表没食子儿茶素没食子酸酯逆转小剂量辐射引起的DNA甲基化

Protective effects of EGCG against methylation changes induced by low dose radiation

目的探讨表没食子儿茶素没食子酸酯（EGCG）对0.5 Gy X射线引起的小鼠Rad23b和Ddit3基因启动子CpG岛甲基化及表达改变的逆转作用。方法 BALB/c雄性小鼠30只按随机数字表法均分为6组：对照组、单纯照射组、EGCG低剂量单纯给药组、EGCG高剂量单纯给药组、EGCG低剂量给药照射组、EGCG 高剂量给药照射组。照射方式为6 MV X 射线分次照射（0.05 Gy/d ×10 d）。小鼠在末次照射后2 h取血后处死，收集肾脏、肝脏、脾脏、脑及肺组织。采用BSP和Real-time PCR法检测各组小鼠外周血单个核细胞（PBMC）及各组织Rad23b和Ddit3启动子CpG岛的甲基化水平和 mRNA 表达变化。结果末次照射后2 h，与对照组比较，单纯照射组Rad23b在PBMC、肝脏、脾脏、脑和肺组织中甲基化水平均升高（ t ＝-20.19、-14.80、-12.05、-28.42、-12.58，P＜0.05），同时mRNA水平在PBMC、肝脏、脑和肺组织中表达降低（t＝25.25、17.43、11.53、22.85，P ＜0.05）；Ddit3甲基化水平在 PBMC、肝脏和肺组织中升高（t ＝-52.89、-20.31和-3.85，P ＜0.05），mRNA 水平在 PBMC 和肝脏中表达降低（t ＝11.89和16.52，P ＜0.05）。与单纯照射组比较，除脾脏中Rad23b外，不同浓度EGCG（10、20 mg/kg）给药照射组均能明显降低X射线引起的Rad23b和Ddit3启动子CpG岛高甲基化（t＝-13.39～7.99，P＜0.05），并诱导mRNA重新表达（t＝-34.02～-2.89，P＜0.05），且转录激活作用在高剂量给药照射组中更加明显。结论 EGCG作为逆转小剂量辐射损伤效应的天然药物，可能通过影响DNA甲基化来发挥作用。

Objective To investigate the role of epigallocatechin gallate （ EGCG） in reversing the CpG island methylation of Rad23b and Ddit3 gene promoter and its mRNA expression induced by 0.5 Gy X-rays. Methods Thirty BALB/c male mice were randomly divided into 6 groups： control group, irradiation group, low/high dose of EGCG group, low/high dose of EGCG with irradiation group. For the irradiation group, mice were fractionally exposed with 6 MV X-rays for 10 d （0.05 Gy/d × 10 d）. 2 hours after the final irradiation, all mice were killed and such tissues as blood, kidney, liver, spleen, brain, and lung were collected. Methylation and expression levels of Rad23b and Ddit3 were measured by bisulfate sequencing primers （ BSP） and Real-time PCR, respectively. Results Compare to the control group, Rad23b was hypermethylated in PBMC, liver, spleen, brain and lung （t= -20.19, -14.80, -12.05,-28.42, -12.58, P〈0.05） in the irradiation group. Meanwhile, its mRNA expression level was down-regulated in PBMC, liver, brain and lung （t=25.25, 17.43, 11.53, 22.85, P〈0.05）. Similarly, a significant hypermethylation change of Ddit3 was observed in PBMC, liver and lung after irradiation （ t=-52.89, -20.31, -3.85, P〈0.05） so that the mRNA expression of Ddit3 decreased in PBMC and liver （ t = 11.89, 16.52, P 〈 0.05 ）. Compared to the irradiation group, EGCG with different concentrations of 10, 20 mg/kg significantly reduced the methylation level of Rad23b and Ddit3 （ t =-13.39-7.99, P〈0.05）, and induced re-expression of mRNA （t= -34.02 - -2.89, P〈0.05）. This change was more notable in the irradiation group with the high dose of EGCG. Conclusions As a natural drug, EGCG may play an important role in affecting DNA methylation and hence protects DNA from radiation damage.