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Red ginseng abrogates oxidative stress via mitochondria protection mediated by LKB1-AMPK pathway
© Dong et al.; licensee BioMed Central Ltd. 2013
Received: 13 September 2012
Accepted: 26 February 2013
Published: 18 March 2013
Korean ginseng (Panax ginseng C.A. Meyer) has been used as a botanical medicine throughout the history of Asian traditional Oriental medicine. Formulated red ginseng (one form of Korean ginseng) has been shown to have antioxidant and chemopreventive effects.
This study investigated the cytoprotective effects and mechanism of action of Korean red ginseng extract (RGE) against severe ROS production and mitochondrial impairment in a cytotoxic cell model induced by AA + iron.
RGE protected HepG2 cells from AA + iron-induced cytotoxicity by preventing the induction of mitochondrial dysfunction and apoptosis. Moreover, AA + iron-induced production of ROS and reduction of cellular GSH content (an important cellular defense mechanism) were remarkably attenuated by treatment with RGE. At the molecular level, treatment with RGE activated LKB1-dependent AMP-activated protein kinase (AMPK), which in turn led to increased cell survival. The AMPK pathway was confirmed to play an essential role as the effects of RGE on mitochondrial membrane potential were reversed upon treatment with compound C, an AMPK inhibitor.
Our results demonstrate that RGE has the ability to protect cells from AA + iron-induced ROS production and mitochondrial impairment through AMPK activation.
During oxidation of fatty acids and phospholipids, phospholipase A2 triggers the release of arachidonic acid (AA), a ω-6 polyunsaturated fatty acid [1, 2]. As a biologically active pro-inflammatory mediator, AA can induce apoptosis through its effects on mitochondria (e.g. calcium uptake into mitochondria, or production of ceramide) [1, 2]. Furthermore, in the presence of iron, which is a catalyst of auto-oxidation, AA stimulates cells to produce excess ROS, resulting in induction of mitochondrial dysfunction [3–7]. AMP-activated protein kinase (AMPK, an important molecule sensing cellular energy status) is activated to reserve cellular energy content, and it plays a function in determining cell survival or death in pathological progression [7, 8]. This crucial role is supported by increases in cell survival upon treatment with the AMPK activators metformin and 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) [9, 10]. Moreover, a line of agents protecting cells has been shown to inhibit radical-induced stress through AMPK activation as well as induction of antioxidant enzymes [11, 12].
Korean ginseng (Panax ginseng C.A. Meyer) is one of the oldest and most frequently used botanicals in the history of traditional Oriental medicine. Korean ginseng extract is recommended for its life-enhancing properties as well as promotion of energy and longevity. Studies have shown that ginseng attenuates free radical-induced oxidative damage [13, 14], prevents carcinogenesis induced by toxicants , and possesses immunostimulating, anti-tumorigenic, and chemopreventive effects [16–18]. These numerous cytoprotective and chemoprotective properties attributed to ginseng might be explained in part by its ability to ameliorate oxidative or nitrosative stress . Korean red ginseng is one form of Korean ginseng that is marinated in an herbal brew (i.e. heating Panax ginseng either by sun-drying or steaming), resulting in the root becoming extremely fragile. It has been shown that red ginseng inhibits oxidative cell death through Nrf2 activation and protects smokers from oxidative DNA damage [20, 21]. Although the biological effects of red ginseng have been well studied, it is not yet clear whether or not its cytoprotective effects against mitochondrial impairment are induced by AA + iron.
In view of the numerous beneficial effects of red ginseng as well as the importance of AMPK in the protection of mitochondria, this study investigated whether or not Korean red ginseng extract (RGE) is capable of protecting mitochondria against the severe oxidative stress induced by AA + iron and, if so, whether or not this extract has the ability to prevent apoptosis. Our work demonstrates that RGE protects cells against severe oxidative burst by inhibiting mitochondrial impairment and ROS production through AMPK activation.
