Twenty adult male and female H. asinina (1–1.5 years of age) were obtained from the Coastal Aquaculture Research and Development Centre, Department of Fisheries, Prachuabkirikhan Province, Thailand. Animals were anesthetized by immersion in an ice bath for 15 min. The HBG and gills were rapidly dissected, washed and frozen in liquid nitrogen for storage at −80°C. Proteins and metabolites were extracted by homogenizing at 4°C in 0.1% trifluoroacetic acid using a Polytron homogenizer (Brinkmann Instruments, Westbury, NY, USA), followed by sonication. The extracts were centrifuged (15,000 × g for 30 min at 4°C) and then the supernatant was lyophilized.

HaCaT cells were purchased from Cell Lines Service (CLS, Eppelheim, Germany). Cells were cultured under standard conditions of 5% CO₂ in air at 37°C with medium renewal every 2 days. Culturing medium was Dulbecco's modified Eagle's medium (DMEM) (Gibco BRL, Gaithersburg, MD, USA) containing 5% fetal bovine serum (FBS), 10 μg/ml of penicillin/streptomycin. Cultured HaCaT cells were plated at a density of 5×10⁵ cells/cm² onto 60×100 cm² tissue culture-treated Petri dishes (SPL Life Sciences Co., Ltd., Pochoen, Korea).

HaCaT cells were trypsinized from subconfluent cultures by adding Trypsin-EDTA (0.25%) solution (Gibco BRL), and incubated for 10 min with regular gentle shaking. The trypsin reaction was stopped by adding 10 ml of DMEM (Gibco BRL) containing 10% FBS. The cell suspension was then centrifuged at 1,000 × g for 5 min at 25°C. The cell pellet was re-suspended in 2 ml of culturing medium with 5% FBS and mixed by vortexing. The cells were counted with a hemocytometer. Then, 1×10⁵ cells were equally seeded into each well of a 96-well black plate and incubated at 37°C for 24 h. After that, the culturing medium was removed and replaced with 200 μl of serial abalone extracts in 1% serum-media. Serial concentrations of abalone extracts were from 0.1 to 50 mg/ml. The cells were then incubated at 37°C for 24 h and 100 μl of a 10% AlamarBlue solution (Invitrogen, Carlsbad, CA, USA) in serum-free medium was directly added to each well. The plate was incubated at 37°C for 4 h and the absorbance was measured at 540 and 630 nm with a fluorescent spectrophotometer (Epoch; BioTek, Winooski, VT, USA). The percentage of cell viability was analyzed by GraphPad Prism version 6 (GraphPad Software, Inc., San Diego, CA, USA) using one-way ANOVA at P<0.001.

BLX-312 ultraviolet radiometer UV-B incubator emitted UV-B light with a wavelength of 300–320 nm. Irradiance was measured by UV sensor. To test UV-B irradiation, cultured HaCaT cells were divided into 4 groups: a control group and 3 experimental groups. In the control group, cells were exposed to serial dosages of UV-B from 10 to 110 mJ/cm². In the experimental groups, cells are incubated with 10, 100 and 200 μg/ml abalone extract prior to and during the serial UV-B exposure. Then, cell viability, morphological changes and inflammatory cytokine expression levels were observed and measured by AlamarBlue, calcein AM-propidium iodide (PI) live-dead staining and western blot analysis.

HaCaT cells were trypsinized from subconfluent cultures by adding trypsin solution, incubated for 10 min with regular gentle shaking. The trypsin reaction was stopped by adding 10 ml of DMEM culture medium containing 10% FBS. The cell suspension was then centrifuged at 1,000 × g for 5 min at 25°C. The cell pellet was re-suspended in 2 ml of culturing-medium with 5% FBS and properly mixed by vortexing. The cells were counted with a hemocytometer. Then, 1×10⁵ cells were equally seeded into each well of a 96-well black-plate and incubated at 37°C for 24 h. After that, the culture medium was removed and replaced with 100 μl of serum-free medium. Ready to test cell plate was covered by a black sticker except one column opened for UV-B exposure. UV-B was emitted at 10 mJ/cm², and then the UV-B exposure was stopped for 5 min and the next column sticker was removed and the next 10 mJ/cm² UV-B dose was administered. The same protocol was repeating and continuing 10 times. Thus, the first column was exposed to 110 mJ/cm² of UV-B, the 11th column received 1 mJ/cm² and the last column was covered and exposed to no UV. Then, 100 μl of 10% AlamarBlue solution in serum-free medium was added to each well. The plate was incubated in 37°C for 4 h and the absorbance was measured at 540 and 630 nm with a fluorescent spectrophotometer (Epoch; BioTek). The number of viable cell was calculated as described previously (41). Next, viability of the cells in the experimental groups was tested in the same way as the control group but either the abalone HBG or gill extract was administered. In the experimental groups, the cells were incubated with abalone peptides at the concentrations of 10, 100, 200 μg/ml for 8 h prior to and during the UV-B exposure. After that the serial UV-B exposure and viability test was carried out in the same way as the control group. The percentage of cell viability was analyzed by GraphPad Prism version 6 (GraphPad Software, Inc.) using one-way ANOVA at P<0.001.

