Cell culture

Human immortalized keratinocytes (HaCaT) were purchased from the Cell Resource Center of the Institute of Basic Medical Sciences, Peking Union Medical College Hospital (IBMS, CAMS/PUMC). The primary melanocytes (MC) were cultured from adult healthy male foreskin tissues in our laboratory, and the second to fourth generations of cultured primary melanocytes were used in subsequent experiments.

Primary melanocytes were acquired from five men who had routine circumcisions performed at Chaoyang Hospital and the PLA Rocket Force Characteristic Medical Center in Beijing, China. Adult male foreskin samples were disinfected with 70% ethanol for disinfection and washed with phosphate-buffered saline (PBS) solution. After the fat and subcutaneous tissue were removed, the prepuce tissue was cut into 3 mm wide strips. After digesting the prepuce in 4 °C refrigerators for 18 h with 0.25% trypsin solution, the epidermis and dermis were separated with a blade. Primary melanocytes were isolated from the prepuce’s middle layer and cultured in the M254-medium (Gibco, USA) with 1% penicillin–streptomycin (Beyotime Biotechnology, Shanghai, China) and 1% human melanocyte growth supplement-2 (HMGS-2, Gibco, USA) at 5% CO2 and 37 °C. HMGS-2 contains essential substances for melanocyte growth but inhibits keratinocytes and fibroblasts. Keratinocytes and fibroblasts were gradually removed from the wall after replacing the culture medium 2–3 times. Similarly, the HaCaT cells were cultivated in the MEM/EBSS (Hyclone, South Logan, UT, USA) containing 1% penicillin–streptomycin and 10% fetal bovine serum (FBS, Hyclone, South Logan, UT, USA) at 5% CO2 and 37 °C.

Animals

The 8-week-old TYR(–/–) and TYR(+/–) knockout C57BL/6 J mice were obtained from the GemPharmatech Company (Jiangsu, China) and the age-matched wild-type TYR(+/+) C57BL/6 J mice from the Charles River Laboratory Animal Center (Beijing, China). The Experimental Animal Welfare Committee of the National Institute for Radiological Protection (NIRP, act no. 2021–009) of the Chinese Center for Disease Control and Prevention (China CDC) approved our animal experiment protocols. The study was conducted following the Chinese regulations for animal experimentation (Ministry of Agriculture, act no. 2001–464, 29 May 2001). We used wild-type TYR(+/+), TYR(–/–), and TYR(+/–) C57BL/6 J mice, three of each type, to explore the effects of TYR on melanin synthesis. In addition, 12 wild-type TYR(+/+) C57BL/6 J mice were randomized into non-treated, UVB, UVB + Vaseline, and UVB + 2.5% MT groups, 3 animals per group (n = 3), to determine the role of melatonin in UVB-induced skin morphological changes and melanin synthesis.

UVB irradiation

Logarithmic cells incubated with 2 mL of PBS were irradiated with 80 mJ/cm2 of UVB for 46 s at 1.5 mW/cm2 power density using a UVB lamp (311–313 nm) (model: SH4B-T UV, SIGMA, Shanghai, China). The dose was calibrated before irradiation using a TN-2340 ultraviolet intensity meter, and the correction coefficient value was 1.16. The light source was placed approximately 40 cm away from the cell. A uniform (1%) homogenous field of 10 cm × 15 cm was prepared for UVB irradiation. Cells were cultivated at 5% CO2 and 37 °C.

The mice were anesthetized and depilated to expose about 3 cm × 5 cm of the skin on the back. After 30 min of pre-treatment with melatonin, the exposed skin on the back and ear was irradiated with a 600 mJ/cm2 dose of UVB for 130 s at 4 mW/cm2 power density using a UVB lamp (311–313 nm), and the changes in the skin on the back and ear were observed at 0, 48, and 96 h after irradiation. A TN-2340 ultraviolet intensity meter was used to calibrate the dose before irradiation, and the correction coefficient value was 1.16. The light source was about 15 cm away from the skin on the back.

