http://informahealthcare.com/dct ISSN: 0148-0545 (print), 1525-6014 (electronic) Drug Chem Toxicol, Early Online: 1–8 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/01480545.2014.928719
ORIGINAL ARTICLE
Protective effect of dieckol against chemical hypoxia-induced cytotoxicity in primary cultured mouse hepatocytes Yu Jin Jeon1, Hyoung Seok Kim1, Kyung-Sik Song2, Ho Jae Han3, Soo Hyun Park4, Woochul Chang5, and Min Young Lee1 Drug and Chemical Toxicology Downloaded from informahealthcare.com by Gazi Univ. on 01/08/15 For personal use only.
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Department of Molecular Physiology, College of Pharmacy, Kyungpook National University, Daegu, Korea, 2College of Pharmacy, Research Institute of Pharmaceutical Sciences, Kyungpook National University, Daegu, Korea, 3Department of Veterinary Physiology, College of Veterinary Medicine, Seoul National University, Seoul, Korea, 4Department of Veterinary Physiology, College of Veterinary Medicine, Chonnam National University, Gwangju, Korea, and 5Department of Biology Education, College of Education, Pusan National University, Busan, Korea Abstract
Keywords
Hepatic ischemic injury is a major complication arising from liver surgery, transplantation, or other ischemic diseases, and both reactive oxygen species (ROS) and pro-inflammatory mediators play the role of key mediators in hepatic ischemic injury. In this study, we examined the effect of dieckol in chemical hypoxia-induced injury in mouse hepatocytes. Cell viability was significantly decreased after treatment with cobalt chloride (CoCl2), a well-known hypoxia mimetic agent in a time- and dose-dependent manner. Pretreatment with dieckol before exposure to CoCl2 significantly attenuated the CoCl2-induced decrease of cell viability. Additionally, pretreatment with dieckol potentiated the CoCl2-induced decrease of Bcl-2 expression and attenuated the CoCl2-induced increase in the expression of Bax and caspase-3. Treatment with CoCl2 resulted in an increased intracellular ROS generation, which is inhibited by dieckol or N-acetyl cysteine (NAC, a ROS scavenger), and p38 MAPK phosphorylation, which is also blocked by dieckol or NAC. In addition, dieckol and SB203580 (p38 MAPK inhibitor) increased the CoCl2-induced decrease of Bcl-2 expression and decreased the CoCl2-induced increase of Bax and caspase-3 expressions. CoCl2-induced decrease of cell viability was attenuated by pretreatment with dieckol, NAC, and SB203580. Furthermore, dieckol attenuated CoCl2-induced COX-2 expression. Similar to the effect of dieckol, NAC also blocked CoCl2induced COX-2 expression. Additionally, CoCl2-induced decrease of cell viability was attenuated not only by dieckol and NAC but also by NS-398 (a selective COX-2 inhibitor). In conclusion, dieckol protects primary cultured mouse hepatocytes against CoCl2-induced cell injury through inhibition of ROS-activated p38 MAPK and COX-2 pathway.
Cobalt chloride, dieckol, hypoxic cell injury, mouse hepatocytes, reactive oxygen species
Introduction Hepatic tissue is quite vulnerable to hypoxic (ischemic) injury compared with other organs (Nakanishi et al., 1995). Hypoxic injury is a common cause of oxidative tissue damage caused by many liver diseases and hepatic surgery (Lehwald et al., 2011). The generation of reactive oxygen species (ROS) during hypoxia as well as in the presence of cobalt chloride (CoCl2) had been described previously (Chandel et al., 1998; Chandel & Schumacker, 1999; Guillemin & Krasnow, 1997; Semenza, 1999). Interestingly, exposure to CoCl2 triggers transcriptional changes that mimic hypoxic response, including up-regulation of the hypoxia-inducible factor-1a (HIF1a), erythropoietin, and glycolytic enzymes (An et al., 1998;
Address for correspondence: Min Young Lee, DVM, PhD, Department of Molecular Physiology, College of Pharmacy, Kyungpook National University, 80 Daehak-ro, Buk-gu, Daegu 702-701, Korea. Tel: +82-53950-8577. Fax: +82-53-950-8557. E-mail: [email protected]
History Received 10 September 2013 Revised 22 March 2014 Accepted 19 May 2014 Published online 18 August 2014
Chandel et al., 1998; Jiang et al., 1997). One common mechanism that may lead to these changes in gene expression is the increased generation of ROS (Chandel et al., 1998). ROS are implicated in the initiation and progression of liver pathologies (Pietrangelo, 1996). Elevated ROS is capable of directly targeting nucleic acids, proteins, and membrane phospholipids, leading to apoptosis of various cell types including hepatocytes (Cao et al., 2001; Chen et al., 2008; Knight et al., 2002; Wang et al., 2000). In addition, ROS produced during oxidative stress have been reported to initiate signaling cascades, which lead to apoptosis (Yu et al., 2008). Available evidence suggests that excessive ROS have been shown to activate members of the MAPK family, including extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun-N-terminal kinase (JNK), and p38 MAPK in various types of cells (Kim et al., 2005; Ouyang & Shen, 2006; Yeo et al., 2008). Especially, the p38 MAPK pathway is known to be stimulated by a variety of environmental stresses and chemicals (Roulston et al., 1998) and is an important
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signaling pathway associated with ROS-induced apoptosis (Jameel et al., 2009). It has been reported that CoCl2 activates p38 MAPK, which is known to be involved in CoCl2-induced apoptosis in other cell types (Zou et al., 2002). It has been reported that ROS can stimulate cyclooxygenase (COX)-2 expression in CoCl2-induced chemical hypoxic condition (Yang et al., 2011; Yeo et al., 2008). Hepatic ischemic injuries result from complex interactions between various inflammatory mediators including COX-2 (Fu et al., 2012). At present, at least two COX isoenzymes, COX-1 and COX-2, have been identified (Yokoyama & Tanabe, 1989). COX-1 is usually expressed constitutively, whereas COX-2 remains undetectable in most tissues in the physiological state but rapidly accumulates under pathological conditions (Dupouy et al., 2006). Although the role of COX-2 overexpression is complicated and remains ambivalent in different cell types, mediating either cytotoxicity or cytoprotection (Booth et al., 2008; Carnieto et al., 2009; Kwak et al., 2010), the inhibition of COX-2 by selective inhibitors, or gene knock-out leads to a significant reduction in ischemiainduced hepatic damage (Hamada et al., 2008; Ozturk & Gezici, 2006). In the present study, we investigated the cytoprotective effect induced by adding dieckol to primary cultured mouse hepatocytes treated with CoCl2, a widely used hypoxia mimetic agent that promotes the accumulation of hypoxia-inducible factor-1 a (HIF-1a), which is a critical regulator for the cellular response to hypoxia (Guo et al., 2006) and which induces oxidative stress (Jung et al., 2007; Zou et al., 2001) and apoptosis in various cells (Kim et al., 2003; Zou et al., 2001). Dieckol is a phloroglucinol derivative isolated from marine brown alga Ecklonia cava and is known to play a variety of biological functions in vitro and in vivo, for example, in antioxidant, antitumor, antihuman immunodeficiency virus (HIV), and anti-inflammatory activities, and also provides protective effects against oxidative stress (Lee et al., 2010; Zhang et al., 2011). Dieckol displays excellent antioxidant properties. However, the detailed molecular mechanisms that play a part in the action of dieckol in hepatocytes on hypoxic injury remain to be clearly elucidated. Therefore, this study assessed the effect of dieckol on hypoxic cellular injury induced by CoCl2 and its associated mechanisms in primary cultured mouse hepatocytes.
