人参皂苷和枸杞多糖对UVB诱导培养的成纤维细胞提早衰老的影响

目的 用重复亚毒性剂量UVB辐射培养的皮肤成纤维细胞，观察衰老相关的生物学标志的表达情况，并研究人参皂苷Rb1、Rg1和枸杞多糖对UVB诱导的提早衰老的影响，及对衰老相关的信号分子p16、p21和p53表达的影响。方法 给予培养的皮肤成纤维细胞10次、每次剂量为15 mJ/cm2的UVB辐射。用光镜观察细胞形态学改变，用透射电镜观察细胞超微结构，用β－半乳糖苷酶化学染色法检测衰老细胞，用流式细胞仪检测细胞周期，用RT-PCR检测p16、p21和p53 mRNA的表达水平。结果 三种中药单体对培养的成纤维细胞形态、β－半乳糖苷酶活性、细胞周期和p16、p21和p53 mRNA的表达均没有影响。UVB辐射后，细胞形态和超微结构发生改变；91.5%细胞β－半乳糖苷酶染色阳性；UVB辐射组的G1期细胞数（88.63% ± 4.67%）上升，与对照组（49.18% ± 5.53%）比较差异有统计学意义（P < 0.05）。UVB + 人参皂苷Rb1、UVB ＋ 人参皂苷Rg1、UVB ＋ 枸杞多糖组G1期细胞数分别为71.04% ± 1.64%、70.38% ± 2.58%、80.09% ± 3.46%，与UVB辐射组比较差异均有统计学意义（P均 < 0.05）。与对照组比较，UVB辐射组的p16、p21和p53 mRNA的表达明显增加（P < 0.05）。与UVB辐射组比较，三种中药单体能明显抑制UVB辐射细胞的p16 mRNA的表达（P均 < 0.05），人参皂苷Rb1和人参皂苷Rg1能明显抑制p21 mRNA的表达（P < 0.05），人参皂苷Rb1和枸杞多糖能明显抑制p53 mRNA的表达（P < 0.05）。结论 人参皂苷Rb1、Rg1和枸杞多糖对UVB诱导培养的成纤维细胞提早衰老具有抑制作用，对p16、p21和p53 mRNA表达的下调作用是抑制作用的机制之一。

Effects of ginsenoside and Lycium barbarum polysaccharide on UVB irradiation-induced premature senescence of skin fibroblasts

Objective To observe the expressions of senescence-associated biomarkers in fibroblasts after repeated exposures to subtoxic doses of ultraviolet B (UVB), and to study the effect of ginsenoside Rb1 and Rg1 as well as Lycium barbarum polysaccharide on the UVB-induced premature senescence and on the expressions of senescence-associated signals including pl6, p21 and pS3. Methods Skin fibroblasts were classified into 8 groups to receive pretreatment with traditional Chinese medicine (TCM) monomers only, UVB irradiation only, no treatment, or both pretreatment and irradiation. UVB was given successively at a dose of 15 mJ/cm~2 for 10 times, and the concentration of three monomers was 50 mg/L. After 5 days of treatment, light microscopy was used to observe the morphology of fibroblasts, transmission electron microscopy to study the cell ultrastructure,β-galactosidase histochemical staining to detect senescent cells, flow cytometry to analyze cell cycle, and RT-PCR to measure the mRNA expressions of p16, p21 and p53 in these skin fibroblasts. Results None of the 3 monomers had any effect on cell morphology, P-galactosidase activity, cell cycle or the mRNA expression of p53, p21 and pl6 in skin fibroblasts. After UVB irradiation, some changes occurred to cell morphology and ultrastructure; 91.5% of the cells were stained positively for P-galactosidase. The proportion of cells in G1 phase was 88.63% ± 4.67% in irradiated fibroblasts, significantly different from that in untreated controls (49.18% ± 5.53%, P< 0.05) and that in irradiated fibroblasts pretreated with ginsenoside Rbl and Rgl as well as Lycium barbarum polysaccharide (71.04% ± 1.64%, 70.38% ± 2.58%, 80.09% ± 3.46%, all P < 0.05). Compared with untreated fibroblasts, the mRNA expression of p53, p21 and pl6 significantly increased in irradiated fibroblasts (P < 0.05), however, the induced increase in the mRNA expression of pl6 was inhibited by all the three monomers (all P< 0.05), that of p2l by ginsenoside Rb1 and Rg1 (P< 0.05), and that of p53 by ginsenoside Rbl and Lycium barbarum polysaccharide (both P < 0.05). Conclusions Ginsenoside Rbl, Rgl and lycium barbarum polysaccharide can inhibit UVB-induced premature senescence, which may be associated with the down-regulation of mRNA expressions of pl6, p21 and p53.