European Journal of Pharmacology 740 (2014) 634–640

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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar

Molecular and cellular pharmacology

AMPK activation by isorhamnetin protects hepatocytes against oxidative stress and mitochondrial dysfunction Guang-Zhi Dong a, Ju-Hee Lee a, Sung Hwan Ki b, Ji Hye Yang b, Il Je Cho a, Seung Ho Kang a,c, Rong Jie Zhao a,d,n, Sang Chan Kim a,n, Young Woo Kim a,e,n a

Medical research center for Globalization of Herbal Formulation, College of Oriental Medicine, Daegu Haany University, Daegu 706-828 Republic of Korea College of Pharmacy, Chosun University, Gwangju 501-759, Republic of Korea c Sunlin University, Pohang, Gyeongbuk, Republic of Korea d Department of Pharmacology, Mudanjiang Medical University, Heilongjiang, China e College of Oriental Medicine, Dongguk University, Gyeongju, Gyeongbuk, Republic of Korea b

art ic l e i nf o

a b s t r a c t

Article history: Received 6 December 2013 Received in revised form 11 June 2014 Accepted 17 June 2014 Available online 24 June 2014

Arachidonic acid (AA) is a ω  6 polyunsaturated fatty acid that is found in the phospholipids of membranes and released from the cellular membrane lipid bilayer by phospholipase A2. During this process, AA could produce excess reactive oxygen species and induce apoptosis and mitochondrial dysfunction by selectively inhibiting complexes I and III. Isorhamnetin, an O-methylated flavonol aglycone, has been shown to have cardio-protective, anti-adipogenic, anti-tumor, and anti-inflammatory effects. In the present study, we investigated the effects of isorhamnetin on hepatotoxicity and the underlying mechanisms involved. Our in vitro experiments showed that isorhamnetin dose-dependently blocked the hepatotoxicity induced by treatment with AA plus iron in HepG2 cells. Furthermore, isorhamnetin inhibited the AA þiron induced generation of reactive oxygen species and reduction of glutathione, and subsequently maintained mitochondria membrane potential in AAþ iron treated HepG2 cells. In addition, isorhamnetin activated AMP-activated protein kinase (AMPK) by Thr-172 phosphorylation of AMPKα, and this was mediated with Ca(2 þ)/calmodulin-dependent protein kinase kinase-2 (CaMKK2), but not liver kinase B1. Experiments using CaMKK2 siRNA or its selective inhibitor, STO-609, revealed the role of CaMKK2 in the isorhamnetin-induced activation of AMPK in HepG2 cells. These results indicate isorhamnetin protects against the hepatotoxic effect of AA plus iron, and suggest that the AMPK pathway is involved in the mechanism underlying the beneficial effect of isorhamnetin in the liver. & 2014 Elsevier B.V. All rights reserved.

Keywords: AMPK Oxidative stress Mitochondria Isorhamnetin Chemical compounds studied in this article: Isorhamnetin (PubChem CID: 5281654)

1. Introduction Oxidative stress promotes cellular damage and is a characteristic feature of several human diseases. For example, it has been shown that modification of membrane phospholipids by excess reactive oxygen species results in cell and tissue injury (Apel and Hirt, 2004; Bergamini et al., 2004; Reddy and Clark, 2004; Shah

Abbreviations: AA, arachidonic acid; ACC, acetyl-CoA carboxylase; AMPK, AMPactivated protein kinase; CaMKK2, Ca(2 þ )/calmodulin-dependent protein kinase kinase-2; DCFH-DA, 20 ,70 -Dichlorofluorescein diacetate; FITC, fluorescein isothiocyanate; GSH, glutathione; IsoRN, Isorhamnetin; LKB1, liver kinase B1; MMP, mitochondrial membrane potential; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; PI, propidium iodide. n Corresponding authors at: Medical research center for Globalization of Herbal Formulation, College of Oriental Medicine, Daegu Haany University, Daegu 706828, Republic of Korea. Tel.: þ82 53 819 1861; fax: þ 82 53 819 1860. E-mail addresses: [email protected] (R.J. Zhao), [email protected] (S.C. Kim), [email protected] (Y.W. Kim). http://dx.doi.org/10.1016/j.ejphar.2014.06.017 0014-2999/& 2014 Elsevier B.V. All rights reserved.