RGE was provided by Korea Tobacco & Ginseng Corporation (Daejeon, Korea) . AA and compound C were purchased from Calbiochem (San Diego, CA). Anti-procaspase-3, anti-phospho-acetyl-CoA carboxylase (ACC), anti-PARP, anti-phospho-LKB1 and anti-phospho-AMPK antibodies were obtained from Cell Signaling Technology (Beverly, MA). Anti-AMPK, anti-ACC and anti-LKB1 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Horseradish peroxidase-conjugated goat anti-rabbit, rabbit anti-goat, and goat anti-mouse IgGs were obtained from Zymed Laboratories (San Francisco, CA). Ferric nitrate, nitrilotriacetic acid , 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), rhodamine 123, 2′,7′-Dichlorofluorescein diacetate (DCFH-DA), anti-β-actin antibody, and other reagents were purchased from Sigma (St. Louis, MO). The solution of iron-NTA complex was prepared as described previously .
HepG2 (human), H4IIE (rat), and AML12 (mouse) hepatocyte-derived cell lines were purchased from ATCC (Rockville, MD). Cells were incubated in Eagle’s minimum essential medium without 10% FBS for 12 h. Then, cells were incubated with 10 μM AA for 12 h, followed by exposure to 5 μM iron after washing with PBS. To assess the effects of RGE, the cells were treated with RGE for 1 h prior to the incubation with AA at the indicated doses .
The MTT assay was performed as previously described . Briefly, HepG2 cells were plated at a density of 1 × 105 cells per well in a 48-well plate. After treatment, viable cells were stained with 0.25 mg/ml MTT for 2 h. The media was then removed, and formazan crystals produced in the wells were dissolved with the addition of 200 μl dimethylsulfoxide. Absorbance at 540 nm was measured using an ELISA microplate reader (Tecan, Research Triangle Park, NC). Cell viability was defined relative to untreated control [i.e. viability (% control) = 100 × (absorbance of treated sample)/ (absorbance of control)].
Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay
The TUNEL assay was performed using the DeadEnd™ Colorimetric TUNEL System, according to the manufacturer’s instruction . HepG2 cells were fixed with 10% buffered formalin in PBS at room temperature for 30 min and were permeabilized with 0.2% Triton X-100 for 5 min. After washing with PBS, each sample was incubated with biotinylated nucleotide and terminal deoxynucleotidyltransferase in 100 μl equilibration buffer at 37°C for 1 h. The reaction was stopped by immersing the samples in 2× saline sodium citrate buffer for 15 min. Endogenous peroxidases were blocked by immersing the samples in 0.3% H2O2 for 5 min. The samples were treated with 100 μl of horseradish peroxidase-labeled streptavidin solution (1:500) and were incubated for 30 min. Finally, the samples were developed using the chromogen, H2O2 and diaminobenzidine for 10 min. The samples were washed and examined under light microscope (200×). The counting of TUNEL-positive cells was repeated three times, and the percentage from each counting was calculated.
Cell lysates and Immunoblot analysis were performed according to previously published methods . Protein bands of interest were developed using an ECL chemiluminescence system (Amersham, Buckinghamshire, UK). Equal protein loading was verified by immunoblotting for β-actin.
Measurement of H2O2 production
DCFH oxidation was determined using a FACS flow cytometer (Partec, Münster, Germany). DCFH-DA is a cell-permeable non-fluorescent probe that is cleaved by intracellular esterases and is turned into the fluorescent DCF upon reaction with H2O2. The level of H2O2 generation was determined by the concomitant increase in DCF fluorescence. After treatment, cells were stained with 10 μM DCFH-DA for 1 h at 37°C. Fluorescence intensity in the cells was measured using FACS. In each analysis, 10,000 events were recorded.
Determination of reduced GSH content
Reduced GSH in the cells was quantified using a commercial GSH determination kit (Oxis International, Portland, OR) . Briefly, the GSH-400 method was a two-step chemical reaction. The first step led to the formation of substitution products (thioethers) between 4-chloro-1-methyl-7-trifluromethyl-quinolinum methylsulfate and all mercaptans present in the sample. The second step included β-elimination reaction under alkaline conditions. This reaction was mediated by 30% NaOH which specifically transformed the substituted product (thioether) obtained with GSH into a chromophoric thione.
Flow cytometric analysis of mitochondrial membrane potential (MMP)
MMP was measured with rhodamine 123, a membrane-permeable cationic fluorescent dye . The cells were treated as specified, stained with 0.05 μg/ml rhodamine 123 for 1 h, and harvested by trysinization. The change in MMP was monitored using a FACS flow cytometer (Partec, Münster, Germany). In each analysis, 10,000 events were recorded.
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.