Live-dead solution was prepared by adding: i) 250 μl of calcein AM (1 mg/ml stock solution); ii) 100 μl PI (1 mg/ml stock solution; iii) 3 ml of 5X binding buffer into serum-free medium to make 15 ml of calcein AM-PI live-dead staining solution (Molecular Probes Life Technologies, Carlsbad, CA, USA). The ready-to-use solution was maintained at 4°C. The fully grown HaCaT cells in 24-well Petri dishes were divided into two groups: i) control group; and ii) experimental group. In the control group, each dish was exposed to the serial UV-B doses started from 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110 mJ/cm². After every 10 mJ/cm², the culture dish was taken out of an incubator and incubated at room temperature for 5 min and then put back in and given the next dose of 10 mJ of UV-B. In the experimental group, 100 μg of abalone HBG and gill extracts (1:1) was incubated with the cultured cells for 8 h prior and during UV-B exposure. Images were captured using phase contrast and fluorescence modes, with an inverted digital microscope (EVOS FLC, Life Technologies, Grand Island, NY, USA).

Fully grown HaCaT cells in Petri dishes were divided into three groups: i) control group in which cells were cultured normally; ii) UV-B group in which cells were exposed to UV-B at 3×10 and 5×10 mJ/cm²; and iii) abalone peptide-treated group in which cells were incubated with 100 μg/ml peptides prior and during UV-B exposure and the cells were exposed to UV-B at 3×10 and 5×10 mJ/cm². After that, confluent cells were scraped and sonicated in 5X lysis buffer containing Protease Inhibitor Cocktail (Merck Millipore, Darmstadt, Germany). Then, the lysates were centrifuged at 15,000 × g at 4°C for 30 min and the supernatants were frozen in liquid nitrogen. The frozen protein samples were lyophilized in a freeze dryer (Scanvac CoolSafe 110-4 PRO). Approximately 25 μg of extract was re-suspended in 0.1 M phosphate-buffered saline (PBS) and mixed with loading dye containing 50 mM Tris-HCl, 10% glycerol, 2% SDS, 100 mM DTT and 0.1% bromophenol blue. Samples were separated using 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were then transferred to 0.45-μm polyvinylidene fluoride membrane. The membranes were blocked for 1 h by blocking solution contained 4% non-fat dry milk, 2% bovine serum albumin (BSA) and 0.1% Tween-20 in Tris-buffered saline (TBS) pH 7.6. Polyclonal antibodies for COX-2 (ab52237), SPK/JNK (ab112501), phospho-SPK/JNK (ab4821), p38 (ab31828) and phospho-p38 (ab4822) were purchased from Abcam (Cambridge, UK) and were used at a 1:1,000 dilution to detect expression levels of the above proteins. The membranes were washed and incubated for 45 min with the secondary antibody (anti-rabbit HRP conjugated; Amersham plc, Amersham, UK) at a 1:5,000 dilution. Then, the protein bands were visualized by enhanced chemiluminescence (Amersham plc) under GENE GNOME (Syngene Bioimaging Private Ltd., Gurgaon, India). The western blot analyses were repeated in triplicate and band density was quantified using densitometry UN-SCAN-IT software (Silk Scientific, Inc., Orem, UT, USA). Protein expression levels were analyzed by GraphPad Prism version 6 (GraphPad Software, Inc.) using Dunnett's multiple comparison test.