and Channon, 2004; Valko et al., 2006; Willner, 2004). During conditions of oxidative and/or inflammatory stress, reactive oxygen species and/or cytokines can induce the oxidative modification of fatty acids within membrane phospholipids, and during this process, arachidonic acid (AA; a ω-6 polyunsaturated fatty acid) is released from the cellular membrane lipid bilayer (Balboa and Balsinde, 2006; Gijon and Leslie, 1999). AA can induce cell death by promoting the uptake of calcium by mitochondria and the production of ceramide (Balboa and Balsinde, 2006; Gijon and Leslie, 1999). In particular, in the presence of iron, released AA stimulates cells to produce more reactive oxygen species, which can induce mitochondrial dysfunction and cell death (Fleming and Bacon, 2005; Galaris and Pantopoulos, 2008; Halliday and Searle, 1996; Neufeld, 2006). Therefore, treatment with AA plus iron offers a treatment model that could be useful for screening agents that protect mitochondria against severe oxidative stress. AMP-activated protein kinase (AMPK) is a multifunctional cytosolic protein that plays important roles in energy homeostasis,

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nutrient metabolism, cell survival, and apoptosis. Although AMPK is activated by cellular stresses including changes in the AMP/ATP ratio (Hardie and Carling, 1997), hypoxia (Sambandam and Lopaschuk, 2003), glucose deprivation (Xing et al., 2003) and osmotic stress (Tian et al., 2001), AMPK activators (e.g., metformin and dithiolethiones) increase cell survival by preventing changes in mitochondrial membrane potential (Detaille et al., 2005; Ido et al., 2002). Furthermore, some beneficial components and extracts of medical herbs protect cells against the severe oxidative stress induced by AA þiron by activating AMPK (Dong et al., 2013; Kim et al., 2009a; Shin and Kim, 2009). In traditional Oriental medicine, water dropwort (Oenanthe javanica) is used to treat disorders in the lung, stomach and liver. Isorhamnetin (IsoRN, 30 -O-methyl quercetin) is an active flavonol aglycone found in the water dropwort, and has been reported to have multiple biological activities, which include cardioprotective, anti-adipogenic, and antitumor activities (Kim et al., 2011; Kong et al., 2009; Sun et al., 2012; Sun et al., 2013; Upadhyay et al., 2010; Zhang et al., 2011). Recently, we isolated IsoRN from the water dropwort and demonstrated its effects on acute inflammation in vivo and in vitro (Yang et al., 2013). In view of the importance of AMPK from the standpoint of cytoprotection and its potential as a cytoprotective, we hypothesized that IsoRN might protect against oxidative stress. Our work demonstrates that IsoRN protects cells against AA þiron induced apoptosis by inhibiting mitochondrial dysfunction and reactive oxygen species production. Furthermore, IsoRN was found by immunoblotting and immunoprecipitation analysis, to activate AMPK in the HepG2 cell line, which could explain in part the mitochondrial protective effect of IsoRN.

2. Materials and methods 2.1. Reagents IsoRN was purified from water dropwort (Oenanthe javanica, Umbelliferae) using successive silica gel column chromatography, as previously described (Yang et al., 2013). The purity of the isolated IsoRN (Z 97%) was confirmed by ultra performance liquid chromatography analysis and the structures was verified on the basis of spectroscopic analyses including HPLC–ESI–MS (Agilent 6120 LC/MS system, Agilent Technologies, Palo Alto, CA) and NMR spectroscopy (data not shown), and compared with reported spectral data (Cao et al., 2009). Arachidonic acid (AA), compound C and STO-609 were purchased from Calbiochem (San Diego, CA, USA). Anti-procaspase-3, anti-phospho-acetyl-CoA carboxylase (ACC), anti-PARP and anti-phospho-AMPK antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). AntiAMPK, horseradish peroxidase-conjugated goat anti-rabbit, rabbit anti-goat, goat anti-mouse IgGs, CaMKK2 siRNA and liver kinase B1 (LKB1) siRNA were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Ferric nitrate, nitrilotriacetic acid, 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), rhodamine 123, 20 ,70 -Dichlorofluorescein diacetate (DCFH-DA), anti-β-actin antibody and other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). 2.2. Cell culture HepG2 (human), AML12 (mouse), and H4IIE (rat) hepatocytederived cell lines were obtained from ATCC (Rockville, MD, USA). HepG2 and H4IIE were cultured in Eagle's minimum essential medium (DMEM) with 10% FBS. AML12 cells were cultured in DMEM/F12 medium. Cells were incubated in medium without 10% FBS for 12 h, and then stimulated with 10 μM AA for 12 h, followed by incubation to 5 μM iron. To determine the effects of IsoRN, the

