Preparation of Pearl Extract (PE)
PE containing 2.1% total protein was kindly provided by Zhejiang Osmum Biological Co., Ltd. (Huzhou, China). The freshwater pearls produced by Hyriopsis cumingii were ground to a nanometre scale (10-100 nm) and hydrolysed with neutral protease to obtain various amino acids, trace elements, and polypeptides.
Cell Culture and Subgroups
HaCaT cells (purchased from Shanghai GeFan Biotechnology Co., Ltd., Shanghai, China) were cultured in RPMI-1640 medium supplemented with 10% foetal bovine serum and 1% penicillin and streptomycin in 5% CO2 at 37°C. Using a biological-inverted microscope, round-shaped newly subcultured HaCaT cells were observed. After 4 h of culture, the cells began to adhere to the culture plate, and after 24 h of growth, the cells were completely adherent. The cells were collected during exponential growth for subsequent experiments and were divided into the following subgroups: control group, photoaging cell group (irradiated with 10 J/cm2 UV), and PE + UV group (pre-treated with PE and then irradiated with 10 J/cm2 UV). According to our previous study, PE was added to the culture medium for 48 h at different concentrations (0, 0.01, 0.1, 1 and 10 μg/mL) and then cells were UV irradiated.
UV Irradiation of Cells
Prior to UV irradiation, the cells were washed with phosphate-buffered saline (PBS) and covered with a thin layer of PBS. The cells were irradiated in ice-cold plates to eliminate UV thermal stimulation. Monolayers of HaCaT cells in a thin layer of PBS were irradiated with 10 J/cm2 UV and incubated with culture medium containing PE for 24 h.
CCK8 Cell Viability Assay
HaCaT cells (1 × 105/mL) were cultured in 96-well plates. Different concentrations of PE (0, 0.01, 0.1, 1 and 10 μg/mL) were added to the cell suspension, and the cells were incubated for 48 h at 37°C in a 5% CO2 incubator. The cells were irradiated with 10 J/cm2 UV, whereas control cells were sham irradiated by covering with tin foil. Furthermore, the cells were incubated for 24 h in a cell incubator at 37°C in 5% CO2. Cell viability was assessed by the CCK8 assay (Jian Cheng Bioengineering Co., Nanjing, China). After the indicated treatments were performed, 10 μL of CCK8 was added to each well for 4 h at 37°C, and the absorbance of each well was measured using a plate reader at a wavelength of 490 nm. Finally, the concentration of PE showing a significant protective effect against UV radiation-induced cell damage was selected for further experiments. The experiment was repeated five times.
Assays of Cellular ROS, Glutathione Peroxidase (GSH-Px), Superoxide Dismutase (SOD), and Malondialdehyde (MDA)
Cells were seeded in 6-well plates (2 × 104 cells/well) and treated with different concentrations of PE (0.1 and 1 μg/mL) for 48 h prior to UV irradiation. Subsequently, the cell suspensions were collected and assayed for ROS and MDA levels and GSH-Px and SOD activity using assay kits in accordance with the manufacturer’s instructions (Jian Cheng Bioengineering Co., Nanjing, China).
Cytokine mRNA Levels
Total RNA was isolated from HaCaT cells via treatment using an RNAiso Plus kit (TaKaRa Bio, Shiga, Japan) according to the manufacturer’s guidelines. For each RT-PCR sample, 1 μg of total RNA was added, and the purity of the RNA was determined as the ratio of the optical density reading at 260 nm relative to that at 280 nm. The ratio of the RNA used for RT-PCR was 1.8 to 2.0. TNF-α, IL-10, and β-actin mRNA levels were determined by real-time quantitative PCR using a SYBR® Premix Ex Taq™ Kit (TaKaRa Bio) according to the manufacturer’s instructions. cDNA amplification of a specific sequence of human TNF-α, IL-10, and β-actin was performed by PCR using the primer sequences shown in Table 1. PCR was conducted at 95°C for 30 s, followed by 40 cycles of 95°C for 5 s and 60°C for 34 s in the StepOne Plus real-time PCR system (Applied Biosystems, Foster City, CA, USA). The qRT-PCR results were analysed and are expressed as the relative mRNA expression of the threshold cycle (CT) value, which was then converted to the fold change. Quantitative real-time RT-PCR assays were performed to detect β-actin expression and normalize the amount of cDNA in each sample.
Cytokine Protein Expression
After being treated as indicated for 48 h, the supernatant of each group of cells in the 6-well plate was collected. TNF-α and IL-10 were detected in accordance with the enzyme-linked immunosorbent assay kit instructions (Jian Cheng Bioengineering Co.). The cytokine concentration was measured three times.
Western Blot Analysis
After appropriate treatment for 48 h, the HaCaT cells were collected using RIPA mixed with phenylmethylsulphonyl fluoride to extract protein (Solarbio Science & Technology Co., Ltd., Beijing, China). Protein levels were measured by the bicinchoninic acid assay (Jian Cheng Bioengineering Co.). Briefly, 50 μg of protein was resolved by 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis for 60 min at 140 V, and the resolved proteins were transferred to a polyvinylidene fluoride membrane for 45 min at 60 V. The membrane was blocked with 5% fat-free dried milk powder in TBST (1×Tris buffered saline, 0.1% Tween-20) at room temperature for 2 h and incubated with primary antibody diluted 1:1000 in fresh blocking buffer overnight at 4°C with gentle shaking. Rabbit anti-caspase-3, rabbit anti-caspase-9, rabbit anti-Bcl-2, rabbit anti-Bax, and rabbit anti-actin antibodies were purchased from Cell Signaling Technology, Danvers, MA, USA. Goat anti-rabbit secondary antibodies (Abmart, Shanghai, China) were diluted at 1:8,000 in fresh blocking buffer and incubated with the membranes for 1 h at room temperature. The membranes were washed five times for 10 min each in TBST, and the bands were detected using an ECL Plus kit (Solarbio Science & Technology Co., Ltd.). The membranes were exposed to Tanon 5200 Multi (Tanon Science & Technology Co., Ltd., Shanghai, China), and TanonImage analysis software was used for quantitative analysis.
Statistical Analysis
All data are expressed as the mean ± SD. Experiments were independently repeated at least three times. P < 0.05 indicated significant differences between the experimental and control groups, which were analysed by one-way analysis of variance. Representative western blots from three independent experiments are shown.