It is known that HSCs spontaneously demonstrate an activated phenotype and begin to proliferate after being plated. To ascertain when the phenotypical activation and proliferation of the HSCs are induced in our experimental conditions, we cultured HSCs for 13 days. The cell number was significantly increased after 7 days of culturing (Table 1). Similarly, the expression of α-smooth muscle actin (α-SMA), the most reliable marker for HSC activation, was observed in control HSCs after 7 days (Figure 1). The staining for α-SMA, using a computer with NIH image, were 15.7 ± 9.1, 16.1 ± 2.9, 22.4 ± 8.7, 37.6 ± 9.1 and 53.5 ± 6.7 pixels at 3, 5, 7, 9, 11 and 13 days after HSC isolation, respectively. Based on cell proliferation and the expression of α-SMA, HSCs that were cultured for 5 days were quiescent.

In our previous study, we found that high glucose levels induced the activation of quiescent HSCs that were cultured for 5 days. To investigate the effect of ECE on high glucose-induced HSC activation, we examined the effect of ECE on the high glucose-induced increase in the expression of type I collagen in HSCs. HSCs were incubated for 24 h with 400 mg/dL glucose and various concentrations of ECE. As shown in Figure 2, treatment with ECE suppressed the high glucose-induced increase in the expression of type I collagen in HSCs. The staining for type I collagen, using a computer with NIH image, were 10.6 ± 3.8, 45.7 ± 5.7, 24.4 ± 7.1, 13.4 ± 3.4 and 14.9 ± 8.1 pixels, in the cells for control (100 mg/dL glucose), 400 mg/dL glucose, 400 mg/dL glucose plus 6.25 μg/mL ECE, 400 mg/dL glucose plus 12.5 μg/mL ECE and 400 mg/dL glucose plus 25 μg/mL ECE, respectively. The most effective concentration by immunostaining was found to be 12.5 µg/mL ECE, and this concentration was used for the following experiments. To quantitatively detect the expression of type I collagen, we measured the levels of intracellular type I collagen by Western blot analysis and showed that treatment of HSCs with 12.5 μg/mL ECE for 24 h markedly suppressed the high glucose-induced increase in the expression of type I collagen (Figure 3).

To investigate the effect of ECE on high glucose-induced HSC activation, we also examined the effect of ECE on the high glucose-induced increase in the proliferation of HSCs. HSCs were incubated for 24 h with 400 mg/dL glucose and 12.5 μg/mL ECE. As shown in Figure 4, treatment with ECE reduced the high glucose-induced cell proliferation to the level of the control cells.

To investigate the relationship between high glucose-induced HSC activation and ROS formation, we measured the level of intracellular ROS using 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA), which is converted to the highly fluorescent DCF in the presence of intracellular ROS. The increase in ROS formation in high glucose-treated HSCs was inhibited by the addition of ECE (Figure 5).

We examined the effect of ECE on the high glucose-induced increase of intracellular lipid peroxidation using a TBARS assay. HSCs were incubated for 6 h with 400 mg/dL glucose and 12.5 μg/mL of ECE. The ECE treatment returned the intracellular MDA level to the level of the control cells (Figure 6).

To investigate the relationship between intracellular GSH levels and high glucose-induced HSC activation, we measured the intracellular GSH levels in HSCs using HPLC. As shown in Figure 7, the levels of intracellular GSH 2 h after a high glucose treatment were lower than those of the control cells. However, ECE not only reversed the decrease in GSH but also induced higher GSH levels than those in control cells.

TGF-β1 is a potent profibrogenic signaling factor that triggers the expression, accumulation and deposition of collagen [19,20]. Therefore, we measured the levels of TGF-β1 in the cell culture medium using a Quantikine TGF-β1 ELISA kit (R&D Systems). As shown in Figure 8, a high glucose concentration (400 mg/dL) stimulated the cells to secrete bioactive TGF-β1, and the stimulation was suppressed upon treatment of the HSCs with ECE.

Nycodenz was obtained from Nycomed Pharma AS (Oslo, Norway). Pronase E was purchased from Merck (Darmstadt, Germany). Deoxyribonuclease I was obtained from Roche LTD (Basel, Switzerland). Collagenase was purchased from Wako Pure Chemical Co., Ltd. (Osaka, Japan). DMEM was obtained from Nissui Pharmaceutical Co. Ltd. (Tokyo, Japan). Fetal bovine serum (FBS) was purchased from Nichirei Biosciences, Inc. (Tokyo, Japan). Monoclonal mouse anti-human smooth muscle actin antibody 1A4, biotinylated goat anti-mouse immunoglobulin and biotinylated goat anti-rabbit immunoglobulin and horseradish peroxidase-labeled streptavidin-biotin complex were obtained from DAKO A/S (Glostrup, Denmark). Rabbit anti-rat collagen type I polyclonal antibody was obtained from Chemicon International, Inc. (Temecula, CA, USA). The Quantikine TGF-β1 ELISA kit was obtained from R&D Systems, Inc. (Minneapolis, Minnesota, MN, USA). Other chemicals used in this study were special-grade commercial products purchased from WAKO Pure Chemical Co., Ltd. (Osaka, Japan).