Optimal doses of abalone HBG and gill extracts were determined using an AlamarBlue cytotoxicity assay (Fig. 1A and B). For extracts derived from HBG, the maximal concentration that enabled cell survival of >50% was at 12.5 μg/ml (Fig. 1A and C). For extracts derived from gills, the concentration was at 12.5 μg/ml (Fig. 1B and D). Based on these results, subsequent experiments were performed using concentrations of 0.5, 2.5, 5.0 μg/ml of abalone tissue extracts.

HaCaT cells were exposed to serial doses of UV-B, starting from 1×10 to 11×10 mJ/cm², and then cell viability was compared between the four groups treated with the HBG and gill extracts (0, 0.1, 2.5 and 5.0 μg/ml with UV-B). At 1×10 to 3×10 mJ/cm² UV-B, there was no difference in cell viability among the four groups. At 4×10 mJ/cm² UV-B, the extract groups showed a significant increase in cell viability compared to the control group (Fig. 2A). In the control group, the cell viability was <50% at 6×10 mJ/cm² UV-B, while in the extract groups it remained at >80%. Moreover, HaCaT cell survival was at >50% following 9×10 mJ/cm² UV-B. HaCaT cell survival rates were further analyzed using a calcein AM-PI live-dead staining assay (Fig. 2B–I). In the 0 and 0.5 μg/ml extract-treated groups, 5×10 mJ/cm² UV-B resulted in almost 50% cell death (Fig. 2B and C), while the survival rate was >90% in the 2.5 and 5 μg/ml extract-treated groups at the same UV-B level (Fig. 2D and E). At 10×10 mJ/cm² UV-B, there was almost complete cell death (0.84% survival) in the control groups (Fig. 2F and G), while there was >30% cell survival when cells were incubated with 2.5 μg/ml abalone extract at the same UV-B level (Fig. 2H and I).

To monitor changes in cell morphology by calcein AM-PI live-dead staining, phase contrast and fluorescence microscopy was used (Fig. 3A–J). HaCaT cell morphological changes and viability following UV-B exposure (6×10 mJ/cm²) were assessed in two groups: i) control group, where HaCaT cells were exposed to UV-B alone; and ii) the experimental group where HaCaT cells received 2.5 μg/ml abalone extracts (1:1 HBG and gill extract) before UV-B exposure. UV-B exposure resulted in three obvious changes in cell morphology: i) cytoplasmic condensation; ii) cell shrinkage and membrane budding; and iii) condensation of chromatin. Fig. 3A–D shows representative micrographs of HaCaT cells with morphological changes after exposure to 6×10 mJ/cm² UV-B alone and with the abalone extract co-treatment. In the presence of 2.5 μg/ml of abalone extract, most cells were viable with only minor morphological changes being observed. At 8×10 mJ/cm² UV-B, most cells in the control group were dead (Fig. 3E and F) while in the experimental group there was >50% cell survival (Fig. 3G and H). This was also observed in the abalone extract-treated groups exposed to a higher UV-B dose (10×10 mJ/cm²). In the negative control (no UV-B exposure) (Fig. 3I and J), HaCaT cells appeared as round or rectangular in shape with an ~40–80 μm diameter. In addition, the nucleus occupied almost half of the total area (with 10–20 μm nucleolus), and the plasma membranes were clear, often attached to neighboring cells.

Expression of inflammation-related proteins (COX-2, SPK/JNK, phospho-SPK/JNK, p38, and phospho-p38) was analyzed by western blot analysis of HaCaT cell extracts following UV-B exposure, with or without treatment with the abalone extracts (Fig. 4A and B). The control represents HaCaT cell extracts with no UV-B and no abalone extract. HaCaT cells were also exposed to UV-B (3×10 and 5×10 mJ/cm²) with or without 2.5 μg/ml abalone extract. No significant change was observed in β-actin protein levels for any treatment. Abalone extract did show more significant inhibition of COX-2 expression when compared to the levels observed at 3×10 and 5×10 mJ/cm² UV-B. The extract also resulted in a more significant inhibition of the inflammatory-related active forms of SPK-JNK and p38, namely phospho-SPK-JNK and phospho-p-38, when compared to these levels following exposure to 5×10 mJ/cm² UV-B without the extract.