Inhibition of AA + iron-induced hepatocyte death
Inhibition of AA + iron-induced ROS generation
Inhibition of MMP dysfunction
Activation of AMPK-ACC pathway via phosphorylation of LKB1
Inhibition of AA + iron-induced stress via AMPK pathway
Korean red ginseng is frequently used as a crude substance in traditional Oriental medicine and is also a well-known, highly used raw medicinal material. RGE have been reported to exhibit various biological activities, including anti-inflammatory and antitumor effects [16–18]. In this study, we report that RGE has cytoprotective effects against AA + iron-induced oxidative burst, as confirmed by inhibition of apoptosis, ROS production, and mitochondrial dysfunction, which were comparable to the efficacies of other known antioxidants (e.g., resveratrol and some flavonoids) [7, 12, 23, 24]. Our results provide evidence that RGE may be beneficial for treatment of liver diseases by protecting cells from radical stress-induced damage.
AA, a representative pro-inflammatory fatty acid derived from cell membranes, stimulates ROS generation, thereby inducing lipid peroxidation. AA is an important mediator of the pathophysiological processes of various diseases, although the role of AA in responding to toxic stress remains controversial. In most cases, AA promotes cellular ROS production and induces decreases in mitochondrial respiratory activity, and ROS generated by metabolism of AA contributes to the process of tissue damage [25, 26]. In addition, AA releases Ca2+ from intracellular stores and increases mitochondrial uptake of Ca2+, which may cause apoptosis . In other cases, prostaglandins, the main byproducts of AA, may be responsible for the protection of some tissues [28, 29]. Nevertheless, AA-stimulated oxidative stress has been shown to have a direct effect on mitochondria [1, 2].
Iron accumulation in specific tissues (e.g. liver) is commonly associated with oxidative and inflammatory damage, including metabolic disease and cancer [3, 30], which enhances oxidant production, lipid peroxidation, protein oxidation, and DNA damage. Since iron is a catalyst of auto-oxidation, the combination of AA and iron increases radical stress and cell death in a synergistic manner [7, 12, 31]. Moreover, HepG2 cells were used to apply the well-established culture conditions of synergism to this model. In fact, a series of cytoprotective and important agents have been evaluated using this model [7, 12, 24, 32]. This cell line was employed to comparatively evaluate the protective effects of RGE in cells and mitochondria. To determine the effects of RGE on oxidative stress, we employed an in vitro approach using a combination treatment with AA and iron to HepG2 cells.
AMPK (an intracellular sensor of energy status) serves as a crucial regulator of cell survival or death in response to pathological stress (e.g., oxidative stress, endoplasmic reticulum stress, and osmotic stress) [8, 33]. This important function of AMPK is supported by the finding that cell viability is increased by treatment with AMPK activators, including AICAR or resveratrol [12, 24]. Moreover, a series of beneficial compounds have shown the ability to protect mitochondria, thereby inhibiting ROS production through activation of AMPK (e.g., oltipraz, resveratrol, isoliquiritigenin, and sauchinone) [7, 12, 23, 24].
In the present study, RGE activated AMPK in hepatocytes. Moreover, AMPK inhibition induced by compound C also prevented the ability of RGE to increase dysfunction of MMP, suggesting that AMPK indeed inhibits AA + iron-induced oxidative stress. In mammalian cells, LKB1 and CaMKKβ are the major upstream kinases of AMPK [34, 35]. RGE phosphorylation of AMPK was inhibited by LKB1 knock-down but not by treatment with CaMMK inhibitor. Overall, it appears that AMPK activation induced by RGE may protect hepatocytes against AA + iron-induced oxidative stress. However, LKB1-AMPK might not be a direct target of RGE. Protein kinase C-ζ or protein kinase A are the kinases that phosphorylate LKB1, an upstream kinase of AMPK . The pharmacological upstream target of RGE remains to be confirmed.
Our results demonstrate that RGE exerts cytoprotective effects by increasing antioxidant capacity and recovery of mitochondrial function, which may be associated with AMPK activation. The present results may be informative in elucidating the action mechanism and efficacy of RGE in hepatocyte protection as well as in determining its potential in treating various diseases related with oxidative stress.
This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government [MEST](No. 2012-0009400). And, this was also partly by a grant from Daegu Haany University Ky. lin Foundation in 2012.
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