Materials and methods Materials Eight-week-old male ICR mice were purchased from Daehan Bio Link Co. (Incheon, Korea). All animal management procedures were conducted in accordance with the standard operation protocols established by Kyungpook National University. Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Laboratories (Japan). CoCl2, N-acetyl-L-cysteine, SB203580, and type IV collagenase were obtained from Sigma-Aldrich (St. Louis, MO). The 5-(and 6)-chloromethyl20 ,70 -dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA) were purchased from Life Technologies (Grand Island, NY). Fetal bovine serum (FBS) was acquired from PAA Laboratories (GmbH, Canada). The phospho-p38 MAPK antibodies were obtained from New England Biolabs
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(Hertfordshire, UK). The HIF-1a, Bcl-2, caspase-3, COX-1, COX-2, goat anti-rabbit IgG, and goat anti-mouse IgG antibodies were supplied by Santa Cruz Biotechnology (Santa Cruz, CA). Extraction and purification of dieckol Freshly collected Ecklonia cava (E. cava) was washed, freezedried, milled into small pieces, and extracted by methanol. The methanol extract of dried E. cava was partitioned with nhexane to remove nonpolar components. The resulting methanol fraction was further fractionated between water and n-buthanol. The n-buthanol fraction was subjected to reverse-phase flash column chromatography and three fractions were given. Each fraction was subjected to High-Pressure Liquid Chromatography (HPLC) to take polyphenolic compounds. The chemical structure of dieckol was determined by spectroscopy (Figure 1). The NMR spectra were recorded on a JEOL ECP-500 FT NMR at 500 MHz for 1H and 125 MHz for 13C, using Tetramethylsilane (TMS) as an internal standard. HPLC was performed on a HP 5980A. Fast Atom Bombardment Mass Spectrometry (FABMS) was obtained with a VG Autospec Ultma mass spectrometer (Waters, Inc., Milford, MA). Isolation of mouse hepatocytes Primary mouse hepatocytes were isolated from mouse liver using the two-step EDTA and collagenase perfusion method. After the mouse was anesthetized, the liver was perfused with Krebs-Henseleit buffer without Ca2+ and SO2 (115 mM 4 NaCl, 25 mM NaHCO3, 5.9 mM KCl, 1.18 mM MgCl2, 1.23 mM NaH2PO4, 6 mM glucose, 0.1 mM EDTA) through the hepatic portal vein to rinse the blood out (flow: at 7–9 ml/ min for 5 min). Then, the liver was perfused with KrebsHenseleit buffer without Ca2+ and SO2 4 containing 0.02% collagenase and 0.1 mM CaCl2 until the liver appeared soft. The liver was then removed and gently minced, and the obtained cells were dispersed in medium (DMEM; Life Technologies) containing 10% FBS and 1% penicillin/streptomycin (Life Technologies). The solution containing the mixed cells and debris was filtered through a 100-mm filter. Subsequently, the filtrate was centrifuged at 50 g for 3 min at 4 C; the cells were washed with DMEM three times and then seeded in collagen-coated plates. The cells were maintained in DMEM high glucose (4.5 g/L) supplemented with 10% FBS, 1% penicillin/streptomycin, 1 mg/ml insulin, and 1012 M dexamethasone for 24 h at 37 C in a humidified atmosphere (5% CO2). The cells were incubated with fresh Williams’ E medium without FBS 24 h prior to the experiments. Cell viability assay Cell viability was detected using CCK-8. Mouse hepatocytes were cultured in 96-well plates with three triplicate wells in each group. The cells were treated with conditioned medium as indicated. The CCK-8 solution was added to each well at a 1:10 dilution followed by further incubation at 37 C for 3 h. Absorbance was measured at 450 nm using a microplate reader (BioTek Instruments, Inc., Winooski, VT). All values are expressed as the mean (±standard error, SE) of triplicate
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experiments. The values were converted from absolute counts to a percentage of the control.
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Western blot analysis The cell homogenates (30 mg of protein) were separated using 10% or 12% SDS-polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride (PVDF) transfer membranes. The blots were then washed with Tris-buffered solution containing Tween-20 (TBST, 10 mM Tris-HCl (pH 7.6), 150 mM NaCl, 0.05% Tween-20), blocked with 5% skimmed milk powder in TBST for 1 h, and incubated for 12 h with the appropriate primary antibody at the dilutions as recommended by the supplier (1:1000). The membranes were then washed with TBST and incubated with horseradish peroxidase-conjugated secondary antibody (1:5000) for 12 h. The bands were visualized using an enhanced chemiluminescence detection system (Thermo Scientific, Waltham, MA) according to the manufacturer’s protocols. Densitometric analysis was performed using image J software from the National Institutes of Health. Detection of intracellular ROS CM-H2DCFDA (DCF-DA), which functions as a ROSsensitive fluorophore, was used to detect the intracellular ROS. The cells were plated on 35-mm cell culture dishes and incubated under the conditions as described previously. The cells were then kept in the dark and treated with 5 mM DCFDA for 30 min at 37 C. After all the treatments were completed, the cells were washed three times with PBS and were imaged using fluorescence microscopy (100; DM IL LED Fluo, Leica, Wetzlar, Germany). Statistical analysis The results are expressed as the mean ± SE. The difference between the two mean values was analyzed by Student’s t test. A p value of 50.05 was considered significant.
Results Effect of dieckol on CoCl2-induced cytotoxicity in primary cultured mouse hepatocytes To examine the effect of CoCl2, the level of cell viability was examined with the CCK-8 assay system. As shown in Figure 2(A), CoCl2 significantly decreased the cell viability levels in a time-dependent manner. Figure 2(B) shows that treatment of mouse hepatocytes with CoCl2 at concentrations ranging from 100 to 1000 mM for 12 h led to a decrease in cell viability in a dose-dependent manner. Additionally, the effect of CoCl2 treatment on the expression levels of hypoxiainducible factor-1a (HIF-1a) was analyzed to verify whether the hypoxic cell responses were induced by CoCl2. The expression of HIF-1a was found to be increased in a timedependent manner (Figure 2C). To determine the effect of dieckol on CoCl2-induced cell injury, the cells were pretreated with various concentrations of dieckol for 30 min and then treated with 500 mM CoCl2. Dieckol significantly attenuated the decreased cell viability and the results indicate that pretreatment with dieckol offers protection against CoCl2induced hypoxic cell injury in primary cultured mouse
Figure 1. Chemical structure of dieckol.