A commercially available polyphenol extract from Ecklonia cava (Seapolynol, Livechem Inc, Jeju, Korea) was used. The total polyphenol content of the Ecklonia cava extract was 99.4%, as measured by the Folin-Ciocalteu reagent using phloroglucinol as a standard. Notable compounds in the Ecklonia cava extract that were identified by HPLC were dieckol (8.2%), 8,8'-bieckol (2.8%), 2-O-(2,4,6-trihydroxyphenyl)-6,6'-bieckol (2.1%), 6,6'-bieckol (1.5%), phlorofurofucoeckol-A (1.4%), eckol (0.6%), 2-phloroeckol (0.4%), 7-phloroeckol (0.4%) and phlorotannin A (0.4%) (Waters, column: CAPCELL PAK ODS column (4.6 × 250 mm); eluent: 30% aqueous MeOH; flow rate: 0.8 mL/min) [11].

Male Wistar rats, which weighed 300–350 g, were purchased from Japan SLC Inc. (Shizuoka, Japan) and housed at constant temperature with free access to water and standard rat chow (Labo MR stock; Japan SLC, Inc., Shizuoka, Japan). Animal experiments followed our institution’s criteria for the care and use of laboratory animals in research, which were in accordance with the guidelines for animal experimentation at Osaka City University.

HSCs were isolated from male Wistar rats as previously described [34]. HSCs were identified by their typical star-like configuration and a vitamin A autofluorescence. The purity was always higher than 95%. In the experiments to ascertain changes in α-smooth muscle actin (α-SMA) expression and cell proliferation after HSC isolation, the cells were plated at 5 × 10⁵ cells/mL on uncoated culture dishes in 1.5 mL of DMEM containing 10% FBS and supplemented with antibiotics (10⁵ U/L of penicillin G and 500 mg/L of streptomycin) until 13 days. The medium was changed to fresh DMEM containing 10% FBS every 2 days. For the experiments on high glucose-induced HSC activation, the cells were plated at 5 × 10⁵ cells/mL on uncoated culture dishes in 1.5 mL of DMEM containing 10% FBS and supplemented with antibiotics for 2 days and then cultured in fresh medium without serum for 24 h. After a pre-incubation, the cells were cultured in DMEM with different concentrations of glucose.

At the end of the incubation, the HSCs were fixed with 4% paraformaldehyde overnight at 4 °C. We used either an anti-α-SMA monoclonal antibody (1:100 dilution) or an anti-type I collagen polyclonal antibody (1:200 dilution) as the primary antibody. The samples were sequentially incubated with 0.3% hydrogen peroxide, followed by normal goat serum, then with the primary antibody for 1 h at room temperature, and afterwards with the biotinylated anti-mouse or anti-rabbit goat immunoglobulins for 30 min. This was then followed by an incubation with the horseradish peroxidase-labeled streptavidin-biotin complex for 30 min. For the peroxidase reaction, 3,3'-diaminobenzidine tetrahydrochloride (DAB) with a nickel chloride color modification was incubated for 5 min until the desired color intensity had developed. Quantification of the intensity of α-SMA and type I collagen expression was analyzed using a computer with the NIH image version 1.62 (National Institutes of Health, Bethesda, MD, USA).

Cells were harvested, washed twice with cold PBS and then dissolved with lysis buffer X (10 mM HEPES (pH 7.6), 10 mM KCl, 0.1 mM EDTA, 0.5% Nonidet P40, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride). After freezing and thawing twice with liquid nitrogen, the cells were sonicated and centrifuged at 800 × g for 10 min at 4 °C. The supernatant was collected for the cytosolic fraction. The precipitant was dissolved with lysis buffer Y (150 mM NaCl, 50 mM tris (pH 7.2), 1 mM EDTA, 1% Nonidet P40, 10 µg/mL leupeptin, 10 µg/mL pepstatin A and 100 µg/mL phenylmethylsulfonyl fluoride) for 30 min. Finally, the solution was sonicated and centrifuged at 2000 × g for 20 min at 4 °C, and the supernatant was collected for the nuclear fraction. Equal amounts of protein were loaded into the lanes of 10% SDS-PAGE gels, and the separated proteins were blotted to 0.45 µm polyvinylidene difluoride (PVDF) membranes (Amersham Pharmacia Biotech, Inc., Uppsala, Sweden). After blocking overnight with 0.1% Tween-20 and 5% non-fat dry milk in TBS, the membrane was incubated with an anti-α-SMA antibody for 1 h at room temperature. After washing, the membrane was re-incubated with biotinylated anti-mouse goat immunoglobulin (diluted 1:1000) for 1 h at room temperature. Next, the membrane was washed several times and then incubated with horseradish peroxidase-coupled streptavidin (diluted 1:200) for 1 h at room temperature. After several washing steps, the color reaction was developed with DAB. Densitometric analysis of the protein bands was performed using the software Scion Image (Scion Corporation, Frederick, MD, USA).

