Abstract: FANG Xiao-bo, ZHOU Bing-rong, LUO Dan, GUO Ze, YIN Hui-bin, HU Yan-yan. Department of Dermatology, First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China Corresponding author: LUO Dan, Email: daniluo2011@gmail.com 【Abstract】 Objective To observe the effect of sirolimus, an autophagy enhancer, on premature senescence in fibroblasts induced by repeated exposure to a subtoxic dose of ultraviolet B （UVB）. Methods Skin fibroblasts from foreskin tissue of healthy adolescents were classified into six groups: control group cultured in Dulbecco′s modified Eagles′ medium （DMEM） containing 1% calf serum, UVB group receiving UVB irradiation only, sirolimus group treated with sirolimus of 10 mg/L （added after daily exchange of culture medium）, and three combined groups receiving UVB irradiation immediately followed by overnight treatment with sirolimus of 0.1, 1.0 and 10.0 mg/L respectively. UVB irradiation was given at a dose of 10 mJ/cm2 once a day for five successive days. After five days of treatment, cell counting kit-8 （CCK-8） was used to evaluate cell viability, β-galactosidase staining to detect senescent cells, Western blot to quantify the expressions of p53, LC3-B and beclin 1 in these fibroblasts. Autophagy level was determined by acridine orange staining followed by fluorescence microscopy and transmission electron microscopy. Data were processed by the SPSS 16.0 software, and statistical analysis was done by one-way analysis of variance, t test and least significance difference. Results Sirolimus significantly increased the proliferative activity of fibroblasts in a dose-dependent manner, with the absorbance value at 450 nm being 0.27 ± 0.02, 0.36 ± 0.04 and 0.39 ± 0.04 for fibroblasts irradiated with UVB followed by treatment with sirolimus of 0.1, 1.0 and 10 mg/L respectively, compared to 0.26 ± 0.01 for fibroblasts irradiated with UVB only （all P < 0.05）. Significant differences were also observed between the fibroblasts irradiated with UVB followed by treatment with sirolimus of 0.1, 1.0 and 10 mg/L and those irradiated with UVB only in the percentage of β-galactosidase-positive fibroblasts （92.50% ± 0.34%, 42.40% ± 0.53% and 6.20% ± 0.39% vs. 95.10% ± 0.32%, all P < 0.05） and intracellular intensity of acridine orange-induced fluorescence （36.43 ± 0.24, 45.25 ± 0.33 and 48.69 ± 0.37 vs. 33.99 ± 0.32, all P < 0.05）. Moreover, the expressions of p53, LC3-B and beclin 1 in the three combined groups differed significantly from those in the UVB group （all P < 0.05）. Conclusion Sirolimus can inhibit UVB-induced premature senescence likely via upregulation of autophagy in fibroblasts. 【Key words】 Fibroblasts; Cell aging; Ultraviolet rays; Sirolimus; Autophagy

Key words: fibrodblast, Cell senescence, Sun protection factor, Autophagy