hepatocytes (Figure 3A). In addition, the effects of dieckol on the expressions of Bax, Bcl-2, and caspase-3 were assessed on treatment with CoCl2. As shown in Figure 3(B), the CoCl2induced decrease in Bcl-2 expression and increase in Bax and caspase-3 levels were potentiated and attenuated by dieckol in a dose-dependent manner. Effect of dieckol on CoCl2-induced oxidative stress in primary cultured mouse hepatocytes To determine whether the dieckol-induced protective effect was associated with its antioxidant capacity in CoCl2-treated mouse hepatocytes, the intracellular ROS levels were observed. As shown in Figure 4(A), CoCl2-induced ROS increase was attenuated by dieckol (100 mM) or NAC, a common ROS scavenger (1 mM). In addition, the levels of signaling molecule, p38 MAPK, associated with CoCl2induced cell injury were elevated. The maximum level of p38 MAPK activation was observed at 120–240 min after treatment with CoCl2 (Figure 3B). Dieckol and NAC also attenuated the CoCl2-induced phosphorylation of p38 MAPK (Figure 4C). Furthermore, we observed that pretreatment with dieckol, NAC, and SB203580 (a p38 MAPK inhibitor, 5 mM) obviously led to attenuation of CoCl2induced decrease in Bcl-2 expression and increase in Bax and capase-3 expressions (Figure 4D). Consistent with these results, the decreased cell viability induced by CoCl2 treatment was partially recovered by pretreatment with dieckol, NAC, and SB203580. Each of dieckol, NAC, and SB203580 alone did not alter cell viability (Figure 4E). Effect of dieckol on CoCl2-induced COX-2 expression in primary cultured mouse hepatocytes After treatment of mouse hepatocytes with CoCl2, we observed that COX-2 expression was significantly augmented, whereas there was no significant change in the COX-1 expression (Figure 5A). To explore whether CoCl2-induced activation of COX-2 was ROS-dependent, the effects of dieckol and NAC on COX-2 were studied. Pretreatment with dieckol and NAC markedly attenuated the increased COX-2
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Figure 2. Effect of CoCl2 on cell viability in primary cultured mouse hepatocytes. (A) Mouse hepatocytes were incubated with 500 mM CoCl2 for various times (0–24 h). (B) Cells were incubated with CoCl2 at indicated concentrations for 12 h. Cell viability was measured by CCK-8 assay. (C) Cells were incubated for various times (0–12 h), and the level of cellular HIF-1a expression was determined through Western blotting. The values are expressed as the mean ± SE of three independent experiments with triplicate dishes. *p50.05 versus control, yp50.05 versus CoCl2 only. ROD, relative optical density.
expression by CoCl2. In this experiment, NS-398, a selective COX-2 inhibitor (10 mM), also attenuated CoCl2-induced COX-2 expression (Figure 5B). Additionally, pretreatment with dieckol and NS-398 repressed the CoCl2-induced increase in the expression of Bax and caspase-3, and potentiated the CoCl2-induced decrease in Bcl-2 expression (Figure 5C). Dieckol and NS-398 also reduced CoCl2-induced cytotoxicity (Figure 5D).
Discussion
Figure 3. Effect of dieckol on CoCl2-induced cytotoxicity. (A) Mouse hepatocytes were pretreated with different concentrations of dieckol for 30 min before exposure to 500 mM CoCl2. Cell viability was measured by CCK-8 assay. (B) Cells were incubated with 500 mM CoCl2 for 12 h in the absence or presence of pretreatment with dieckol at the indicated concentrations for 30 min. Cell lysates were subjected to Western blot analysis using Bax, Bcl-2-, and caspase-3-specific antibodies. The values are expressed as the mean ± SE of three independent experiments with triplicate dishes. *p50.05 versus control, yp50.05 versus CoCl2 only. ROD, relative optical density.
Ischemic liver injury is often induced by diseases such as trauma, metabolic diseases, sepsis, surgery, or transplantation (Jaeschke, 2000, 2006). Oxidative stress is found to be a key risk factor of these diseases. Cobalt chloride (CoCl2) has been reported to replace ferrous ions in prolyl-4-hydroxylase (P4H), thereby causing a conformational change in the P4H protein, which consequently leads to a hypoxic condition, characterized by intranuclear accumulation of hypoxia inducible factor-1a (HIF-1a) (Goldberg et al., 1988; Sharp & Bernaudin, 2004; Yuan et al., 2003). CoCl2 is also known to induce apoptosis in various types of cells (Gotoh et al., 2012; Lan et al., 2013; Talwar et al., 2011). In the present study, we demonstrated that chemical hypoxia-induced hepatocellular injury is markedly reduced by the addition of dieckol. In our results, the exposure of mouse hepatocytes to CoCl2-induced cytotoxicity was evidenced by the decreased cell viability, and we observed that dieckol significantly attenuated CoCl2induced decrease of cell viability. Additionally, addition of CoCl2 led to an increase in Bax expression, caspase-3 activation, and a decrease in Bcl-2 expression. The Bcl-2 family, which includes Bcl-2, Bcl-xL, Bad, and Bax, is an
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Figure 4. Effect of dieckol on the CoCl2-induced ROS generation, p38 MAPK activation, and cell injury. (A) Dichlorofluorescein (DCF)-sensitive cellular ROS was assessed. (a) Control. (b) Treatment with 500 mM CoCl2 for 2 h. (c) Pretreatment with 100 mM dieckol for 30 min before exposure to 500 mM CoCl2 for 2 h. (d) Treatment with 100 mM dieckol for 30 min followed by 2 h culture. (e) Pretreatment with 1000 mM N-acetyl cysteine (NAC, a ROS scavenger) for 30 min before exposure to 500 mM CoCl2 for 2 h. (f) Treatment with 1000 mM NAC for 30 min followed by 2 h culture. (B) Cells were incubated for various times (0–240 min) with 500 mM CoCl2. (C) Cells were pretreated with 100 mM dieckol, 1000 mM NAC, or 1 mM SB203580 (a p38 MAPK inhibitor) for 30 min and then treated with 500 mM CoCl2 for 120 min. Cell lysates were subjected to Western blotting to determine the levels of phospho-p38 MAPK. (D) Cells were pretreated with 100 mM dieckol, 1000 mM NAC, or 1 mM SB203580 for 30 min and then treated with 500 mM CoCl2 for 12 h. Cell lysates were subjected to Western blotting to determine the levels of Bax, Bcl-2, and caspase-3 expression. (E) Cells were pretreated 100 mM dieckol, 1000 mM NAC, or 1 mM SB203580 for 30 min followed by incubation in the presence or absence of 500 mM CoCl2 for 12 h. Cell viability was measured by CCK-8 assay. The values are expressed as the mean ± SE of three independent experiments with triplicate dishes. *p50.05 versus control, yp50.05 versus CoCl2 only. ROD, relative optical density.
important regulator of various apoptotic pathways (Sarada et al., 2008). They can activate or inhibit the activation of caspase-3 in the execution of apoptosis (Susnow et al., 2009). In general, Bax exerts pro-apoptotic activity whereas Bcl-2 exerts an anti-apoptotic activity (Wang, 2001). In the present study, we showed that pretreatment of mouse hepatocytes with dieckol resulted in a significant decrease of Bax expression and caspase-3 activation and a potentiation of decreased Bcl-2 expression induced by CoCl2.
These findings suggest that dieckol plays an important role in suppressing apoptotic cell death induced by chemical hypoxia in mouse hepatocytes. Previous studies suggest that hypoxia or hypoxia-mimicking CoCl2-induced cellular damage is associated with generation of excessive ROS (Guillemin & Krasnow, 1997; Zou et al., 2002). ROS participate and regulate a diverse number of downstream signaling pathways, leading to specific cellular functions (Aviram, 2000; Halliwell, 1994)
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Figure 5. Effect of dieckol on CoCl2-induced COX-2 expression and cell injury. (A) Mouse hepatocytes were incubated with 500 mM CoCl2 for various times (0–12 h). (B) Cells were pretreated with 100 mM dieckol, 1000 mM NAC, or 10 mM NS-398 (a selective COX-2 inhibitor) for 30 min and then treated with 500 mM CoCl2 for 12 h. Cell lysates were subjected to Western blotting to determine the level of COX-1 or COX-2 expression. (C) Cells were pretreated with 100 mM dieckol or 10 mM NS-398 for 30 min and then treated with 500 mM CoCl2 for 12 h. Cell lysates were subjected to Western blotting to determine the levels of Bax, Bcl-2, and caspase-3 expression. (D) Cells were pretreated 100 mM dieckol or 10 mM NS-398 for 30 min followed by incubation in the presence or absence of 500 mM CoCl2 for 12 h. Cell viability was measured by CCK-8 assay. The values are expressed as the mean ± SE of three independent experiments with triplicate dishes. *p50.05 versus control, yp50.05 versus CoCl2 only. ROD, relative optical density.
