Abstract:

Wang Shen, Zhou Bingrong, Luo Dan, Zhang Jia′an, Liu Juan, Zhang Lichao, Yi Fei, Wu Hongjin, Li Dan, Hu Yanyan. Department of Dermatology, First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China Corresponding authors: Luo Dan, Email: daniluo2013@njmu.edu.cn; Zhou Bingrong, Email: bingrong.2002@163.com 【Abstract】 Objective To investigate the effects of conditioned medium of prematurely senescent fibroblasts induced by ultraviolet B （UVB） on the proliferation, aging and autophagy of human dermal fibroblasts. Methods Human dermal fibroblasts were isolated from the circumcised foreskin of healthy adolescent males, and subjected to primary culture. Premature senescence was induced in some fibroblasts by UVB radiation at 10 mJ/cm2 once daily for 5 consecutive days. Some fibroblasts were classified into two groups: an experimental group cultured in conditioned medium of UVB-induced prematurely senescent fibroblasts, and a control group cultured in conditioned medium of normal fibroblasts. After treatment for 20 consecutive days, cell counting kit-8 （CCK8） assay and 5-ethynyl-2′.-deoxyuridine （EDU） staining were performed to evaluate cellular proliferation, flow cytometry was conducted to estimate cell cycle, β-galactosidase staining to determine the percentage of senescent cells, accridine orange staining to detect the autophagy level, and Western blot and indirect immunofluorescence assay were carried out to determine the expression level of the autophagy-related protein LC3-B. Statistical analysis was done by using a two-sample t test with the Graphpad Prism 5 software. Results Compared with the control group, the proliferative activity of fibroblasts was significantly decreased （0.831 ± 0.017 vs. 0.973 ± 0.017, t = 5.850, P < 0.05）, while EDU staining and flow cytometry both showed a significant increase in the percentage of S-phase cells （both P < 0.05）, in the experimental group. The percentage of β-galactosidase-positive fibroblasts was significantly higher in the experimental group than in the control group （25.710% ± 0.304% vs. 5.257% ± 1.023%, t = 19.170, P < 0.05）. Accridine orange staining revealed that the red fluorescence intensity of fibroblasts was significantly lower （14.287 ± 2.269 vs. 29.614 ± 2.650, t = 4.390, P < 0.05）, while Western blot and indirect immunofluorescence assay both showed a significant elevation in the expression level of LC3-B （both P < 0.05）, in the experimental group compared with the control group. Conclusions The conditioned medium of prematurely senescent fibroblasts induced by UVB can downregulate autophagy and proliferation of fibroblasts, but accelerate their aging.