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cells were treated with IsoRN for 1 h prior to the exposure with AA (Dong et al., 2013). 2.3. MTT assay The MTT assay was performed as previously described (Dong et al., 2013). HepG2 cells were cultured in a 48-well plate with density of 1  105 cells/well. HepG2 cells were stained with 0.25 mg/ml MTT for 2 h after treatment of IsoRN or AA þiron or combination of AA þiron and IsoRN. The formazan crystals products were dissolved with the addition of 200 μl dimethylsulfoxide after remove media. Then, take 100 μl into 96-well plate and read the absorbance at 540 nm by ELISA microplate reader (Tecan, Research Triangle Park, NC, USA). 2.4. Annexin V and propidium iodide (PI) double staining HepG2 cells were treated with IsoRN or AA þiron or combination of AA þ iron and IsoRN, and then stained with fluorescein isothiocyanate (FITC) Annexin V and PI staining kit (Life Technology, Grand Island, USA) by manual. The 2  105 cells were counted by flow cytometer (FACS, Partec, Münster, Germany) and measured Fluorescence intensity of individual cell. 2.5. Immunoblot analysis Lysis of cells and immunoblot analysis were performed as previously described (Dong et al., 2013). Cells were lysed in RIPA lysis buffer, and heated with SDS loading dye. The proteins were loaded onto SDS-PAGE gels, and transfer to NC membrane. The protein bands of interest were developed using an ECL chemiluminescence system (Amersham, Buckinghamshire, UK) after incubation with 1st and 2nd antibody. 2.6. Measurement of H2O2 production The level of reactive oxygen species generation was determined by the concomitant increase in DCF fluorescence by a Fluorescence spectrometer (Partec, Münster, Germany). Cells were cultured in multi-well plates and treated with IsoRN or AA. After treatment, the cells were incubated with 10 μM DCFH-DA for 30 min, then incubated with iron for 1 h at 37 1C. Fluorescence intensity in the cells was measured using the Fluorescence spectrometer (Tecan, Research Triangle Park, NC, USA). 2.7. Determination of reduced GSH content Glutathione (GSH) concentration in the HepG2 cells was measured by using a two-step chemical GSH #400 kit (Oxis International, Portland, OR, USA) (Dong et al., 2013). After treatments, cells were homogenized and measured the concentration of GSH by a spectrometer (Tecan, Research Triangle Park, NC, USA). 2.8. Flow cytometric analysis of mitochondrial membrane potential (MMP) MMP was measured by FACS after staining with rhodamine 123, a membrane-permeable cationic fluorescent dye. After treatment of iron for 1 h, cells were stained with 0.05 μg/ml rhodamine 123 for 1 h, and harvested by trysinization. The change in MMP was measured the fluorescence intensity of cells by a FACS. In each analysis, 10,000 events were recorded.

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2.9. Transient transfection

3. Results

Cells were transiently transfected with siRNA for CaMKK2 or LKB1 for 3 h in the presence of Lipofectamines reagent.

3.1. IsoRN inhibits AA þiron-induced hepatocyte death

2.10. Immunoprecipitation Cells were lysed by immunoprecipitation lysis buffer after treatment of IsoRN (Anderson et al., 2008). Protein concentration was determined using a bicinchoninic acid protein assay kit (Thermo Scientific, Rockford, IL, USA), then equal amount of proteins (1 mg) were incubated with CaMKK2 or AMPK antibody in 1 ml immunoprecipitation lysis buffer overnight at 4 1C. Protein A/G magnetic beads were added to the complex, and precipitated the complex by magnetic field. After washing the beads, the complex was heated with 1 X SDS loading dye and run onto SDS-PAGE gels. The protein bands were developed using an ECL chemiluminescence system (Amersham, Buckinghamshire, UK) after incubation with HRPlabeled 1st antibody for overnight (Lightning-LinkTM HRP conjugation kit, Innova Bioscience, Babraham, UK).