such as growth, metabolic rate, cell division, necrosis, apoptosis, and aging processes (de Magalhaes & Church, 2006; Kim & Lee, 2010; Menon & Goswami, 2007). But an imbalance in the formation and neutralization of ROS leads to oxidative stress (Chen et al., 2007). In the present study, we used N-acetyl cysteine (NAC), a potent ROS scavenger, as a positive control to understand the role of ROS in the induction of cytotoxicity by CoCl2 (Sun, 2010). Our results showed that CoCl2-induced ROS generation was prevented by the addition of dieckol or NAC. p38 MAPK activation is implicated in cellular damage associated with hepatic hypoxia (Lee et al., 2008). p38 MAPK is a member of the MAPK family and is preferentially activated by inflammatory cytokines and environmental stress, such as osmotic stress, heat shock, and hypoxia (Lan et al., 2013). In the present study, we observed that the treatment of mouse hepatocytes with CoCl2 significantly upregulated phosphorylation of p38 MAPK and that pretreatment of cells with dieckol and NAC markedly blocked CoCl2-induced p38 MAPK phosphorylation. These results suggest that elevated intracellular ROS levels can lead to an activation of p38 MAPK. Indeed, these results are supported by previous reports that ROS interact with p38 MAPK in CoCl2-induced hypoxic injury (Lan et al., 2012, 2013). In the present study, we found that dieckol, NAC, and SB203580 attenuated the CoCl2-induced decrease in the expression of Bcl-2 as well as the CoCl2-induced increase in the expression of Bax and caspase-3. In addition, dieckol, NAC, and SB203580 (an inhibitor of p38 MAPK) blocked CoCl2-induced decrease in cell viability, indicating that
activation of p38 MAPK by ROS mediates the hepatic cell damage induced by CoCl2. These results are supported by the evidence that superoxide-induced apoptotic cell death in rat hepatic stellate cells is associated with p38 MAPK activation (Jameel et al., 2009). Therefore, the results of this study suggest that hepatoprotective effects of dieckol are mediated by reduction not only of CoCl2-induced ROS but also of p38 MAPK activated by CoCl2-induced ROS. In the present study, the expression of COX-2 was increased by the treatment of mouse hepatocytes with CoCl2. At present, there are at least two forms of COX: COX-1 is constitutively expressing and COX-2 is induced by multiple factors including cytokines, hormones, mitogens, oxidative stresses, and so forth (Jung et al., 2009; Smith et al., 2000). Our results showed that increased COX-2 expression by CoCl2 is attenuated by pretreatment with dieckol and NAC. This result indicates that COX-2 expression is enhanced by CoCl2-induced intracellular ROS and dieckol represses COX-2 expression by its antioxidant activity. Previous studies showed that inhibition of COX-2 exerts hepatoprotective effects on liver damage (Vadiraja et al., 1998). COX-2 expression is upregulated by ischemia or reperfusion in the rat liver, and the inhibition of COX-2 is found to improve liver function and viability (Kim et al., 2003; Oshima et al., 2009). Through this study, we observed that dieckol and NS-398, a selective inhibitor of COX-2, increased CoCl2-induced decrease in the expression of Bcl-2 and attenuated the CoCl2-induced increase in the expression of Bax and caspase3. In addition, dieckol and NS-398 attenuated CoCl2-induced
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cytotoxicity. Overall, these findings suggest that oxidative stress plays a pivotal role in hepatocellular injuries induced by CoCl2 and ROS mediate CoCl2-induced injury through p38 MAPK and COX-2 pathway in mouse hepatocytes. In conclusion, the present study demonstrated that dieckol has a cytoprotective effect against chemical hypoxia-induced cell injury through inhibition of the ROS, p38 MAPK, and COX-2 pathway in primary cultured mouse hepatocytes.
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Acknowledgments This work was supported by a grant from the Next-Generation BioGreen 21 Program (No. PJ009090), Rural Development Administration; National Research Foundation (NRF) grant funded by the Korean government (MSIP) (NRF2012R1A4A1028835); Kyungpook National University Research Fund, 2013, Republic of Korea.
Declaration of interest None declared.
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Guo M, Song LP, Jiang Y, et al. (2006). Hypoxia-mimetic agents desferrioxamine and cobalt chloride induce leukemic cell apoptosis through different hypoxia-inducible factor-1alpha independent mechanisms. Apoptosis 11:67–77. Halliwell B. (1994). Free radicals, antioxidants, and human disease: curiosity, cause, or consequence? Lancet 344:721–724 Hamada T, Tsuchihashi S, Avanesyan A, et al. (2008). Cyclooxygenase-2 deficiency enhances Th2 immune responses and impairs neutrophil recruitment in hepatic ischemia/reperfusion injury. J Immunol 180: 1843–1853. Jaeschke H. (2000). Reactive oxygen and mechanisms of inflammatory liver injury. J Gastroenterol Hepatol 15:718–724. Jaeschke H. (2006). Mechanisms of liver injury. II. Mechanisms of neutrophil-induced liver cell injury during hepatic ischemia-reperfusion and other acute inflammatory conditions. Am J Physiol Gastrointest Liver Physiol 290:G1083–1088. Jameel NM, Thirunavukkarasu C, Wu T, et al. (2009). p38-MAPK- and caspase-3-mediated superoxide-induced apoptosis of rat hepatic stellate cells: reversal by retinoic acid. J Cell Physiol 218:157–166. Jiang BH, Zheng JZ, Leung SW, et al. (1997). Transactivation and inhibitory domains of hypoxia-inducible factor 1alpha. Modulation of transcriptional activity by oxygen tension. J Biol Chem 272: 19253–19260. Jung JY, Mo HC, Yang KH, et al. (2007). Inhibition by epigallocatechin gallate of CoCl2-induced apoptosis in rat PC12 cells. Life Sci 80: 1355–1363. Jung WK, Heo SJ, Jeon YJ, et al. (2009). Inhibitory effects and molecular mechanism of dieckol isolated from marine brown alga on COX-2 and iNOS in microglial cells. J Agric Food Chem 57: 4439–4446. Kim HJ, Yang SJ, Kim YS, Kim TU. (2003). Cobalt chloride-induced apoptosis and extracellular signal-regulated protein kinase activation in human cervical cancer HeLa cells. J Biochem Mol Biol 36: 468–474. Kim KC, Lee C. (2010). Curcumin induces downregulation of E2F4 expression and apoptotic cell death in HCT116 human colon cancer cells; involvement of reactive oxygen species. Korean J Physiol Pharmacol 14:391–397. Kim SD, Moon CK, Eun SY, et al. (2005). Identification of ASK1, MKK4, JNK, c-Jun, and caspase-3 as a signaling cascade involved in cadmium-induced neuronal cell apoptosis. Biochem Biophys Res Commun 328:326–334. Knight TR, Ho YS, Farhood A, Jaeschke H. (2002). Peroxynitrite is a critical mediator of acetaminophen hepatotoxicity in murine livers: protection by glutathione. J Pharmacol Exp Ther 303:468–475. Kwak HJ, Park KM, Choi HE, et al. (2010). The cardioprotective effects of zileuton, a 5-lipoxygenase inhibitor, are mediated by COX-2 via activation of PKC delta. Cell Signal 22:80–87. Lan A, Xu W, Zhang H, et al. (2013). Inhibition of ROS-activated p38MAPK pathway is involved in the protective effect of H2S against chemical hypoxia-induced inflammation in PC12 cells. Neurochem Res 38:1454–1466. Lan AP, Xiao LC, Yang ZL, et al. (2012). Interaction between ROS and p38MAPK contributes to chemical hypoxia-induced injuries in PC12 cells. Mol Med Rep 5:250–255. Lee MY, Jung SC, Lee JH, Han HJ. (2008). Estradiol-17beta protects against hypoxia-induced hepatocyte injury through ER-mediated upregulation of Bcl-2 as well as ER-independent antioxidant effects. Cell Res 18:491–499. Lee SH, Han JS, Heo SJ, et al. (2010). Protective effects of dieckol isolated from Ecklonia cava against high glucose-induced oxidative stress in human umbilical vein endothelial cells. Toxicol In Vitro 24: 375–381. Lehwald N, Tao GZ, Jang KY, et al. (2011). Wnt-beta-catenin signaling protects against hepatic ischemia and reperfusion injury in mice. Gastroenterology 141:707–718, 718 e701–705. Menon SG, Goswami PC. (2007). A redox cycle within the cell cycle: ring in the old with the new. Oncogene 26:1101–1109. Nakanishi K, Tajima F, Nakamura A, et al. (1995). Effects of hypobaric hypoxia on antioxidant enzymes in rats. J Physiol 489: 869–876. Oshima K, Yabata Y, Yoshinari D, Takeyoshi I. (2009). The effects of cyclooxygenase (COX)-2 inhibition on ischemia-reperfusion injury in liver transplantation. J Invest Surg 22:239–245.
