. The present results showed that hesperidin inhibited t-BuOOH-induced hepatocytes ROS generation. In addition, we also found that the accumulation of MDA induced by t-BuOOH was remarkably suppressed by hesperidin. This observation suggested that hesperidin exerted its protective effects against t-BuOOH cytotoxicity through the scavenging of free radicals and inhibiting lipid peroxidation. More and more evidence suggests that HO-1 provides cytoprotection in L02 cells to neutralize oxidative stress and HO-1 gene activation is an important adaptive mechanism to preserve homeostasis at the sites of liver injury (Choi et al., 2003; Nakahira et al., 2003; Tsuchihashi et al., 2007). HO-1 is induced by a wide range of stimuli, including various antioxidants (Balogun et al., 2003).

Many studies have demonstrated that hesperidin enhanced endogenous antioxidative defense activity (Garg et al., 2001; Loguercio and Federico, 2003). In the present study, hesperidin upregulated HO-1 gene expression in mRNA levels, protein levels, and enzyme activity levels. Previous reports have shown that Nrf2 plays a key role in regulating HO-1 expression (Balogun et al., 2003; Chen et al., 2005; Chow et al., 2005). Nrf2, a basic leucine zipper redox-sensitive transcriptional factor, plays a center role in ARE-mediated phase II detoxifying and antioxidant enzymes and has been reported to be a critical regulator in cell survival mechanisms (Motohashi and Yamamoto, 2004). It upregulates the expression of cytoprotective and antioxidant genes that attenuate liver injury (Okawa et al., 2006). It has been predicted that specific inducers of Nrf2 would make good chemoprotective reagents against ROS and chemical carcinogens. Indeed, induction of Nrf2 target genes enhanced protection against cancer, chemical toxicity, and certain chronic disorders (Hwanga et al., 2009). Plant-derived inducers including sulforaphane, 6-methylsulfinylhexyl isothiocyanate and curcumin were found to induce the activation of Nrf2 nuclear translocation (Chen and Kong, 2004; Morimitsu et al., 2002; Tsuchihashi et al., 2007). Our results indicated that the transcriptional activation of Nrf2 was directly involved in the regulation of HO-1 expression. To verify the cytoprotective effect of hesperidin on the cell signal transduction system, we performed western blot analysis upon upregulation of HO-1 and Nrf2 translocation, with a focus on the MAPK pathway. MAPK pathways have been reported to be involved in Nrf2-dependent translocation (Shen et al., 2004). In vertebrates, the three major kinase cascades are represented by ERK,

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JNK, and p38 MAPK (Johnson and Lapadat, 2002). All of these kinases appear to be involved to some extent in the activation of Nrf2 nuclear translocation in response to diverse stimulus. It is generally acknowledged that MAPK can be differentially regulated by the same stimuli in diverse cell types. ERK pathway is thought to mediate cellular responses to growth and differentiation factors, whereas JNK and p38 pathway are activated by distinct and overlapping sets of stress-related stimuli (Alam et al., 2000). For instance, quercetin induces Nrf2 nuclear translocation and HO1upregulation via the ERK/MAPK and p38/MAPK pathway but not the JNK/MAPK pathway in human hepatocytes (Yao et al., 2007). 3H-1,2-dithiole-3-thione increase nuclear Nrf2 accumulation via the ERK/MAPK pathway but not the p38/MAPK and JNK/MAPK pathway in murine keratinocytes (Manandhar et al., 2007). In PC12 cells, 15-Deoxy-D12, 14-prostaglandin J2 induces HO-1 expression via the ERK/MAPK and Akt/PI3K pathway but not the p38/MAPK and JNK/MAPK pathway (Kim et al., 2008). In the present study, hesperidin dose dependently facilitated the phosphorylation of ERK1/2, but neither p38 nor JNK. In addition, upregulation of HO-1as well as Nrf2 nuclear translocation by hesperidin was remarkably inhibited by PD98059, a highly selective inhibitor of ERK/MAPK pathway, suggesting that the activation of ERK/MAPK pathway by hesperidin contributed to HO-1 expression and Nrf2 nuclear translocation. In conclusion, t-BuOOH-induced hepatic L02 cell oxidative injury was prevented by hesperidin, which was accompanied by upregulation of HO-1. Hepatocytes treated with hesperidin exhibited elevated activation of ERK/MAPK, which appears to be responsible for nuclear translocation of Nrf2, thereby inducing HO-1 gene expression. As our study was limited to the cell level, further study is needed to confirm that hesperidin has a liver protective effect in organs or humans. However, these antioxidant properties, inhibitory effects against t-BuOOH -induced oxidative damage, and promotive endogenous antioxidative defense systems suggest that hesperidin might prove to be a promising therapeutic approach in the treatment of oxidative stress-related liver injury.

Conflict of Interest The authors declare that there are no conflicts of interest.

Acknowledgments This work was financially supported by the National Special Fund for State Key Laboratory of Bioreactor Engineering (No. 2060204) and the 111 Project (No.B07023) as well as Shanghai Committee of Science and Technology (No. 09JC1404500).

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