Preparation and treatment of melatonin formulations

The Sigma-Aldrich Company (Merck, Darmstadt, Germany) supplied melatonin powder. After completely dissolving 50 mg of melatonin powder in 0.5 mL absolute ethanol, PBS was added until the total volume of the solution was 2.15 mL to obtain the 0.1 mol/L melatonin storage solution. Diluting the concentrations with PBS yielded the other concentrations. The cells were pretreated with a 10−5 mol/ L melatonin solution for 12 h before irradiation. The composition of the melatonin ointment was given in weight per weight (W/W) percentages. First, solution 1 was prepared by mixing 100 µL of anhydrous ethanol and 200 µL of Tween-20 (Solarbio Company, Beijing, China). Then, a 1% melatonin ointment was prepared by adding 1 mg of melatonin powder to solution 1, and Vaseline was added to increase the total weight of the mixture to 1 g. Also, 2.5% and 5% melatonin ointments were prepared using this method and the corresponding required percentage of the individual components. The exposed back and ear skin of mice was evenly and thinly coated with melatonin ointment for 30 min before irradiation.

shRNA transfection

The pLKD-CMV-EGFP-2A-Puro-U6-TYR lentivirus vector was purchased from OBIO Technology (Shanghai, China) and transfected into the primary melanocytes at MOI = 40 using pLKD-CMV-EGFP-2A-Puro-U6-NC as the negative control (NC). A fluorescent microscope was used to detect the GFP protein level at 200 × and determine the lentivirus transfection efficiency (Thermo Scientific, Waltham, MA, USA) after 24 h. Then, the M-PER® Mammalian Protein Extraction Reagent (Thermo Scientific, Waltham, MA, USA) was used to extract the total protein in the subsequent assays. Three duplications of cell samples per group for each experiment, and this experiment was repeated three times.

Automated capillary electrophoresis Western blotting analysis

After cell lysis for 30 min at 4 °C using the RIPA buffer (Beyotime Biotechnology, Shanghai, China) containing protease and phosphatase inhibitors (Roche, Basel, Switzerland), the cells were centrifuged at 12,000 r/min using the cell lysates. The supernatant protein content was determined using the Bicinchoninic acid (BCA) kit (Thermo Fisher Scientific, Waltham, MA, USA). After extraction, a 5 × master mix with 0.1 × sample buffer was used to dilute cellular proteins using the relevant experimental kit. Then, the primary antibody was diluted using antibody diluent II provided in the kit. The diluted protein, diluted primary antibody, HRP-labeled secondary antibody, antibody diluent II, washing solution, and luminol-conjugate mix were then poured into each well of the plate provided in the kit. Finally, the proteins were fractionated, immobilized, and immunologically detected using the automatic capillary electrophoresis Western System (ProteinSimple, San Jose, CA, USA). The Compass for SW 4.0 software (ProteinSimple, San Jose, CA, USA) was used to quantify and visualize the proteins. The antibodies used were mouse anti-p53 (1C12) mAb (#2524S, CST, 1 : 50), rabbit anti-phospho-p53 (Ser15) antibody (#9284, CST, 1 : 50), the mouse anti-Tyrosinase antibody (T311) (sc-20035, Santa Cruz Biotechnology, 1 : 10), and mouse anti-β-actin mAb (#3700, CST, 1 : 50). Three duplications of cell samples per group for each experiment, and this experiment was repeated three times.

Quantitative real-time PCR (qRT-PCR)

The TRIzol reagent (Ambion, Austin, TX, USA) was used to extract the total RNA, which was later used to prepare cDNA using the PrimeScript™ II 1st Strand cDNA Synthesis Kit (TaKaRa, Tokyo, Japan) by reverse transcription. After that, 7500-Fast Real-time PCR (Thermo Company, USA) was used to conduct qRT-PCR. The expression of target genes was determined using the ∆∆CT approach, with β-actin as the reference. The sequences of tyrosinase (TYR) primers were 5′-TTGTGAGCTTGCTGTGTCGT-3′ (forward) and 5′-GTCAGGCTTTTTGGCCCTAC-3′ (reverse).

