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目的探讨羟基酪醇(hydroxytyrosol,HT)对苏丹红Ⅰ号(SudanI)所致的HepG2遗传毒性的化学防护作用及机制。方法采用微核(micronucleus,MN)试验检测细胞染色体损伤程度。为探讨机制,以2′,7′-二氢二氯荧光素二乙酸酯(DCFH-DA)为荧光探针检测细胞内活性氧(reactive oxygen species,ROS)水平;以荧光法检测细胞内的谷胱甘肽(glutathione,GSH)的水平;以Western blot法来检测γ-谷氨酰半胱氨酸合成酶(gamma-glutamylcysteine synthetase,γ-GCS)的表达水平。结果100 mol/L苏丹红Ⅰ号引起HepG2细胞的微核形成率、ROS水平和GSH水平明显增加(P<0.01),γ-GCS表达水平较对照组明显减少(P<0.01);不同浓度的HT(0、25、50和100 umol/L)预处理HepG2细胞30 min,再加入100umol/L苏丹红Ⅰ号后,HT预处理组微核形成率、ROS水平和GSH水平较单独接触苏丹红Ⅰ号组明显减轻(P<0.01或P<0.05),并且存在剂量依赖关系,而γ-GCS表达水平HT预处理组较对照组明显增加。结论在本试验条件下,HT能够通过降低细胞内ROS的水平以及增加细胞内的古胱甘肽的水平而抑制苏丹红Ⅰ号所致的遗传毒性。
Objective To investigate the chemoprotective effect of hydroxytyrosol (HT) on HepG2 genetic toxicity induced by Sudan I and its mechanism. Methods The degree of cell chromosome damage was detected by micronucleus (MN) test. In order to explore the mechanism, the level of reactive oxygen species (ROS) in cells was detected by using 2 ’, 7’-dichlorofluorescein diacetate (DCFH-DA) as fluorescent probe. Glutathione (GSH). The expression of γ-glutamylcysteine synthetase (γ-GCS) was detected by Western blot. Results 100 nmol / L Sudan II induced micronucleus formation, ROS and GSH levels in HepG2 cells (P <0.01), while the expression of γ-GCS in HepG2 cells was significantly decreased compared with the control group (P <0.01) HT (0, 25, 50 and 100 umol / L) pretreatment HepG2 cells for 30 min, then add 100umol / L Sudan I, micronuclei formation rate, ROS levels and GSH levels in HT pretreatment group compared with Sudan Ⅰ group was significantly reduced (P <0.01 or P <0.05), and there is a dose-dependent manner, while γ-GCS expression levels of HT pretreatment group was significantly increased compared with the control group. Conclusion Under the experimental conditions, HT can inhibit the genotoxicity caused by Sudan I by decreasing the level of intracellular ROS and increasing the level of intracellular glutathione.