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Wang G, Hazra TK, Mitra S, et al. (2000). Mitochondrial DNA damage and a hypoxic response are induced by CoCl(2) in rat neuronal PC12 cells. Nucleic Acids Res 28:2135–2140. Wang X. (2001). The expanding role of mitochondria in apoptosis. Genes Dev 15:2922–2933. Yang C, Ling H, Zhang M, et al. (2011). Oxidative stress mediates chemical hypoxia-induced injury and inflammation by activating NF-kappab-COX-2 pathway in HaCaT cells. Mol Cells 31:531–538. Yeo JE, Kim JH, Kang SK. (2008). Selenium attenuates ROS-mediated apoptotic cell death of injured spinal cord through prevention of mitochondria dysfunction; in vitro and in vivo study. Cell Physiol Biochem 21:225–238. Yokoyama C, Tanabe T. (1989). Cloning of human gene encoding prostaglandin endoperoxide synthase and primary structure of the enzyme. Biochem Biophys Res Commun 165:888–894. Yu F, Wang Z, Ju B, et al. (2008). Apoptotic effect of organophosphorus insecticide chlorpyrifos on mouse retina in vivo via oxidative stress and protection of combination of vitamins C and E. Exp Toxicol Pathol 59:415–423. Yuan Y, Hilliard G, Ferguson T, Millhorn DE. (2003). Cobalt inhibits the interaction between hypoxia-inducible factor-alpha and von HippelLindau protein by direct binding to hypoxia-inducible factor-alpha. J Biol Chem 278:15911–15916. Zhang C, Li Y, Qian ZJ, et al. (2011). Dieckol from Ecklonia cava regulates invasion of human fibrosarcoma cells and modulates MMP-2 and MMP-9 expression via NF-kappaB pathway. Evid Based Complement Alternat Med 2011:140462. Zou W, Yan M, Xu W, et al. (2001). Cobalt chloride induces PC12 cells apoptosis through reactive oxygen species and accompanied by AP-1 activation. J Neurosci Res 64:646–653. Zou W, Zeng J, Zhuo M, et al. (2002). Involvement of caspase-3 and p38 mitogen-activated protein kinase in cobalt chloride-induced apoptosis in PC12 cells. J Neurosci Res 67:837–843.
ORIGINAL ARTICLE
Protective effect of dieckol against chemical hypoxia-induced cytotoxicity in primary cultured mouse hepatocytes Yu Jin Jeon1, Hyoung Seok Kim1, Kyung-Sik Song2, Ho Jae Han3, Soo Hyun Park4, Woochul Chang5, and Min Young Lee1 Drug and Chemical Toxicology Downloaded from informahealthcare.com by Gazi Univ. on 01/08/15 For personal use only.
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Department of Molecular Physiology, College of Pharmacy, Kyungpook National University, Daegu, Korea, 2College of Pharmacy, Research Institute of Pharmaceutical Sciences, Kyungpook National University, Daegu, Korea, 3Department of Veterinary Physiology, College of Veterinary Medicine, Seoul National University, Seoul, Korea, 4Department of Veterinary Physiology, College of Veterinary Medicine, Chonnam National University, Gwangju, Korea, and 5Department of Biology Education, College of Education, Pusan National University, Busan, Korea Abstract
Keywords
Hepatic ischemic injury is a major complication arising from liver surgery, transplantation, or other ischemic diseases, and both reactive oxygen species (ROS) and pro-inflammatory mediators play the role of key mediators in hepatic ischemic injury. In this study, we examined the effect of dieckol in chemical hypoxia-induced injury in mouse hepatocytes. Cell viability was significantly decreased after treatment with cobalt chloride (CoCl2), a well-known hypoxia mimetic agent in a time- and dose-dependent manner. Pretreatment with dieckol before exposure to CoCl2 significantly attenuated the CoCl2-induced decrease of cell viability. Additionally, pretreatment with dieckol potentiated the CoCl2-induced decrease of Bcl-2 expression and attenuated the CoCl2-induced increase in the expression of Bax and caspase-3. Treatment with CoCl2 resulted in an increased intracellular ROS generation, which is inhibited by dieckol or N-acetyl cysteine (NAC, a ROS scavenger), and p38 MAPK phosphorylation, which is also blocked by dieckol or NAC. In addition, dieckol and SB203580 (p38 MAPK inhibitor) increased the CoCl2-induced decrease of Bcl-2 expression and decreased the CoCl2-induced increase of Bax and caspase-3 expressions. CoCl2-induced decrease of cell viability was attenuated by pretreatment with dieckol, NAC, and SB203580. Furthermore, dieckol attenuated CoCl2-induced COX-2 expression. Similar to the effect of dieckol, NAC also blocked CoCl2induced COX-2 expression. Additionally, CoCl2-induced decrease of cell viability was attenuated not only by dieckol and NAC but also by NS-398 (a selective COX-2 inhibitor). In conclusion, dieckol protects primary cultured mouse hepatocytes against CoCl2-induced cell injury through inhibition of ROS-activated p38 MAPK and COX-2 pathway.
