Red ginseng abrogates oxidative stress via mitochondria protection mediated by LKB1-AMPK pathway

RGE was provided by Korea Tobacco & Ginseng Corporation (Daejeon, Korea) [22]. 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 [9], 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 [7].

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 [12].

The MTT assay was performed as previously described [12]. 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)].

The TUNEL assay was performed using the DeadEnd™ Colorimetric TUNEL System, according to the manufacturer’s instruction [23]. 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 [23]. Protein bands of interest were developed using an ECL chemiluminescence system (Amersham, Buckinghamshire, UK). Equal protein loading was verified by immunoblotting for β-actin.

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[23]. 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.

Reduced GSH in the cells was quantified using a commercial GSH determination kit (Oxis International, Portland, OR) [12]. 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.

MMP was measured with rhodamine 123, a membrane-permeable cationic fluorescent dye [12]. 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.

AA + iron-induced cytotoxicity model is an effective experimental model for screening drugs for liver disease [7]. To determine whether or not RGE protects liver cells from AA + iron-induced injury, HepG2 cell viability was measured by MTT assay after treatment with different doses of RGE. Treatment with AA + iron significantly reduced cell viability compared with the control group as shown in Figure 1A. However, RGE treatment inhibited AA + iron treatment-induced cell death in a dose-dependent manner, and cell viability was completely recovered by treatment with 1 mg/ml of RGE (Figure 1A). To further investigate the cytoprotective effects of RGE on AA + iron-induced liver cell injury, TUNEL assay was performed at a dose of 1 mg/ml. Treatment with 1 mg/ml of RGE alone did not induce hepato-cytotoxicity, whereas the same dose (1 mg/ml) of RGE significantly reduced AA + iron-induced cell death (Figure 1B). To confirm the cytoprotective effects of RGE on AA + iron-induced cell death, the levels of PARP and procaspase-3 were measured by immunoblot analysis. Treatment with AA + iron induced cleavage of PARP and procaspase-3, resulting in cell death. In contrast, decreases in the levels of PARP and procaspase-3 induced by AA + iron were inhibited by treatment with RGE (Figure 1C). These results indicate that RGE has cytoprotective effects against apoptosis in hepatocytes induced by AA + iron.

To investigate the mechanism underlying the protective effects of RGE on AA + iron-induced liver cell death, ROS generation was measured by FACS with or without RGE treatment. There was no ROS generation in RGE alone-treated cells comparable to control cells. AA + iron treatment significantly induced ROS generation, whereas RGE treatment completely inhibited ROS production (Figure 2A). To further investigate the anti-oxidative effects of RGE on AA + iron-treated cells, GSH was measured by the colorimetric method. The intracellular concentration of GSH in HepG2 cells was reduced by treatment with AA + iron (Figure 2B). Treatment with RGE increased the intracellular concentration of GSH and inhibited the AA + iron-induced reduction of GSH. Taken together, these data indicate that RGE inhibits AA + iron-induced ROS generation and GSH reduction.

Next, we determined whether or not AA + iron-induced liver cell death is mediated by mitochondrial dysfunction. We measured fluorescence intensity in HepG2 cells stained with rhodamine 123 by FACS. MMP was not altered by treatment with RGE alone compared with the control group (Figure 3A). The number of rhodamine 123-negative cells increased AA + iron treatment, whereas it was significantly reduced by RGE co-treatment (Figure 3B). These results indicate that RGE prohibits AA + iron-induced ROS generation and dysfunction of MMP to protect liver cells.

To further investigate the mechanism of RGE during hepatocyte protection, the AMPK pathway was analyzed by immunoblot analysis. The phosphorylation levels of AMPK and ACC increased upon RGE treatment, and protein levels reached their maximums at 0.5-1 h and 1-3 h, respectively (Figure 4A and B). AMPK and ACC were also phosphorylated upon RGE treatment in both H4IIE and AML12 immortalized hepatocyte cell lines (Figure 4C).

LKB1, the upstream kinase of AMPK, was also phosphorylated by RGE treatment in HepG2, H4IIE, and AML12 cell lines (Figure 5A). Phosphorylation of AMPK and ACC was not detectable in LKB1-null HeLa cells (Figure 5B). Furthermore, phosphorylation of AMPK and ACC was decreased by knock-down of LKB1 in RGE-treated HepG2 cells (Figure 5C). Additionally, STO609 (1 μg/ml), an inhibitor of calcium/calmodulin-dependent kinase kinase (CaMKK) β, another upstream kinase of AMPKα, had no effect in reversing RGE-induced AMPKα phosphorylation (Figure 5D). These data indicate that RGE treatment activates the AMPK-ACC pathway in hepatocytes via activation of LKB1.

To determine the involvement of AMPK in RGE-induced protection of hepatocytes, MMP was measured after treatment with Compound C, an AMPK inhibitor (Figure 6A). There was no protective effect of RGE against AA + iron-induced mitochondrial dysfunction in AMPK inhibitor-treated cells (Figure 6B), verifying that AMPK is the key protein protecting liver cells upon RGE treatment. All of these data indicate that RGE activates the AMPK pathway to protect hepatocytes against AA + iron (Figure 6C).