2.11. Data analysis One way analysis of variance procedures were used to assess significant differences among treatment groups. For each significant treatment effect, the Newman–Keuls test was utilized to compare multiple group means.

Increasing concentrations (3–100 μM) of IsoRN were used to investigate its protective effect against the hepatotoxic effect of AA þiron, which significantly reduced cell viability, as assessed by MTT assay (Fig. 1A). IsoRN was found to protect HepG2 cells in a dose-dependent manner with a peak effect at 30 μM. Subsequent studies were conducted by treating HepG2 wells with IsoRN at this concentration. To confirm the protective effect of IsoRN, cells were stained with annexin V and PI (Fig. 1B), which do not stain live cells (PI-negative and annexin V-negative). As shown in Fig. 1C, pretreatment with IsoRN completely prevented cell death caused by AA þiron. To further examine the protective effects of IsoRN on AA þiron induced cell toxicity, we measured levels of procaspase-3 and BclXL by immunoblot analysis. Treatment of AA þiron markedly reduced the levels of procaspase-3 and BclXL, and these effects were completely blocked by IsoRN pretreatment (Fig. 1D). Taken together, these results show that IsoRN protects HepG2 cells against AA þ iron induced apoptosis. 3.2. IsoRN inhibits AA þiron-induced reactive oxygen species generation and GSH reduction Next, we investigated whether IsoRN inhibits reactive oxygen species generation in AA þiron treated hepatocytes by using a spectrometer. There was no increase in reactive oxygen species

Fig. 1. Effect of isorhamnetin (IsoRN) on hepatocytotoxicity. (A) Cell viability was measured by the MTT assay. HepG2 cell, a human derived hepatocyte cell line, was incubated with 10 μM arachidonic acid (AA) for 12 h and then additionally treated with 5 μM iron for 4 h. Data represent the mean 7 S.E.M. of four replicates. The statistical significance of differences was compared with the vehicle-treated control group (nn indicates Po 0.05), or compared with AA þ iron-treated group (# and ## indicate Po 0.05 and P o0.01, respectively). (B) HepG2 cells were treated AA þ iron with or without 30 μM IsoRN as described in the above, and then stained with annenxin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI), and counted the fluorescence intensity by FACS. (C) The percentage of annenxin V-FITC positive cells or PI positive or both positive or both negative was quantified. Data represent the mean7 S.E.M. of three separate experiments. (nn indicates P o0.01 as compared with fluorescence negative fraction of vehicle-treated control; ## indicates Po 0.01 as compared with fluorescence negative fraction of AA þiron-treated group). (D) Immunoblot analysis of procaspase 3, BclXL and β-actin were performed on the lysates of HepG2 cells that had been treated as described in the above. Equal protein loading was verified by β-actin. Results were confirmed by repeated experiments.

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generation in cells treated with IsoRN alone, but treatment with AA þiron significantly increased reactive oxygen species levels (Fig. 2A). However, IsoRN completely inhibited reactive oxygen species produced by AA þiron. To confirm the anti-oxidative effect of IsoRN, we measured GSH contents using a colorimetric method. AA þiron reduced the intracellular concentration of GSH in HepG2 cells (Fig. 2B), and pretreatment with IsoRN inhibited this reduction. On the other hand, treatment with IsoRN alone had no effects on cellular GSH levels. These results indicate that IsoRN inhibits the production of reactive oxygen species and the reduction of GSH induced by AA þ iron.

3.3. IsoRN inhibits mitochondrial dysfunction Mitochondria are a major source of reactive oxygen species generation and oxidative stress. Therefore, we also examined the effect of AA þiron on mitochondrial dysfunction and the protection afforded by IsoRN in HepG2 cells stained with rhodamine 123. Rhodamine fluorescence intensity was not altered in IsoRN treated cells versus untreated controls (Fig. 3A), whereas AA þiron reduced the rhodamine fluorescence, meaning AA þiron increased the population of rhodamine 123-negative cells, RN1 fraction. As was expected, pretreatment with IsoRN successfully prevented AA þiron induced reduction in fluorescence (Fig. 3B). These results

