1. 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.
We acquired primary melanocytes from five individuals who received routine circumcision at the PLA Rocket Force Characteristic Medical Center, Beijing, China. Adult male foreskin samples were soaked in 70% ethanol for disinfection and washed with phosphate-buffered saline (PBS) solution. After removing the fat and subcutaneous tissue, prepuce tissue was cut into 3 mm wide strips. The epidermis and dermis were separated with a blade after the prepuce was digested using 0.25% trypsin solution in 4°C refrigerators for 18 h. Primary melanocytes were extracted from the middle layer of the prepuce and cultivated in the M254-medium (Gibco, USA) containing 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.
2. Animals
We obtained eight-week-old TYR(–/–) knockout C57BL/6J mice from the GemPharmatech Company (Jiangsu, China) and the age-matched wild-type (WT) C57BL/6J mice from the Charles River Laboratory Animal Center (Beijing, China). Our protocols for animal experiments were approved by the Experimental Animal Welfare Committee of the National Institute for Radiological Protection (NIRP, act no. 2021–009) under the Chinese Center for Disease Control and Prevention (China CDC). The study was conducted following the Chinese regulations for animal experimentation (Ministry of Agriculture, act no. 2001–464, 29 May 2001). Wild-type TYR(–/–) and TYR(+/–) knockout C57BL/6J mice showed the effect of the differences in TYR on morphological features and melanin synthesis in hair, whiskers, skin, eyes, and paws of mice (n = 3). In total, 12 C57BL/6J wild-type mice, were randomized into non-treated, UVB, UVB + Vaseline, and UVB + 2.5% MT groups (n = 3) to determine the role of melatonin in UVB-induced skin morphological changes and melanin synthesis.
3. 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). 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 kept about 40 cm away from the cell. A uniform (1%) homogenous field of 10 cm× 15 cm was prepared for UVB irradiation. We cultivated cells 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.
4. Preparation and treatment of melatonin formulations
Melatonin powder was obtained from the Sigma-Aldrich Company (Merck, Darmstadt, Germany). After 50 mg of melatonin powder was completely dissolved 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. Other concentrations were prepared by diluting them with PBS. The cells were pretreated with 10 − 5 mol/ L melatonin solution for 12 h before irradiation. The composition of the melatonin ointment was given as 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.
5. shRNA Transfection
The pLKD-CMV-EGFP-2A-Puro-U6-TYR lentivirus vector was purchased from the 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.
6. 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 Bicinchoninic acid (BCA) kit (Thermo Fisher Scientific, Waltham, MA, USA) was used to determine the supernatant protein contents. 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. After that, the diluted protein, diluted primary antibody, HRP-labeled secondary antibody, antibody diluent II, washing solution, and luminol-conjugate mix were poured into every well of plate provided in the kit. Finally, the automatic capillary electrophoresis Western System (ProteinSimple, San Jose, CA) was used for fractionating, immobilizing, and immunologically detecting the proteins. The "Compass for SW" software (ProteinSimple, San Jose, CA) 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).
7. 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).
8. DOPA staining
The cells were added to each well with 1 mL of 4% paraformaldehyde (PFA) (Solarbio Company, China) after being washed with PBS. Then, they were incubated on a shaking table for 15 min for fixation. Next, PBS was used to rinse the cells for 3 min thrice and 1 mL of 0.3% TritonX-100 (Sigma Company, USA) was added to the cells per well for 30 min, followed by washing thrice with PBS.The cells in the staining group were combined 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 a magnification of 400×. The cells incubated with PBS were used as a control. Melanin formation was confirmed when the cells turned black. The optical density ratio to total area (IOD/ARE) of the cells stained black was quantified using the Image-Pro Plus software (Media Cybernetics, USA).
9. Melanin content assay
After UVB irradiation for 72 h, we harvested and rinsed the cells with PBS thrice 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.
10. Measurement of tyrosinase activity
After seeding the cells into the 96-well plates and exposing them to UVB irradiation for 72 h, 1% Triton X-100 buffer (100 µL) was added to each well and shaken for 15 min. Next, 0.1% 3, 4-Dihydroxy-L-phenylalanine (L-DOPA) buffer (100) was added to each well, followed incubation at 37°C for 2 h. Similarly, we determined the OD492 value to assess tyrosinase activity.
11. 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 cells that were stained blue. 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.
12. Giemsa staining
The primary melanocytes were fixed with methanol for 15 min after they were irradiated with 80 mJ/cm2 UVB for 72 h. After removing, the primary melanocytes were stained with a Giemsa working solution for 15 min and washed with a PBS solution. The size of the cells and nuclei was recorded using a microscope.
13. Lyso-Tracker Red Staining
The primary melanocytes were incubated with a working solution of Lyso-Tracker Red 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).
14. Statistical analysis
The GraphPad Prism 9.0 software (San Diego, CA) was used for conducting statistical analyses and plotting. A one-way ANOVA was performed, followed by LSD tests for comparing several groups, whereas a two-tailed Student's t-test was conducted 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.