黄芪甲苷对长波紫外线影响人成纤维细胞的干预研究

Intervention of astragaloside to human skin fibroblasts affected by ultraviolet A

目的探讨黄芪甲苷对于长波紫外线(UVA)辐射导致人皮肤成纤维细胞老化的保护作用的机制。方法体外培养人皮肤成纤维细胞,以10 J/cm2UVA进行照射,并加入20 mg/L黄芪甲苷干预处理。在照射后24、72 h,流式细胞仪测定细胞周期分布变化;在照射后24 h,实时定量聚合酶链式反应法(RT-PCR)检测细胞衰老相关基因p53 mRNA、p16 mRNA和c-myc mRNA表达水平。结果与对照组相比,UVA照射作用下成纤维细胞出现了显著G1期阻滞(P<0.01),照射后24 h,G1期细胞比率由对照组的59.31%升高至81.36%,至照射后72 h,G1期细胞比率进一步升高至89.09%。黄芪甲苷可明显减轻UVA引起的细胞周期阻滞(P<0.01),G1期细胞比率下降为69.14%。RT-PCR检测显示UVA诱导p53 mRNA和p16 mRNA表达水平升高,而c-myc mRNA表达下降(P<0.01);黄芪甲苷干预处理可抑制UVA引起的p53 mRNA和p16 mRNA表达(P<0.01);黄芪甲苷干预可下调c-myc mRNA表达,UVA照射合并黄芪甲苷干预可进一步降低c-myc mRNA表达水平(P<0.01)。结论 UVA可诱导皮肤成纤维细胞出现G1期阻滞而启动老化进程,加入黄芪甲苷可有效减轻细胞周期阻滞延缓光老化进程,其具体机制可能与调控p53、p16、c-myc等老化相关基因表达有关。

Objective To discuss the mechanism of astragaloside intervening the ageing of human skin fibroblasts induced by the radiation of ultraviolet A （UVA）. Methods Skin fibroblasts were cultured in vitro, irradiated with UVA （10 J/cm2） and then intervened with astragaloside （20 rag/L）. 48 h and 72 h, the changes of cell cycle distribution were detected by using flow cytometer, After 24 h, and after 24 h, the expressions of ageing-related genes p53, pl6 and c-myc were detected by using real-time fluorescence quantitative polymerase chain reaction （RT-PCR）. Results Compared with control group, there were obvious blocks in G~ phase of fibroblasts in UVA group （ P 〈 0. 01 ）. After 24 h, the proportion of fibroblasts in G~ phase increased from 59.31% in control group to 81.36% in UVA group, and after 72 h, the proportion increased further to 89.09%. In astragaloside group, the blocks in G1 phase of fibroblasts were significantly alleviated （ P 〈 0.01 ）, and proportion of fibroblasts in G1 phase decreased to 69.14%. The results of RT-PCR showed that the expressions of p53 mRNA and pl6 mRNA increased and expression of c-myc mRNA decreased in UVA group （P 〈0.01 ）. The expressions of p53 mRNA and p16 mRNA were inhibited （P 〈 0.01 ） and expression of c-myc mRNA was down-regulated in astragaloside group. The expression of c-myc mRNA was down-regulated further in UVA combining astragaloside group （combining group, P 〈 0.01 ）. Conclusion The radiation of UVA can start the ageing process of skin fibroblasts through inducing the blocks in G1 phase. Astragaloside can inhibit this ageing process, and the mechanism may be related to regulating the expressions of ageing-related genes.