The intracellular GSH levels were determined according to the method of Sack et al. [35]. Cells were collected with Tris-HCl buffer (25 mM Tris adjusted to pH 7.5 with HCl). After sonication and centrifugation, the supernatant was used for an HPLC assay. For assaying GSH, 300 µL of supernatant was mixed with 300 µL of borate buffer (0.56 N boric acid adjusted to pH 10.4 with NaOH) and 50 µL of an OPA solution (10 mg/mL OPA in 10% methanol). GSH was separated on an ODS-II column (4.6 × 150 mm; particle size 5 µm; Shimadzu Techno-Research, Kyoto, Japan) using solvents A (30 mM sodium acetate adjusted to pH 6.0 with acetic acid) and B (92.3% methanol/7.7% acetonitrile, v/v). The sample was eluted with 96% solvent A and 4% solvent B and then with a programmed solvent gradient using a linear gradient curve. The gradient changed from 4 to 10% of solvent B from 0 to 5 min, from 10 to 14.9% of solvent B from 5 to 15 min, and from 14.9 to 4% of solvent B from 20 to 21 min at a flow rate of 0.45 mL/min. The fluorescence of the eluted fractions was measured using an RF1520 fluorescence monitor at excitation and emission wavelengths of 230 and 445 nm, respectively, to assay the GSH levels (JASCO Corporation, Tokyo, Japan).

The MTT [3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide] assay, an index of cell viability and growth, is based on the ability of viable cells to reduce a yellow water-soluble dye to a dark-blue insoluble formazan product. After 24 h of culture, the cells were incubated with the MTT solution for 2 h. The cells showing an MTT reaction were quantified at 600 nm using a multilabel counter (Wallac 1420 ARVOsx, Perkin Elmer Inc., Waltham, MA, USA). Cell survival was estimated as a percentage of the value of the untreated controls.

A relatively specific probe for the presence of hydrogen peroxide, 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA), was used to analyze the intracellular ROS formation [36,37]. Cells were incubated with 2.4 mM DCFH-DA (5 µL) for the last 30 min of the glucose treatment. Cells were washed with PBS twice and resuspended in Hank’s solution. The fluorescence intensities were measured using a multilabel counter (Wallac 1420 ARVOsx, Perkin Elmer Inc.) with an excitation wavelength of 485 nm and an emission wavelength of 535 nm. The amount of intracellular ROS was calculated from a standard curve derived from 2',7'-dichlorofluorescein (DCF). The protein concentration was measured using the Bradford method [38]. For visualization of the intracellular fluorescence, the cells were observed with an FSX100 Bio Imaging Navigator, which is an all-in-one fluorescence imaging system (Olympus Corporation, Tokyo, Japan).

Lipid peroxidation was assessed by determining the rate of production of thiobarbituric acid (TBA)-reactive components, which mainly detect malonaldehyde (MDA) [39]. After incubating for 6 h, the HSCs were collected and resuspended in 500 µL of Hank’s solution. After freezing and thawing, the 250 µL suspension was diluted to 500 µL with distilled water and then mixed with 15 µL of 50 mM butylated hydroxytoluene (BHT) and 1 mL of the thiobarbituric acid (TBA) solution. The mixtures were heated at 95 °C for 15 min. The fluorescence intensities were measured using a multilabel counter (Wallac 1420ArVOsx, Perkin Elmer Inc.) with an excitation wavelength of 485 nm and an emission wavelength of 535 nm. Tetramethoxypropane was used for a standard, and the results are expressed as nmol equivalents of MDA.

TGF-β1 was analyzed using a Quantikine TGF-β1 ELISA kit (R&D Systems, Inc.) according to the manufacturer’s instructions. The assay employs the quantitative sandwich enzyme immunoassay technique. The intensity of the color was measured at 450 nm by a multilabel counter (Wallac 1420 ARVOsx, Perkin Elmer Inc.). The concentrations of the total TGF-β1 was calculated from a standard curve derived from various defined concentrations of recombinant TGF-β1.

Results are presented as the means ± S.D. Statistical comparisons were performed between groups by a one-way analysis of variance and a post-hoc multiple comparison using a Tukey’s test. A p-value less than 0.05 was considered significant.