DOPA staining

The cells were added to each well with 1 mL of 4% paraformaldehyde (PFA) (Solarbio Company, China) after being washed with PBS. They were then fixed on a shaking table for 15 min. After that, PBS was used to rinse the cells three times for 3 min, and 1 mL of 0.3% TritonX-100 (Sigma Company, USA) was added to the cells per well for 30 min before washing three times with PBS. The staining group cells were mixed with 1 mL of 0.1% concentration L-DOPA solution (Sigma Company, USA) per well, preheated at 37 °C, and incubated for 4 h at 37 °C. The control cells were treated with 1 mL of PBS per well and photographed at 400 × magnification, and the PBS-incubated cells were used as a control. When the cells turned black, this confirmed the formation of melanin. The optical density ratio to total area (IOD/ARE) of the stained black cells was quantified using the Image-Pro Plus software (Media Cybernetics, USA). Three duplications of cell samples per group for this experiment.

Melanin content assay

Seventy two hours after UVB irradiation, we harvested and rinsed the cells three times with PBS and added 1 mol/L of NaOH. Then, the resultant mixture (100 µL) was added to the 96-well plates, followed by incubation at 37 °C for 60 min. Finally, a microplate reader (Multiskan MK3, Thermo Electron Corporation, MA, USA) was used to measure the absorbance (OD) value at 492 nm (OD492) to determine the melanin content. Three duplications of cell samples per group for this experiment.

Measurement of tyrosinase activity

Seventy two hours after cells were seeded into 96-well plates and exposed to UVB irradiation, 1% Triton X-100 buffer (100 µL) was added to each well and shaken for 15 min. After that, 0.1% 3, 4-Dihydroxy-L-phenylalanine (L-DOPA) buffer (100) was added to each well, followed by incubation at 37 °C for 2 h. Similarly, we determined the OD492 value to assess tyrosinase activity. Three duplications of cell samples per group for this experiment.

Analysis of senescence-associated beta-galactosidase (SA-β-gal) activity

The cells were stained following specific instructions using the SA-β-gal staining kit (Beyotime Biotechnology, Shanghai, China). Briefly, after washing with PBS, the cells were fixed using the fixation solution at ambient temperature for 15 min followed by overnight incubation at 37 °C with the staining solution. An optical microscope was used to analyze the results from three randomly selected fields at 200 × magnification and count the number of stained blue cells. Finally, the Image-Pro Plus 6.0 software (Media Cybernetics, Silver Spring, USA) was used to calculate the proportion of SA-β-gal-positive cells. Three duplications of cell samples per group for this experiment.

Giemsa staining

Seventy two hours after the primary melanocytes were irradiated with 80 mJ/cm2 UVB, they were fixed with methanol for 15 min. The primary melanocytes were removed and stained with Giemsa working solution for 15 min and washed with PBS solution. A microscope was used to measure the size of the cells and nuclei. Three duplications of cell samples per group for this experiment.

Lyso-Tracker Red staining

The primary melanocytes were incubated with Lyso-Tracker Red working solution for 30 min. Then, the Lyso-Tracker Red staining working solution was removed, and a normal cell culture medium was added to every well. The fluorescence intensity was then measured and photographed using a fluorescence microscope and imaging system (Olympus). Three duplications of cell samples per group for this experiment.

Statistical analysis

The GraphPad Prism 9.0 software (San Diego, CA, USA) was used for statistical analysis and plotting. A one-way ANOVA was used to compare several groups, followed by LSD tests, and a two-tailed Student’s t-test was used to compare two groups. The data homogeneity of the data was confirmed based on the variances and the normal distribution. The data were presented as the mean ± SD. All differences among and between groups were considered to be statistically significant at P < 0.05.