野黄芩苷对苯并芘致人胚肺成纤维细胞DNA损伤的保护作用

Protective Effect of Scutellarin on DNA Damage Induced by Benzopy- rene in Human Embryonic Lung Fibroblasts

目的探讨野黄芩苷对苯并芘（benzo[a]pyrene，B（a）P）诱导的DNA损伤的保护作用。方法①采用稳定转染抑癌基因p53-GFP质粒的p53-/-MEF细胞（p53-GFP细胞）筛选野黄芩苷等小分子化合物对p53启动子的激活情况；②将MRC-5细胞分为DMSO对照组，B（a）P处理组，B（a）P和野黄芩苷共处理组，用Western印迹检测不同处理组MRC-5细胞中p-p53（Ser15）和γH2AX的蛋白表达情况。⑧将MRC-5细胞分为DMSO对照组，B（a）P处理组，B（a）P和野黄芩苷共处理组，Western印迹检测不同处理组MRC-5细胞中凋亡相关蛋白PARP的变化。结果荧光结果显示，10μmol／L的B（a）P能够明显激活p53-GFP荧光，其中野黄芩苷和B（a）P共处理后荧光表达被明显抑制；用Western印迹检测发现，与DMSO对照组相比，B（a）P处理的MRC-5细胞中p-p53（Ser15）和γH2AX的蛋白表达增加，PARP的切割增强，而野黄芩苷和B（a）P共处理的MRC-5细胞中p-p53（Ser15）和γH2AX蛋白表达水平较B（a）P处理组明显降低，同时PARP的切割明显减弱，并且具有浓度依赖性。结论本研究采用稳定转染p53．GFP质粒的p53-/-MEF细胞初步观察到野黄芩苷可能对苯并芘诱导的DNA损伤具有保护作用。在MRC-5细胞中的进一步研究证明，野黄芩苷与B（a）P共处理能降低MRC-5中p-p53（Ser15）和7H2AX的表达水平，同时能降低凋亡相关蛋白的表达，提示野黄芩苷对苯并芘致MRC-5细胞DNA损伤具有保护作用，其机制可能与抑制DNA损伤和凋亡通路有关。

Objective To study the protective effect of scuteHarin on DNA damage induced by benzo [a] pyrene （ B （a） P） . Methods ①The p53-/-MEF cells （p53-GFP cells） were successfully transfected with p53-GFP plasmid to screen the activation of p53 promoter induced by small molecule compounds such as scutellarin. ② The MRC-5 cells were divided into DMSO control group, B （a） P treatment group, B （a） P ＋ scutellarin group; p-p53 （Serl5） and γH2AX pro- tein expression levels were detected by Western blotting. ③ The MRC-5 cells were divided into DM-SO control group, B （a） P treatment group, B （a） P ＋ scutellarin group; Western blotting was used to detect the changes of apoptosis-related protein PARP in MRC-5 ceils. Results Fluorescence Results showed that 10 μmoL/L of B （a） P significantly activated p53-GFP fluorescence, while the fluorescence activation was significantly inhibited by scutellarin ＋ B （a） P. Western blotting showed that in comparison with DMSO group, the expression of p-p53 （Ser15） and γH2AX in MRC-5 cells in B （a） P group was increased, PARP cleavage enhanced, whereas p-p53 andγH2AX were significantly lower than those in the B （a） P ＋ scutellarin group, at the same time, the cleavage of PARP was significantly decreased in a concentration-dependent manner. Conclu- sion In this study, by stable transfection with p53-GFP plasmid in p53-/- MEF cells, it was ini- tially observed that scutellarin could protect against DNA damage induced by benzopyrene. Further studies in MRC-5 cells demonstrated that the combination of scutellarin and B （a） P can reduce the expression of p-p53 （ Ser15 ） and γH2AX in MRC-5 and decrease the expression of apoptosis-related proteins in MRC-5 cells, suggesting that scutellarin has a protective effect on DNA damage induced by benzopyrene in MRC-5 cells, and its mechanism may be related to inhibition of DNA damage and apoptosis pathway.