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indicate IsoRN protects hepatocytes by inhibiting the mitochondrial dysfunction caused by AA þiron. 3.4. IsoRN activates AMPK pathway To investigate the mechanism responsible for the effects of IsoRN, AMPK activation was examined by immunoblotting. Treatment of HepG2 cells with 30 μM IsoRN significantly induced the phosphorylation of AMPK, which peaked at 0.25–1 h (Fig. 4A and B). In addition, ACC, a primary downstream target of AMPK, was phosphorylated when the hepatocyte derived cell lines HepG2, H4IIE and AML12 were treated with IsoRN (Fig. 4A). To determine the pathway of AMPK activation by IsoRN, we used siRNAs targeting CaMKK2 or LKB1, the major upstream kinases of AMPK. AMPK phosphorylation by IsoRN was decreased by CaMKK2 knock-down (Fig. 4C), but not by LKB1 knock-down (data not shown). Pretreatment with STO609 (1 μg/ml), a chemical inhibitor of CaMKK2, also blocked the ability of IsoRN to activate AMPK (Fig. 4D). It has been previously shown that CaMKK2 regulates AMPK by forming a physical binding complex between CaMKK2 and AMPK (Anderson et al., 2008; Green et al., 2011), and thus, we explored the interaction between CaMKK2 and AMPK. In HepG2 cells, IsoRN increased the association between CaMKK2 and AMPK (as determined by immunoprecipitation and immunoblot assays by using antibodies against CaMKK2 and AMPK; Fig. 4E). These results

Fig. 2. Effect of IsoRN on oxidative stress. HepG2 cells were treated as descripted in the method section, and cellular reactive oxygen species production was monitored by measuring intensity of dichlorofluorescein fluorescence (A), and cellular GSH content was assessed in cells by using GSH assay kit (B). Data represent the mean7 S.E.M. of three separate experiments. The statistical significance of differences between treatments and either the vehicle-treated control (nnP o0.01) or cells treated with AA þiron (##P o 0.01) was determined.

Fig. 3. Effect of IsoRN on mitochondrial dysfunction. (A) HepG2 cells were treated with IsoRN for 1 h, followed by incubation with AA (12 h) and iron (1 h). The cells were stained with rhodamine 123 for 1 h and measured rhodamine 123 intensity by FACS. Treatment of AA þiron increased the subpopulation of RN1 fraction (low rhodamine 123 fluorescence), as indicated by the left shift. (B) Relative MMP (%) represents the mean 7 S.E.M. of three separate experiments. The statistical significance of differences was compared with vehicle-treated control (nnPo 0.01) or AA þiron-treated group (##Po 0.01).

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Fig. 4. The effects of IsoRN on AMPK activation. (A) Immunoblot analysis of p-AMPK, AMPK, p-ACC and β-actin were performed in lysates of HepG2, H4IIE and AML12 cells that had been treated with 30 μM of IsoRN for the indicated time periods. (B) Relative protein level of AMPKα phosphorylation in HepG2 cells was determined by repeated experiments. Data represent the mean 7 S.E.M. (treatment mean significantly different from vehicle-treated control, nP o0.05, nnPo 0.01). (C) AMPKα phosphorylation was inhibited by knock-down of CaMKK2. (D) AMPKα phosphorylation was reduced by STO609 (CaMKK2 inhibitor). HepG2 cells were treated with STO609 (1 μg/ml) for 1 h and treated IsoRN (30 μM). (E) Co-immunoprecipitation of CaMKK-β and AMPK was accessed after treatment with or without IsoRN.

Fig. 5. The effect of AMPK activation on protection of mitochondria. (A) IsoRN-induced AMPKα phosphorylation was decreased by treatment of compound C (AMPK inhibitor). HepG2 cells were incubated with IsoRN for 15 min following treatment of 5 μM compound C for 30 min. (B) The effect of IsoRN on MMP was revered by compound C. After treatment with 5 μM compound C for 30 min, HepG2 cells were treated with IroRN and/or ironþ AA, and stained with rhodamine 123 as described in the legend of Fig. 3A. The statistical significance of differences was compared with AA þiron-treated group (nnPo 0.01) or compoun C þ AAþ iron-treated group (##P o0.01).

indicate the AMPK pathway is activated by IsoRN, and that CaMKK2 is responsible for AMPK activation by IsoRN. 3.5. IsoRN inhibits mitochondrial dysfunction via AMPK pathway To determine the role of AMPK activation in hepatoprotective effect by IsoRN, we measured MMP levels after treating with Compound C (an AMPK inhibitor). Compound C inhibited AMPK phosphorylation by IsoRN (Fig. 5A), and inhibited the mitochondrial protective effect of IsoRN in the presence of AA þiron (Fig. 5B). These data indicate that IsoRN activates the AMPK pathway, and that this activation is responsible for its protection of mitochondria against oxidative stress.

