关键词: 芦丁, 成纤维细胞, 衰老, 黑素细胞, 黑素生成, 黄褐斑

Abstract: 【Abstract】 Objective To investigate effects of rutin on ultraviolet irradiation （UVR）-induced human dermal fibroblast （FB） senescence and melanogenesis in mouse ear skin. Methods The third- to fifth-passage FBs were divided into 4 groups: a blank control group, a UVR group, a rutin group, and a combined treatment group. In the UVR group, FBs were irradiated using an ultraviolet irradiator at a single dose of 0.6 J/cm2 UVA combined with 0.03 J/cm2 UVB once daily for 5 consecutive days; FBs in the rutin group were cultured in Dulbecco's modified Eagle medium containing 50 μmol/L rutin for 5 days; the combined treatment group received both UVR and the treatment with 50 μmol/L rutin for 5 days; the blank control group underwent no treatment. β-Galactosidase staining was performed to assess the senescence of FBs, real-time quantitative PCR （qPCR） to measure the telomere length in FBs, and Western blot analysis to detect the expression levels of stem cell factor （SCF） in FB cell lysates and culture supernatants. FB culture supernatants were collected from each group, and mixed with M254 medium at a ratio of 3∶1 to prepare conditioned medium, which was then used to treat PIG1 melanocytes for 24 hours. Western blot analysis was conducted to determine the tyrosinase （TYR） expression in PIG1 melanocytes in each group, while the 5-ethynyl-2′-deoxyuridine （EdU） incorporation assay was applied to assess the proliferative activity of PIG1 cells in each group. Ten Dct-LacZ transgenic mice were divided into a control group and a UVR group. For each mouse, 5% rutin-containing cream was topically applied to the right ear after UVR, while the left ear treated with the cream base alone served as a control. Skin biopsies were performed after 4 weeks, followed by X-gal staining and Avidin/fluorescein isothiocyanate （FITC） staining to count the numbers of melanocytes and mast cells in mouse ear skin. Results In the UVR group, the number of senescent FBs （25.67 ± 2.89）, relative protein expression levels of SCF （1.95 ± 0.22）, and relative levels of SCF in the cell culture supernatant （1.52 ± 0.34） were all significantly higher than those in the blank control group （5.67 ± 1.56, 0.95 ± 0.11, 1.01 ± 0.31, respectively）, while these indicators were significantly lower in the combined treatment group （12.00 ± 1.63, 1.32 ± 0.19, 1.15 ± 0.32, respectively） than in the UVR group （all P < 0.05）. The relative telomere length in FBs was significantly shorter in the UVR group （0.49 ± 0.12） than in the blank control group （0.94 ± 0.11; LSD-t = 3.15, P = 0.021）, but significantly longer in the combined treatment group （0.81 ± 0.13） than in the UVR group （LSD-t = 4.30, P = 0.034）. After the treatment with FB conditioned medium, the relative expression level of TYR in PIG1 melanocytes and the number of EdU-positive cells were significantly higher in the UVR group （2.54 ± 0.21, 33.54 ± 3.21, respectively） than in the blank control group （0.97 ± 0.19, 21.45 ± 2.51, respectively; both P < 0.001）, but significantly lower in the combined treatment group （1.63 ± 0.12, 18.54 ± 3.87, respectively） than in the UVR group （both P < 0.001）. X-gal staining and Avidin/FITC staining showed that the numbers of melanocytes and mast cells in the mouse left ear skin in the UVR group （5.00 ± 1.22, 98.60 ± 8.47, respectively） were significantly higher than those in the mouse left ear skin in the control group （1.80 ± 0.45, 53.80 ± 5.76, respectively） and those in the mouse right ear skin treated with the rutin-containing cream in the UVR group （2.80 ± 0.45, 69.60 ± 8.89, respectively） （all P < 0.05）. Conclusion Rutin may inhibit UVR-induced skin melanogenesis by suppressing the senescence of dermal FBs and paracrine secretion of SCF.

Key words: Rutin, Fibroblast, Aging, Melanocytes, Melanogenesis, Melasma