连翘花黄色素对秀丽隐杆线虫在氧化应激下的保护作用

Yellow Pigment from Forsythia Flower Promotes Resistance to Oxidative Stress in Caenorhabditis elegans

自由基过度引起的氧化应激是多种疾病发生的因素。连翘花黄色素(forsythia flower yellow pigment, FFYP)中含有大量的抗氧化活性物质,但其对氧化应激的抵抗性仍不清楚。本文首先通过化学方法检测FFYP的体外抗氧化活性;用细胞内抗氧化活性(cellular antioxidant activity,CAA)方法检测FFYP细胞内抗氧化活性;然后以秀丽隐杆线虫(Caenorhabditis elegans,C.elegans)为模型,检测FFYP对线虫氧化应激抵抗力及体内抗氧化指标的影响;用Daf-16和Skn-1突变体线虫和qRT-PCR实验探究其作用机制。研究结果表明,FFYP具有1,1-二苯基-2-三硝基苯肼(1,1-diphenyl-2-picrylhydrazyl, DPPH)自由基清除能力,铁离子还原能力和活性氧自由基(reactive oxygen species, ROS)清除能力,并且具有浓度依赖性。用500μmol/L的胡桃醌提供氧化应激压力时,FFYP能显著提高线虫在氧化应激下的寿命,表明FFYP可以提高线虫对氧化应激的抵抗力。进一步研究发现,FFYP可显著降低线虫体内ROS自由基含量,提高超氧化物歧化酶(superoxide dismutase, SOD)和过氧化氢酶(catalase, CAT)活性,增加还原型谷胱甘肽(glutathione, GSH)含量,表明FFYP通过提高线虫体内抗氧化防御系统活性清除自由基来提高线虫对氧化应激的抵抗力。突变体线虫实验显示,FFYP对线虫延长氧化应激下寿命的效应在Skn-1突变体线虫中完全消失,在Daf-16突变体中效应被减弱。qRT-PCR实验也显示,Daf-16和Skn-1靶基因的表达量均被提高。表明FFYP对线虫氧化应激抵抗力提高的作用是通过Daf-16和Skn-1共同作用。这预示着FFYP具有很好的抗氧化及抗应激药用价值,有潜力成为一种新的有生物活性的天然色素。

Oxidative stress caused by excessive free radicals is a factor in the occurrence of various diseases. A large number of components in forsythia flower yellow pigment (FFYP) have antioxidant activity, but its resistance mechanism to oxidative stress is still unclear. In this paper, the antioxidant activity of FFYP in vitro was detected by chemical method. The anti-oxidation activity of FFYP in cells was detected by Cellular Antioxidant Activity (CAA) assay. The Caenorhabditis elegans (C. elegans) model was used to detect the effects of FFYP on oxidative stress resistance and antioxidant index in vivo;the mechanism of action was analyzed by Daf-16 and Skn-1 mutant nematodes and qRT-PCR. The experimental results showed that FFYP could remove 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radicals, reduce iron ions and scavenge reactive oxygen species (ROS). All the above effects were concentration dependent. FFYP significantly increased the lifespan of nematodes under oxidative stress when 500 μmol/L of juglone was added. This indicated that FFYP increased the resistance of nematodes to oxidative stress. Further studies have found that FFYP could significantly reduce the content of ROS in nematodes, increase the activity of superoxide dismutase (SOD) and catalase (CAT), and increase the levels of glutathione (GSH). This study demonstrated that FFYP enhanced the resistance of nematodes to oxidative stress by increasing the activity of the antioxidant defense system to scavenge free radicals. Mutant nematode experiments showed that the promotion effect of FFYP in the lifespan of nematodes under oxidative stress completely disappeared in the Skn-1 mutant nematode, and was attenuated in the Daf-16 mutant. qRT-PCR experiments also showed that the expression levels of Daf-16 and Skn-1 target genes were both increased. These results indicated that FFYP enhanced the resistance of nematodes to oxidative stress through the interaction of Daf-16 and Skn-1. This indicated that FFYP, as an antioxidant that could prevent oxidative damage, has the potential to become a new bioactive natural pigment.