Cobalt chloride, dieckol, hypoxic cell injury, mouse hepatocytes, reactive oxygen species
Introduction Hepatic tissue is quite vulnerable to hypoxic (ischemic) injury compared with other organs (Nakanishi et al., 1995). Hypoxic injury is a common cause of oxidative tissue damage caused by many liver diseases and hepatic surgery (Lehwald et al., 2011). The generation of reactive oxygen species (ROS) during hypoxia as well as in the presence of cobalt chloride (CoCl2) had been described previously (Chandel et al., 1998; Chandel & Schumacker, 1999; Guillemin & Krasnow, 1997; Semenza, 1999). Interestingly, exposure to CoCl2 triggers transcriptional changes that mimic hypoxic response, including up-regulation of the hypoxia-inducible factor-1a (HIF1a), erythropoietin, and glycolytic enzymes (An et al., 1998;
Address for correspondence: Min Young Lee, DVM, PhD, Department of Molecular Physiology, College of Pharmacy, Kyungpook National University, 80 Daehak-ro, Buk-gu, Daegu 702-701, Korea. Tel: +82-53950-8577. Fax: +82-53-950-8557. E-mail: [email protected]
History Received 10 September 2013 Revised 22 March 2014 Accepted 19 May 2014 Published online 18 August 2014
Chandel et al., 1998; Jiang et al., 1997). One common mechanism that may lead to these changes in gene expression is the increased generation of ROS (Chandel et al., 1998). ROS are implicated in the initiation and progression of liver pathologies (Pietrangelo, 1996). Elevated ROS is capable of directly targeting nucleic acids, proteins, and membrane phospholipids, leading to apoptosis of various cell types including hepatocytes (Cao et al., 2001; Chen et al., 2008; Knight et al., 2002; Wang et al., 2000). In addition, ROS produced during oxidative stress have been reported to initiate signaling cascades, which lead to apoptosis (Yu et al., 2008). Available evidence suggests that excessive ROS have been shown to activate members of the MAPK family, including extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun-N-terminal kinase (JNK), and p38 MAPK in various types of cells (Kim et al., 2005; Ouyang & Shen, 2006; Yeo et al., 2008). Especially, the p38 MAPK pathway is known to be stimulated by a variety of environmental stresses and chemicals (Roulston et al., 1998) and is an important
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signaling pathway associated with ROS-induced apoptosis (Jameel et al., 2009). It has been reported that CoCl2 activates p38 MAPK, which is known to be involved in CoCl2-induced apoptosis in other cell types (Zou et al., 2002). It has been reported that ROS can stimulate cyclooxygenase (COX)-2 expression in CoCl2-induced chemical hypoxic condition (Yang et al., 2011; Yeo et al., 2008). Hepatic ischemic injuries result from complex interactions between various inflammatory mediators including COX-2 (Fu et al., 2012). At present, at least two COX isoenzymes, COX-1 and COX-2, have been identified (Yokoyama & Tanabe, 1989). COX-1 is usually expressed constitutively, whereas COX-2 remains undetectable in most tissues in the physiological state but rapidly accumulates under pathological conditions (Dupouy et al., 2006). Although the role of COX-2 overexpression is complicated and remains ambivalent in different cell types, mediating either cytotoxicity or cytoprotection (Booth et al., 2008; Carnieto et al., 2009; Kwak et al., 2010), the inhibition of COX-2 by selective inhibitors, or gene knock-out leads to a significant reduction in ischemiainduced hepatic damage (Hamada et al., 2008; Ozturk & Gezici, 2006). In the present study, we investigated the cytoprotective effect induced by adding dieckol to primary cultured mouse hepatocytes treated with CoCl2, a widely used hypoxia mimetic agent that promotes the accumulation of hypoxia-inducible factor-1 a (HIF-1a), which is a critical regulator for the cellular response to hypoxia (Guo et al., 2006) and which induces oxidative stress (Jung et al., 2007; Zou et al., 2001) and apoptosis in various cells (Kim et al., 2003; Zou et al., 2001). Dieckol is a phloroglucinol derivative isolated from marine brown alga Ecklonia cava and is known to play a variety of biological functions in vitro and in vivo, for example, in antioxidant, antitumor, antihuman immunodeficiency virus (HIV), and anti-inflammatory activities, and also provides protective effects against oxidative stress (Lee et al., 2010; Zhang et al., 2011). Dieckol displays excellent antioxidant properties. However, the detailed molecular mechanisms that play a part in the action of dieckol in hepatocytes on hypoxic injury remain to be clearly elucidated. Therefore, this study assessed the effect of dieckol on hypoxic cellular injury induced by CoCl2 and its associated mechanisms in primary cultured mouse hepatocytes.
Materials and methods Materials Eight-week-old male ICR mice were purchased from Daehan Bio Link Co. (Incheon, Korea). All animal management procedures were conducted in accordance with the standard operation protocols established by Kyungpook National University. Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Laboratories (Japan). CoCl2, N-acetyl-L-cysteine, SB203580, and type IV collagenase were obtained from Sigma-Aldrich (St. Louis, MO). The 5-(and 6)-chloromethyl20 ,70 -dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA) were purchased from Life Technologies (Grand Island, NY). Fetal bovine serum (FBS) was acquired from PAA Laboratories (GmbH, Canada). The phospho-p38 MAPK antibodies were obtained from New England Biolabs
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(Hertfordshire, UK). The HIF-1a, Bcl-2, caspase-3, COX-1, COX-2, goat anti-rabbit IgG, and goat anti-mouse IgG antibodies were supplied by Santa Cruz Biotechnology (Santa Cruz, CA). Extraction and purification of dieckol Freshly collected Ecklonia cava (E. cava) was washed, freezedried, milled into small pieces, and extracted by methanol. The methanol extract of dried E. cava was partitioned with nhexane to remove nonpolar components. The resulting methanol fraction was further fractionated between water and n-buthanol. The n-buthanol fraction was subjected to reverse-phase flash column chromatography and three fractions were given. Each fraction was subjected to High-Pressure Liquid Chromatography (HPLC) to take polyphenolic compounds. The chemical structure of dieckol was determined by spectroscopy (Figure 1). The NMR spectra were recorded on a JEOL ECP-500 FT NMR at 500 MHz for 1H and 125 MHz for 13C, using Tetramethylsilane (TMS) as an internal standard. HPLC was performed on a HP 5980A. Fast Atom Bombardment Mass Spectrometry (FABMS) was obtained with a VG Autospec Ultma mass spectrometer (Waters, Inc., Milford, MA). Isolation of mouse hepatocytes Primary mouse hepatocytes were isolated from mouse liver using the two-step EDTA and collagenase perfusion method. After the mouse was anesthetized, the liver was perfused with Krebs-Henseleit buffer without Ca2+ and SO2 (115 mM 4 NaCl, 25 mM NaHCO3, 5.9 mM KCl, 1.18 mM MgCl2, 1.23 mM NaH2PO4, 6 mM glucose, 0.1 mM EDTA) through the hepatic portal vein to rinse the blood out (flow: at 7–9 ml/ min for 5 min). Then, the liver was perfused with KrebsHenseleit buffer without Ca2+ and SO2 4 containing 0.02% collagenase and 0.1 mM CaCl2 until the liver appeared soft. The liver was then removed and gently minced, and the obtained cells were dispersed in medium (DMEM; Life Technologies) containing 10% FBS and 1% penicillin/streptomycin (Life Technologies). The solution containing the mixed cells and debris was filtered through a 100-mm filter. Subsequently, the filtrate was centrifuged at 50 g for 3 min at 4 C; the cells were washed with DMEM three times and then seeded in collagen-coated plates. The cells were maintained in DMEM high glucose (4.5 g/L) supplemented with 10% FBS, 1% penicillin/streptomycin, 1 mg/ml insulin, and 1012 M dexamethasone for 24 h at 37 C in a humidified atmosphere (5% CO2). The cells were incubated with fresh Williams’ E medium without FBS 24 h prior to the experiments. Cell viability assay Cell viability was detected using CCK-8. Mouse hepatocytes were cultured in 96-well plates with three triplicate wells in each group. The cells were treated with conditioned medium as indicated. The CCK-8 solution was added to each well at a 1:10 dilution followed by further incubation at 37 C for 3 h. Absorbance was measured at 450 nm using a microplate reader (BioTek Instruments, Inc., Winooski, VT). All values are expressed as the mean (±standard error, SE) of triplicate
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experiments. The values were converted from absolute counts to a percentage of the control.
