Uva lamp

Before UVA irradiation, HDFs cells were rinsed and submerged under a thin layer of PBS to prevent UVA absorption by components of the medium, such as VB1. Cells were then irradiated using a Philips UVA lamp with an emission spectrum between 320 and 400 nm. Mock-irradiated cells were manipulated identically, except that they were not exposed to UVA. The dose of UVA irradiation was 10 J/cm² per day, as verified with a UV light meter (Sigma, Shanghai, China) for 3 days. Following each UVA irradiation, cells were incubated in complete medium, supplemented with indicated compounds.

Eight-week-old female FVB mice were obtained from the National Key Laboratory of Genetics (Changsha, Hunan, China). Animals were housed at 23 ± 1°C and 50 ± 10% relative humidity in a specific pathogen-free environment. Animal experiments were approved by the Animal Research Committee at the Xiang Ya Hospital of Central South University. The dorsal skin area of mice was shaved before and during experiments. Mice were divided into control, UVA,vehicle gel and VB1 groups, with 10 mice in each group. All Mice except control group were irradiated 3 times/week for 12 weeks with 20 J/cm² doses under a Philips UVA lamp placed 20 cm away (emission spectrum: 320–400 nm). The dorsal skin of mice was washed with 75% ethanol before each irradiation exposure to avoid blocking or absorption of UVA rays by previous applications of the VB1 gel. UVA doses were verified with a UV light meter. A Carbomer substrate gel containing 2% VB1 or vehicle gel lacking VB1 was applied dorsally to the mice accordingly every day. No topical application or irradiation was performed in the control group.

To prevent UVA absorption by factors within the growth medium, when reached 80% confluence, HDF cells were rinsed in phosphate-buffered saline (PBS), and submerged under a thin layer of PBS prior to UVA irradiation. Cells were then irradiated three times with a UVA dose of 10 J/cm² per day for 3 days, as verified with a UV light meter (Sigma, Shanghai, China), using a Philips UVA lamp (emission spectrum320 to 400 nm). Mock-irradiated cells underwent identical procedures, but were not exposed to UVA. The time interval of these three UVA irradiations is 24 hours. Following each UVA irradiation, cells were incubated in complete medium as described above.

Determination kits for malondialdehyde (MDA), glutathione (GSH) content, superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-PX) were purchased from Nanjing Jiancheng Bioengineering Institute. The other suppliers are DMEM high glucose medium (HyClone); P/S antibiotic (HyClone); fetal bovine serum (Hangzhou Tianhang Biotechnology Co., Ltd.); trypsin EDTA (Jinuo Biomedical Technology Co., Ltd.); Cell Counting Kit-8 (CCK8), cell proliferation detection kit (Biosharp); reactive oxygen species (ROS) detection kit (Beyotime); Annexin V-FITC/PI apoptosis detection kit (Tianjin Sanjian Biotechnology Co., Ltd.); a UVA lamp purchased from Philips Lighting (China) Investment Co., Ltd. (the lamp power is 9W and the spectral wave is 364–366 nm); Dr-200bs enzyme labeling detector (Diatek); FACSCalibur flow meter (BD); Sco6we CO₂ constant temperature incubator (Shell Lab); Cx-21 ordinary optical microscope (Olympus); and a Tgl-16 freezing centrifuge (Instrument of Hunan Xiangyi Laboratory).