自噬促进剂西罗莫司对中波紫外线诱导成纤维细胞提早衰老的影响

Effects of sirolimus on ultraviolet B irradiation-induced premature senescence of skin fibroblasts

目的研究自噬促进剂西罗莫司对反复亚毒性中波紫外线诱导提早衰老（UVB—SIPS）的影响。方法人皮肤成纤维细胞分为6组，对照组、10mg／L西罗莫司组、UVB组、UVB＋0．1mg／L西罗莫司组、UVB＋1．0mg／L西罗莫司组、UVB＋10．0mg／L西罗莫司组。UVB照射剂量10mJ／cm2每日1次共5次，对照组用含1％小牛血清的DMEM培养基培养5d；西罗莫司组在每次换液后加入西罗莫司；西罗莫司＋UVB组在每次照射UVB后加入西罗莫司过夜。CCK-8检测细胞活性，β半乳糖苷酶化学染色法检测衰老细胞，吖啶橙染色检测细胞自噬，Western印迹检测衰老相关分子信号p53及自噬相关蛋白LC3-B及beclin1的表达水平。数据用SPSS16．0软件分析，多组间均数行单因素方差分析，结合t检验和LSD法。结果UVB＋0．1、1．0、10mg／L西罗莫司组的细胞活性（A450值）分别为0．2±0．02、0．36±0．04、0．39±0．04，呈浓度依赖性升高，与UVB组（0．26±0．01）比较，差异均有统计学意义（均P〈0．05）；3个组B半乳糖苷酶染色阳性细胞分别为92．50％±0．34％、42．40％±0．53％、6．20％±0．39％，与UVB组（95．10％±0．32％）比较，差异均有统计学意义（P〈0．05）；3组吖啶橙染色荧光定量分别为36．43±0．24、45．25±0．33、48．69±0．37，与UVB组（33．99±0．32）比较，差异均有统计学意义（P〈0．05）；3组p53、LC3-B及beclin1的表达与UVB组比较，差异均有统计学意义（P〈0．05）。结论自噬促进剂西罗莫司在提高细胞自噬率的同时，可抑制UVB诱导的成纤维细胞提早衰老。

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 fihroblasts in a dose-dependent manner, with the absorhance 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 fibrohlasts 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.