Fucoxanthin, the constituent of Laminaria japonica, triggers AMPK-mediated cytoprotection and autophagy in hepatocytes under oxidative stress

AA and Compound C (C.C) were purchased from Calbiochem (San Diego, CA, USA). Anti-phospho-ACC, phospho-LKB1, procaspase-3, PARP, Bcl_XL, LC3 I/II, beclin-1, AMPK, and phospho-AMPK antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA). Bal-A1 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Horseradish peroxidase-conjugated goat anti-rabbit, rabbit anti-goat, and goat anti-mouse IgGs were obtained from Zymed Laboratories (San Francisco, CA, USA). FX, acrydine orange hemi zinc chloride salt, 3-methyladenine (3-MA), anti-ß-actin antibody and other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA).

L. japonica was purchased from Daewon pharmacy (Daegu, Korea), which is standardized with a standard herb of L. japonica in Korea Food and Drug Administrations. The L. japonica (100 g) were extracted as previously described [20, 21]. The yield of lyophilized LJE was estimated to be 1.19% based on the dried weight.

HepG2 cells, a human hepatocyte-derived cell line, were provided by American Type Culture Collection (Rockville, MD, USA), and cultured as previously described [20]. To simulate oxidative stress, cells were incubated with 10 μM AA for 12 h, followed by exposure to 5 μM iron for 1 h. The cells were treated with FX or LJE for 1 h prior to the incubation with AA at the indicated doses.

The cells were plated at a density of 1 × 10⁵ cells per well in a 48-well for 24 h as previously described [20]. The media was incubated with 0.25 mg/ml MTT for 2 h, and formazan crystals were dissolved with the addition of 200 μl DMSO.

The TUNEL assay was performed using the DeadEnd™ Colorimetric TUNEL System, according to the manufacturer’s instruction. The samples were washed and examined under light microscope.

The cells were plated at a density of 5 × 10⁵ cells per well in a 6-well plate for 24 h. After the treatment designated, cells were lysed in RIPA buffer (Thermo Scientific, Rockford, IL, USA) as previously described [20, 21]. The protein bands were detected using Fusion Solo scanning system (Vilber Lourmat, Paris, France), and quantified using Image J ver 1.42 software (NIH, Bethesda, USA).

DCFH-DA, a cell-permeable non-fluorescent probe, has been used as a substrate for quantitation of intracellular oxidant production in HepG2 cells [21]. After treatment of reagents, cells were stained with 10 μM DCFH-DA for 30 min at 37 °C. The fluorescence intensity in the cells was measured at an excitation/emission wavelength of 485/535 nm, using in the cells measured in a microplate reader.

Intracellular GSH content was quantified using a commercial GSH BIOXYTECH GSH-400 kit (Oxis International, Portland, OR) according to the manufacturer’s protocol, and the absorbance level was measured at 405 nm.

ΔΨm was measured using rhodamine123 (Rh123). Following treatment, cells were stained with 0.05 μg/ml of Rh123 for 1 h and then harvested by trypsinization. The change in ΔΨm was monitored using a FACS flow cytometer (Partec, Münster, Germany). In each analysis, a total of 10,000 events were recorded as previous described [20].

HepG2 cells were plated on 18-mm cover glasses and incubated for 24 h to reach at approximately 70% confluence. They were then incubated either in the presence or absence of 30 μM FX, washed twice with PBS and fixed with ice-cold 4% paraformaldehyde for 10 min at room temperature. Subsequently, the cells were stained with AO (1 μg/ml) for 15 min at room temperature, washed, and examined under a fluorescence microscope (Nikon, Japan).

Water ACQUITYTM ultraperformance LC system (USA) was used to assess UPLC analysis as previously described [20]. Waters ACQUITYTM BEH C18 column (1.7 μm, 2.1 mm × 100 mm) was used as Waters ACQUITYTM PDA and HPLC Column, and the fucoxanthin was analyzed at 330 nm. The standard fucoxanthin was melted by methanol and diluted to make solution containing 1 μg/ml. L. japonica 1 g was also added with methanol 10 ml, and then sonication was perfomed for 3 h. After then, it was filtered through a 0.2 μm filter (Nalgene, NY, USA). A mobile phase was a mixed liquid of the acetonitrile and water, and the analysis condition was as in Table 1. The sample was injected with 2 μl, and a flow rate was 0.4 ml/min.