4. Discussion Oxidative and inflammatory stress are the most common causes of cellular dysfunctions in liver, heart and the circulatory system and in diabetes, ageing, and infection (Apel and Hirt, 2004;

Bergamini et al., 2004; Reddy and Clark, 2004; Shah and Channon, 2004; Valko et al., 2006; Willner, 2004). Previously, we demonstrated that IsoRN inhibited the acute phase of inflammation induced by carrageenan injection in rats, and the protein expression of inducible nitric oxide synthase and productions of inflammatory cytokines in macrophages (Yang et al., 2013). In addition, we previously reported that IsoRN inhibits cyclooxygenase-2 expression in response to inflammation caused by heme oxygenase-1 induction and reactive oxygen species production (Seo et al., 2014). In the present study, we found IsoRN has protective effects against severe oxidative stress and mitochondrial dysfunction. In an effort to confirm the anti-oxidant effect of IsoRN, we used two approaches, that is, by examining its effects on HepG2 cells treated with AA þiron and by determining its underlying molecular mechanism using a line of hepatocytes. Under normal conditions, a balance is maintained between reactive oxygen species and intercellular antioxidants, and reactive oxygen species act as intracellular signals (Hawley et al., 2005; Nohl et al., 2003; Scandalios, 2002). However, reactive oxygen species overproduction can damage cell components, such as

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nucleic acids, lipids and proteins (Apel and Hirt, 2004; Bergamini et al., 2004; Reddy and Clark, 2004; Shah and Channon, 2004; Valko et al., 2006; Willner, 2004). In addition, free iron induces cell death by inducing a reactive oxygen species accumulation in organs (Reddy and Clark, 2004; Valko et al., 2006). In liver, oxidative stress induced by iron accumulation can cause the pathological process associated with various chronic liver diseases, such as fibrosis, cirrhosis and hepatocellular carcinoma (Thursz, 2007; Price and Kowdley, 2009; Hino et al., 2013). Furthermore, it has been shown that excess iron causes the release of AA by increasing oxidative stress and changing the levels of membrane phospholipids (Mattera et al., 2001; Tadolini and Hakim, 1996). Although prostaglandins produced from AA may protect cells in some tissues, AA also induces cellular and mitochondrial reactive oxygen species production and reduces mitochondrial respiration. In addition, AA may increase cellular Ca2 þ contents and stimulate the uptake of Ca2 þ by mitochondria (Scorrano et al., 2003). Caro and Cederbaum (2001) and our group (Kim et al., 2009b) previously showed that treatment of AA þ iron caused substantial cellular toxicity and mitochondrial damage. In the present study, we used AA þiron treated hepatocytes as an in vitro model to study the effects of IsoRN, a therapeutic anti-oxidant candidate. Recently, several studies have been conducted to determine the antioxidant capacities of natural products, and as result, a large number of natural compounds have been reported to possess good antioxidant properties (Ndhlala et al., 2010). IsoRN is a flavonol aglycone that is found in herbs and plants used as traditional medicines, such as the water dropwort. Furthermore, the molecular mechanisms responsible for the effects of IsoRN have been the focus of study. Yang et al. (2013) reported IsoRN inhibited the activation of macrophages by blocking the mitogen-activated protein kinase/nuclear factor-κB signaling pathway. Seo et al. (2014) found IsoRN blocks the generations of reactive oxygen species and/or reactive nitrogen species and prevents cell death caused by bacteria. In the present study, IsoRN protected hepatocytes against AA þiron induced oxidative stress and mitochondrial dysfunction, and significantly inhibited excessive hydrogen peroxide production and maintained cellular GSH levels. AMPK is a crucial mediator of cellular energy homeostasis and determines survival/death in the presence of oxidative stress and endoplasmic reticulum stress (Hayashi et al., 2000; Terai et al., 2005). Furthermore, AMPK is emerging as an attractive therapeutic target for the treatment of hepatic disorders (Viollet et al., 2009). AMPK activity is modulated by various pharmacological and natural drugs, such as adiponectin, leptin, 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR), A-769662, polyphenols, and two major classes of antidiabetic drugs biguanides (e.g., metformin and phenformin) and thiazolidinediones (TZDs) (Viollet et al., 2009). Recently, AMPK was also found to be a kinase that controls mitochondrial function (Sid et al., 2013). The importance of the role of AMPK is strengthened by previous studies. Oltipraz (a cancer chemopreventive agent) was found to have a cytoprotective effect against the severe oxidative stress elicited by AAþ iron, in a manner mediated by the AMPK-dependent inhibition of mitochondrial impairment, ER stress, and reactive oxygen species production (Shin and Kim, 2009). Resveratrol, the bioactive component of red wine, also protected mitochondria from AAþ iron induced oxidative stress via the AMPK pathway (Shin et al., 2009), and the activation of AMPK by isoliquiritigenin, a flavonoid found in licorice, also protected mitochondria against radical stress (Choi et al., 2010). Therefore, AMPK is a potential target for mitochondrial protection in the presence of oxidative stress. In the present study, IsoRN activated AMPK in a line of hepatocytes, and this activation protected hepatocytes against the cytotoxic effect of AAþ iron. In addition, the beneficial effect of IsoRN on mitochondria was confirmed by the inhibition of AMPK by compound C.

