绞股蓝总皂苷对光老化人皮肤成纤维细胞Caspase-3信号通路的影响

摘要

Objective To investigate the protective effects of Gyp on caspase-3 signaling pathway of the injured hu-man skin fibroblasts ( HSF ) exposed to ultraviolet A ( UVA ) radiation. Methods HSF were randomly divided into control group,UVA model group,normal serum group,low dose drug serum group,middle dose drug serum group and high dose drug serum group. DCF was used to detect the amount of ROS in cells induced by ultraviolet. MTT assay was used to detect the cell vi-ability of HSF. RT-PCR and Western-blotting assays were used to detect the expression of Caspase-3 mRNA and its protein re-spectively. Results Compared with the control group,the amount of ROS in cells and OD were decreased significantly in the UVA model group,normal serum group,low dose drug serum group,middle dose drug serum group and high dose drug serum group,but the expression of Caspase -3 mRNA and its protein were increased significantly(P <0. 01). Compared with the UVA model group and normal serum group,the amount of ROS in cells and the expression of Caspase-3 mRNA and its protein were decreased significantly in low dose drug serum group,middle dose drug serum group and high dose drug serum group,but the OD was increased significantly in middle dose drug serum group and high dose drug serum group(P<0. 01). Compared with the low dose drug serum group,the expression of Caspase-3 mRNA was decreased significantly in middle dose drug serum group and high dose drug serum group(P<0. 01). Compared with the middle dose drug serum group,the expression of Caspase-3 mRNA was decreased significantly in high dose drug serum group ( P<0. 01 ) . Compared with the low dose drug serum group and middle dose drug serum group,the expression of Caspase -3 mRNA was decreased significantly in high dose drug serum group(P<0. 01). Conclusion Gyp can protect HSF from UVA-induced injury. Its protective effect on the cells may come from the suppression of the caspase-3 mRNA and protein expression and then prevent mitochondrial signaling pathway from acti-vation,thus reducing the apoptosis of HSF. Therefore,the mechanism of the protective effects of Gyp on UVA-injured HSF may closely related to its gene repair function on the cells.%目的：观察绞股蓝总皂苷（ Gyp ）含药血清对光老化人皮肤成纤维细胞（ HSF 细胞）凋亡现象及Caspase-3信号通路的影响。方法 HSF细胞分为：空白对照组、长波紫外线（ UVA）模型组、正常血清干预组、低剂量含药血清干预组、中剂量含药血清干预组和高剂量含药血清干预组。采用二氯荧光素（ DCF）法检测UVA诱导的细胞活性氧表达；采用四甲基偶氮唑盐微量酶反应比色法（ MTT法）检测各组HSF细胞活性；采用反转录-聚合酶链反应（ RT-PCR）和Western-blotting方法，分别检测各组HSF细胞Caspase-3基因和蛋白表达。结果与空白对照组比较，UVA模型组、正常血清干预组、低剂量含药血清干预组、中剂量含药血清干预组、高剂量含药血清干预组HSF细胞活性氧（平均荧光强度）及活性氧水平升高、OD 值降低、Caspase -3 mRNA 和蛋白表达水平升高（ P ＜0.01）；与UVA模型组和正常血清干预组比较，低剂量含药血清干预组、中剂量含药血清干预组、高剂量含药血清干预组HSF细胞活性氧（平均荧光强度）及活性氧水平降低，Caspase-3 mRNA和蛋白表达水平降低，中剂量含药血清干预组、高剂量含药血清干预组OD值升高（P＜0.01）；与低剂量含药血清干预组比较，中剂量含药血清干预组、高剂量含药血清干预组Caspase-3 mRNA表达水平降低（ P＜0.01）；与中剂量含药血清干预组比较，高剂量含药血清干预组Caspase-3 mRNA表达水平降低（P＜0.01）；与低剂量含药血清干预组和中剂量含药血清干预组比较，高剂量含药血清干预组Caspase-3表达水平降低（P＜0.01）。结论 Gyp含药血清可以帮助HSF细胞增强对UVA照射引起的光老化损伤的抵御能力，能够缓解HSF细胞光老化现象，其作用机制包括有效抑制细胞内活性氧产生和抑制Caspase-3信号通路的激活，使光老化细胞凋亡数目显著下降。Gyp对光老化HSF细胞的药理作用与减轻及修复HSF细胞光老化中DNA所受的损伤有关。