GraphPad Prism software version 5.01 (Graph Pad Software, La Jolla, CA) was used for all statistical analyses as previously described [20, 21]. Significance levels were calculated by repeated measures of ANOVA with the Dunnett post hoc test under 95% confidence interval. Data were presented as the mean with standard deviation (mean ± S.D.). Within figures, the P values were displayed with asterisks (***P < 0.001, **P < 0.01, *P < 0.05).

An MTT assay for cell viability indicated that LJE pretreatment (3, 10, 30, and 50 μg/ml) significantly protected cells from the potential injury induced by AA + iron. Since the maximum cell viability was achieved at 30 μg/ml of LJE, the same concentration was applied in subsequent experiments (Fig. 1a). In western blot analysis, treatment of AA + iron markedly induced decreases in the protein levels of procaspase-3 and Bcl_XL, verifying AA + iron induction of apoptosis, which was completely blocked by LJE pretreatment (Fig. 1b). Morphological examination by light microscopy and TUNEL assay (Fig. 1c) confirmed the cytoprotective effect of LJE against the synergized toxicity of AA + iron. After treatment with LJE, positive staining located in the nucleus by AA + iron were apparently decreased (Fig. 1c). To further examine the antioxidative effects of LJE, we measured the contents of GSH and ROS. The intracellular concentration of GSH was substantially decreased by AA + iron, but was recovered by LJE treatment. In contrast, treatment with LJE alone had no effects on cellular GSH levels (Fig. 1d). The ROS generation assay using DCFH-DA indicated that LJE treatment effectively abrogated increases in ROS production caused by AA + iron (Fig. 1e). The effect of LJE on AA + iron-induced loss of mitochondrial membrane potential (ΔΨm) monitored by FACS analysis of Rho123 staining (Fig. 1f). Rho123 fluorescence intensity was not significantly altered in LJE-treated cells compared to untreated controls, though AA + iron markedly reduced rhodamine fluorescence, indicating the loss of ΔΨm (Fig. 1f). LJE treatment significantly restrored the loss of ΔΨm caused by AA + iron (Fig. 1f).

Next, we determined the effects of FX against oxidative stress induced by AA + iron (Fig. 2a). FX treatment inhibited death of cell induced by AA + iron, and this decrease in cell viability was recovered by pre-treatment with 30 μM of FX (Fig. 2b). In western blot analysis, cleavage of PARP and caspase-3 were strongly observed in AA + iron-treated cells, which were blocked by FX pretreatment (Fig. 2c). FX treatment significantly inhibited the change in ΔΨm caused by AA + iron (Fig. 2d). These results indicate that FX remarkably suppressed AA + iron-induced collapse of ΔΨm, consequently protecting liver cells.

Stimulation of FX (30 μM) markedly induced the phosphorylation of AMPK (Fig. 3a). This compound also induced the phosphorylation of LKB1, an upstream kinase of AMPK, and ACC, a primary downstream target of AMPK (Fig. 3a). To determine the role of AMPK in protection of HepG2 cells by FX, we measured ΔΨm levels after treating with a chemical inhibitor of AMPK, C.C. C.C inhibited the protective effect of FX on AA + iron-induced the loss of ΔΨm in HepG2 cells (Fig. 3b). Collectively, these results suggest that FX activates the LKB1-AMPK signaling pathway, and that this activation of beneficial molecules is responsible for FX’s inhibition of mitochondria damage induced by oxidative stress.

Treatment with 30 μM FX upregulated beclin-1 and promoted the conversion of LC3 I to LC3 II as compared to the control (Fig. 4a). The AVOs were clearly observed in the HepG2 cells (red fluorescence) following treatment with FX (Fig. 4b). As shown in Fig. 4c, inhibition of AMPK activity by C.C markedly attenuated FX-induced accumulation of beclin-1, suggesting that AMPK is critical in the regulation of FX-induced autophagy. In addition, western blotting revealed that FX rapidly downregulated the phosphorylation of mTORC1 (Ser2448), which is known to negatively modulate autophagy and be inhibited by AMPK (Fig. 5a). In contrast, the phosphorylation levels of ULK1 (Ser555) was increased during FX treatment in time-dependent manner (Fig. 5a). Furthermore, we manipulated autophagy activity using 3-MA and Baf-A1 to suppress autophagy. FACS analysis of ΔΨm showed that 3-MA and Baf-A1 partially blocked the effect of FX on mitochondrial protection (Fig. 5b).