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AMPK can be activated by changes in intracellular AMP/ATP ratio or increases in intracellular Ca2 þ concentration (Viollet et al., 2009). In mammalian cells, AMPK activation is regulated by LKB1 and CaMKK2, the major upstream kinases that phosphorylate Thr172 in the activation loop of the catalytic α-subunit of AMPK. LKB1 is mainly involved in AMPK activation after a change in AMP/ ATP ratio. On the other hand, CaMKK2 is viewed as an alternate upstream kinase that could also activate AMPK in intact cells in an AMP-independent manner in response to increased intracellular Ca2 þ concentrations (Hawley et al., 2005; Hurley et al., 2005). AMPK activation by resveratrol-activated SIRT1 is mediated by the upstream kinase, LKB1, but not CaMKK2 (Shin et al., 2009). Sauchinone, a cytoprotective lignan in Saururus chinensis, also induced LKB1-dependent AMPK activation (Kim et al., 2009b), and epigallocatechin-3-gallate (EGCG; a green tea polyphenol) has been recently reported to potently activate AMPK. Furthermore, it has been shown that EGCG-induced AMPK phosphorylation is mediated by CaMKK activation via the production of reactive oxygen species (Collins et al., 2007). In the present study, the activation of AMPK by IsoRN was inhibited by CaMKK2 knockdown but not by LKB1 knock-down, which shows its activation of AMPK may depend on upstream CaMKK2. In addition, pretreatment with STO609 (a commercial CaMMK2 inhibitor) also blocked the ability of IsoRN to induce the phosphorylation of AMPK. Moreover, immunoprecipitation assays revealed that IsoRN increased AMPK to CaMKK2 binding, which is consistent with previous reported findings regarding the CaMKK2 activation of AMPK via AMPK-CaMKK2 complex formation (Anderson et al., 2008; Green et al., 2011). Taken together, it appears that IsoRN activates AMPK in a CaMKK2 mediated manner, and that this activation facilitates its protection of hepatocytes against AA þiron induced oxidative stress. However, it remains to be determined its direct target, the pharmacological upstream kinases in the aspect of cytoprotection.

5. Conclusion In conclusion, the present study shows that IsoRN inhibits reactive oxygen species generation, mitochondrial dysfunction, and the decrease in GSH levels induced by AA þiron. In addition, the cellular protective effects of IsoRN were found to involved activation of the AMPK-CaMKK2 pathway. We believe these results demonstrate showing IsoRN is a potential therapeutic candidate for the prevention of liver disease.

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AMPK activation by isorhamnetin protects hepatocytes against oxidative stress and mitochondrial dysfunction.

Arachidonic acid (AA) is a ω-6 polyunsaturated fatty acid that is found in the phospholipids of membranes and released from the cellular membrane lipi...
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