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Western blot analysis The cell homogenates (30 mg of protein) were separated using 10% or 12% SDS-polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride (PVDF) transfer membranes. The blots were then washed with Tris-buffered solution containing Tween-20 (TBST, 10 mM Tris-HCl (pH 7.6), 150 mM NaCl, 0.05% Tween-20), blocked with 5% skimmed milk powder in TBST for 1 h, and incubated for 12 h with the appropriate primary antibody at the dilutions as recommended by the supplier (1:1000). The membranes were then washed with TBST and incubated with horseradish peroxidase-conjugated secondary antibody (1:5000) for 12 h. The bands were visualized using an enhanced chemiluminescence detection system (Thermo Scientific, Waltham, MA) according to the manufacturer’s protocols. Densitometric analysis was performed using image J software from the National Institutes of Health. Detection of intracellular ROS CM-H2DCFDA (DCF-DA), which functions as a ROSsensitive fluorophore, was used to detect the intracellular ROS. The cells were plated on 35-mm cell culture dishes and incubated under the conditions as described previously. The cells were then kept in the dark and treated with 5 mM DCFDA for 30 min at 37 C. After all the treatments were completed, the cells were washed three times with PBS and were imaged using fluorescence microscopy (100; DM IL LED Fluo, Leica, Wetzlar, Germany). Statistical analysis The results are expressed as the mean ± SE. The difference between the two mean values was analyzed by Student’s t test. A p value of 50.05 was considered significant.
Results Effect of dieckol on CoCl2-induced cytotoxicity in primary cultured mouse hepatocytes To examine the effect of CoCl2, the level of cell viability was examined with the CCK-8 assay system. As shown in Figure 2(A), CoCl2 significantly decreased the cell viability levels in a time-dependent manner. Figure 2(B) shows that treatment of mouse hepatocytes with CoCl2 at concentrations ranging from 100 to 1000 mM for 12 h led to a decrease in cell viability in a dose-dependent manner. Additionally, the effect of CoCl2 treatment on the expression levels of hypoxiainducible factor-1a (HIF-1a) was analyzed to verify whether the hypoxic cell responses were induced by CoCl2. The expression of HIF-1a was found to be increased in a timedependent manner (Figure 2C). To determine the effect of dieckol on CoCl2-induced cell injury, the cells were pretreated with various concentrations of dieckol for 30 min and then treated with 500 mM CoCl2. Dieckol significantly attenuated the decreased cell viability and the results indicate that pretreatment with dieckol offers protection against CoCl2induced hypoxic cell injury in primary cultured mouse
Figure 1. Chemical structure of dieckol.
hepatocytes (Figure 3A). In addition, the effects of dieckol on the expressions of Bax, Bcl-2, and caspase-3 were assessed on treatment with CoCl2. As shown in Figure 3(B), the CoCl2induced decrease in Bcl-2 expression and increase in Bax and caspase-3 levels were potentiated and attenuated by dieckol in a dose-dependent manner. Effect of dieckol on CoCl2-induced oxidative stress in primary cultured mouse hepatocytes To determine whether the dieckol-induced protective effect was associated with its antioxidant capacity in CoCl2-treated mouse hepatocytes, the intracellular ROS levels were observed. As shown in Figure 4(A), CoCl2-induced ROS increase was attenuated by dieckol (100 mM) or NAC, a common ROS scavenger (1 mM). In addition, the levels of signaling molecule, p38 MAPK, associated with CoCl2induced cell injury were elevated. The maximum level of p38 MAPK activation was observed at 120–240 min after treatment with CoCl2 (Figure 3B). Dieckol and NAC also attenuated the CoCl2-induced phosphorylation of p38 MAPK (Figure 4C). Furthermore, we observed that pretreatment with dieckol, NAC, and SB203580 (a p38 MAPK inhibitor, 5 mM) obviously led to attenuation of CoCl2induced decrease in Bcl-2 expression and increase in Bax and capase-3 expressions (Figure 4D). Consistent with these results, the decreased cell viability induced by CoCl2 treatment was partially recovered by pretreatment with dieckol, NAC, and SB203580. Each of dieckol, NAC, and SB203580 alone did not alter cell viability (Figure 4E). Effect of dieckol on CoCl2-induced COX-2 expression in primary cultured mouse hepatocytes After treatment of mouse hepatocytes with CoCl2, we observed that COX-2 expression was significantly augmented, whereas there was no significant change in the COX-1 expression (Figure 5A). To explore whether CoCl2-induced activation of COX-2 was ROS-dependent, the effects of dieckol and NAC on COX-2 were studied. Pretreatment with dieckol and NAC markedly attenuated the increased COX-2
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Figure 2. Effect of CoCl2 on cell viability in primary cultured mouse hepatocytes. (A) Mouse hepatocytes were incubated with 500 mM CoCl2 for various times (0–24 h). (B) Cells were incubated with CoCl2 at indicated concentrations for 12 h. Cell viability was measured by CCK-8 assay. (C) Cells were incubated for various times (0–12 h), and the level of cellular HIF-1a expression was determined through Western blotting. The values are expressed as the mean ± SE of three independent experiments with triplicate dishes. *p50.05 versus control, yp50.05 versus CoCl2 only. ROD, relative optical density.
expression by CoCl2. In this experiment, NS-398, a selective COX-2 inhibitor (10 mM), also attenuated CoCl2-induced COX-2 expression (Figure 5B). Additionally, pretreatment with dieckol and NS-398 repressed the CoCl2-induced increase in the expression of Bax and caspase-3, and potentiated the CoCl2-induced decrease in Bcl-2 expression (Figure 5C). Dieckol and NS-398 also reduced CoCl2-induced cytotoxicity (Figure 5D).
Discussion
Figure 3. Effect of dieckol on CoCl2-induced cytotoxicity. (A) Mouse hepatocytes were pretreated with different concentrations of dieckol for 30 min before exposure to 500 mM CoCl2. Cell viability was measured by CCK-8 assay. (B) Cells were incubated with 500 mM CoCl2 for 12 h in the absence or presence of pretreatment with dieckol at the indicated concentrations for 30 min. Cell lysates were subjected to Western blot analysis using Bax, Bcl-2-, and caspase-3-specific antibodies. The values are expressed as the mean ± SE of three independent experiments with triplicate dishes. *p50.05 versus control, yp50.05 versus CoCl2 only. ROD, relative optical density.
Ischemic liver injury is often induced by diseases such as trauma, metabolic diseases, sepsis, surgery, or transplantation (Jaeschke, 2000, 2006). Oxidative stress is found to be a key risk factor of these diseases. Cobalt chloride (CoCl2) has been reported to replace ferrous ions in prolyl-4-hydroxylase (P4H), thereby causing a conformational change in the P4H protein, which consequently leads to a hypoxic condition, characterized by intranuclear accumulation of hypoxia inducible factor-1a (HIF-1a) (Goldberg et al., 1988; Sharp & Bernaudin, 2004; Yuan et al., 2003). CoCl2 is also known to induce apoptosis in various types of cells (Gotoh et al., 2012; Lan et al., 2013; Talwar et al., 2011). In the present study, we demonstrated that chemical hypoxia-induced hepatocellular injury is markedly reduced by the addition of dieckol. In our results, the exposure of mouse hepatocytes to CoCl2-induced cytotoxicity was evidenced by the decreased cell viability, and we observed that dieckol significantly attenuated CoCl2induced decrease of cell viability. Additionally, addition of CoCl2 led to an increase in Bax expression, caspase-3 activation, and a decrease in Bcl-2 expression. The Bcl-2 family, which includes Bcl-2, Bcl-xL, Bad, and Bax, is an
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Figure 4. Effect of dieckol on the CoCl2-induced ROS generation, p38 MAPK activation, and cell injury. (A) Dichlorofluorescein (DCF)-sensitive cellular ROS was assessed. (a) Control. (b) Treatment with 500 mM CoCl2 for 2 h. (c) Pretreatment with 100 mM dieckol for 30 min before exposure to 500 mM CoCl2 for 2 h. (d) Treatment with 100 mM dieckol for 30 min followed by 2 h culture. (e) Pretreatment with 1000 mM N-acetyl cysteine (NAC, a ROS scavenger) for 30 min before exposure to 500 mM CoCl2 for 2 h. (f) Treatment with 1000 mM NAC for 30 min followed by 2 h culture. (B) Cells were incubated for various times (0–240 min) with 500 mM CoCl2. (C) Cells were pretreated with 100 mM dieckol, 1000 mM NAC, or 1 mM SB203580 (a p38 MAPK inhibitor) for 30 min and then treated with 500 mM CoCl2 for 120 min. Cell lysates were subjected to Western blotting to determine the levels of phospho-p38 MAPK. (D) Cells were pretreated with 100 mM dieckol, 1000 mM NAC, or 1 mM SB203580 for 30 min and then treated with 500 mM CoCl2 for 12 h. Cell lysates were subjected to Western blotting to determine the levels of Bax, Bcl-2, and caspase-3 expression. (E) Cells were pretreated 100 mM dieckol, 1000 mM NAC, or 1 mM SB203580 for 30 min followed by incubation in the presence or absence of 500 mM CoCl2 for 12 h. Cell viability was measured by CCK-8 assay. The values are expressed as the mean ± SE of three independent experiments with triplicate dishes. *p50.05 versus control, yp50.05 versus CoCl2 only. ROD, relative optical density.
important regulator of various apoptotic pathways (Sarada et al., 2008). They can activate or inhibit the activation of caspase-3 in the execution of apoptosis (Susnow et al., 2009). In general, Bax exerts pro-apoptotic activity whereas Bcl-2 exerts an anti-apoptotic activity (Wang, 2001). In the present study, we showed that pretreatment of mouse hepatocytes with dieckol resulted in a significant decrease of Bax expression and caspase-3 activation and a potentiation of decreased Bcl-2 expression induced by CoCl2.
These findings suggest that dieckol plays an important role in suppressing apoptotic cell death induced by chemical hypoxia in mouse hepatocytes. Previous studies suggest that hypoxia or hypoxia-mimicking CoCl2-induced cellular damage is associated with generation of excessive ROS (Guillemin & Krasnow, 1997; Zou et al., 2002). ROS participate and regulate a diverse number of downstream signaling pathways, leading to specific cellular functions (Aviram, 2000; Halliwell, 1994)
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Figure 5. Effect of dieckol on CoCl2-induced COX-2 expression and cell injury. (A) Mouse hepatocytes were incubated with 500 mM CoCl2 for various times (0–12 h). (B) Cells were pretreated with 100 mM dieckol, 1000 mM NAC, or 10 mM NS-398 (a selective COX-2 inhibitor) for 30 min and then treated with 500 mM CoCl2 for 12 h. Cell lysates were subjected to Western blotting to determine the level of COX-1 or COX-2 expression. (C) Cells were pretreated with 100 mM dieckol or 10 mM NS-398 for 30 min and then treated with 500 mM CoCl2 for 12 h. Cell lysates were subjected to Western blotting to determine the levels of Bax, Bcl-2, and caspase-3 expression. (D) Cells were pretreated 100 mM dieckol or 10 mM NS-398 for 30 min followed by incubation in the presence or absence of 500 mM CoCl2 for 12 h. Cell viability was measured by CCK-8 assay. The values are expressed as the mean ± SE of three independent experiments with triplicate dishes. *p50.05 versus control, yp50.05 versus CoCl2 only. ROD, relative optical density.
such as growth, metabolic rate, cell division, necrosis, apoptosis, and aging processes (de Magalhaes & Church, 2006; Kim & Lee, 2010; Menon & Goswami, 2007). But an imbalance in the formation and neutralization of ROS leads to oxidative stress (Chen et al., 2007). In the present study, we used N-acetyl cysteine (NAC), a potent ROS scavenger, as a positive control to understand the role of ROS in the induction of cytotoxicity by CoCl2 (Sun, 2010). Our results showed that CoCl2-induced ROS generation was prevented by the addition of dieckol or NAC. p38 MAPK activation is implicated in cellular damage associated with hepatic hypoxia (Lee et al., 2008). p38 MAPK is a member of the MAPK family and is preferentially activated by inflammatory cytokines and environmental stress, such as osmotic stress, heat shock, and hypoxia (Lan et al., 2013). In the present study, we observed that the treatment of mouse hepatocytes with CoCl2 significantly upregulated phosphorylation of p38 MAPK and that pretreatment of cells with dieckol and NAC markedly blocked CoCl2-induced p38 MAPK phosphorylation. These results suggest that elevated intracellular ROS levels can lead to an activation of p38 MAPK. Indeed, these results are supported by previous reports that ROS interact with p38 MAPK in CoCl2-induced hypoxic injury (Lan et al., 2012, 2013). In the present study, we found that dieckol, NAC, and SB203580 attenuated the CoCl2-induced decrease in the expression of Bcl-2 as well as the CoCl2-induced increase in the expression of Bax and caspase-3. In addition, dieckol, NAC, and SB203580 (an inhibitor of p38 MAPK) blocked CoCl2-induced decrease in cell viability, indicating that
activation of p38 MAPK by ROS mediates the hepatic cell damage induced by CoCl2. These results are supported by the evidence that superoxide-induced apoptotic cell death in rat hepatic stellate cells is associated with p38 MAPK activation (Jameel et al., 2009). Therefore, the results of this study suggest that hepatoprotective effects of dieckol are mediated by reduction not only of CoCl2-induced ROS but also of p38 MAPK activated by CoCl2-induced ROS. In the present study, the expression of COX-2 was increased by the treatment of mouse hepatocytes with CoCl2. At present, there are at least two forms of COX: COX-1 is constitutively expressing and COX-2 is induced by multiple factors including cytokines, hormones, mitogens, oxidative stresses, and so forth (Jung et al., 2009; Smith et al., 2000). Our results showed that increased COX-2 expression by CoCl2 is attenuated by pretreatment with dieckol and NAC. This result indicates that COX-2 expression is enhanced by CoCl2-induced intracellular ROS and dieckol represses COX-2 expression by its antioxidant activity. Previous studies showed that inhibition of COX-2 exerts hepatoprotective effects on liver damage (Vadiraja et al., 1998). COX-2 expression is upregulated by ischemia or reperfusion in the rat liver, and the inhibition of COX-2 is found to improve liver function and viability (Kim et al., 2003; Oshima et al., 2009). Through this study, we observed that dieckol and NS-398, a selective inhibitor of COX-2, increased CoCl2-induced decrease in the expression of Bcl-2 and attenuated the CoCl2-induced increase in the expression of Bax and caspase3. In addition, dieckol and NS-398 attenuated CoCl2-induced
DOI: 10.3109/01480545.2014.928719
cytotoxicity. Overall, these findings suggest that oxidative stress plays a pivotal role in hepatocellular injuries induced by CoCl2 and ROS mediate CoCl2-induced injury through p38 MAPK and COX-2 pathway in mouse hepatocytes. In conclusion, the present study demonstrated that dieckol has a cytoprotective effect against chemical hypoxia-induced cell injury through inhibition of the ROS, p38 MAPK, and COX-2 pathway in primary cultured mouse hepatocytes.
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Acknowledgments This work was supported by a grant from the Next-Generation BioGreen 21 Program (No. PJ009090), Rural Development Administration; National Research Foundation (NRF) grant funded by the Korean government (MSIP) (NRF2012R1A4A1028835); Kyungpook National University Research Fund, 2013, Republic of Korea.
Declaration of interest None declared.
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