大黄素-8-O-β-D-葡萄糖苷的体内外遗传毒性评价

Study on in vitro and in vivo Genotoxicity of Emodin-8-O-β-D-glucoside

目的:评价大黄素-8-O-β-D-葡萄糖苷(EG)的体内外遗传毒性,并比较体外细胞试验及大鼠体内实验评价结果的差异。方法:采用体外二维(2D)、三维(3D)细胞培养法分别构建2D、3D HepaRG细胞模型,造模成功后,分别将2D、3D HepaRG细胞分为空白对照组[0.5%二甲基亚砜(DMSO)]、丝裂霉素C组(阳性对照,0.1μg/mL)和EG低、中、高剂量组(10、50、200μg/mL),然后检测各组HepaRG细胞的微核形成率和尾DNA百分含量。将SD大鼠分为空白对照组(0.5%羧甲基纤维素钠)、甲磺酸乙酯组(阳性对照,200 mg/kg)和EG低、中、高剂量组(100、300、1 000 mg/kg),每组6只,连续灌胃给药15 d,每天1次;15 d后检测各组大鼠骨髓嗜多染红细胞、肝细胞的微核形成率及外周血淋巴细胞、肝细胞的尾DNA百分含量、尾距。结果:在体外2D HepaRG细胞模型中,与空白对照组比较,丝裂霉素C组HepaRG细胞的微核形成率和尾DNA百分含量均显著升高(P<0.01),EG各剂量组HepaRG细胞的微核形成率和尾DNA百分含量差异无统计学意义(P>0.05);在3D HepaRG细胞模型中,与空白对照组比较,丝裂霉素C组HepaRG细胞的微核形成率和尾DNA百分含量均显著升高(P<0.01或P<0.001),EG高剂量组HepaRG细胞的尾DNA百分含量显著升高(P<0.01)。在大鼠体内实验中,与空白对照组比较,甲磺酸乙酯组大鼠骨髓嗜多染红细胞、肝细胞的微核形成率和外周血淋巴细胞、肝细胞的尾DNA百分含量、尾距均显著升高(P<0.01),EG高剂量组大鼠外周血淋巴细胞尾DNA百分含量显著升高(P<0.01),EG各剂量组大鼠骨髓嗜多染红细胞、肝细胞的微核形成率和肝细胞尾DNA百分含量、尾距差异无统计学意义(P>0.05),但随剂量增加有升高趋势。结论:本研究结果提示在2D细胞模型中,EG未导致染色体断裂及DNA损伤,但3D细胞模型长期给药和体内重复给药结果均显示EG存在一定DNA损伤风险,故3D HepaRG细胞模型的评价结果更接近大鼠体内实验结果。

OBJECTIVE:To evaluate the in vitro and in vivo genotoxicity of emodin-8-O-β-D-glucoside(EG),and to compare the difference of in vitro cell test and in vivo test of rats. METHODS:2D and 3D hepatocyte models were established by in vitro two-dimensional(2D) and three-dimensional(3D) cell culture. After modeling,2D and 3D hepatocyte were divided into blank control group(0.5% DMSO),mitomycin C group(positive control,0.1 μg/m L),EG low-dose,medium-dose and high-dose groups(10,50,200 μg/m L),respectively. The micronucleus ratio and tail DNA% of Hepa RG cells were detected. SD rats were divided into blank control group(0.5% sodium carboxymethyl cellulose),ethyl methanesulfonate group(positive control,200 mg/kg),EG low-dose,medium-dose and high-dose groups(100,300,1 000 mg/kg),with 6 rats in each group. They were given medicine intragastrically for consecutive 15 d, once a day. 15 days later, the micronucleus formation rate of bone marrow polychromatic erythrocytes and hepatocytes,the tail DNA% and tail distance of peripheral blood lymphocytes and hepatocytes were measured. RESULTS:In the in vitro 2D Hepa RG hepatocyte model,compared with blank control group,the micronucleus formation rate and tail DNA% of Hepa RG cell were increased significantly in mitomycin C group(P<0.01). There was no statistical significance in micronucleus formation rate and tail DNA% of Hepa RG cell among EG groups(P>0.05). In 3D Hepa RG cell model, compared with blank control group,micronucleus formation rate and tail DNA% of Hepa RG cell were increased significantly in mitomycin C group(P<0.01 or P<0.001), while tail DNA% of Hepa RG cell was increased significantly in EG high-dose group(P<0.01). In the in vivo test,compared with blank control group,the micronucleus formation rate of bone marrow polychromatic erythrocytes and hepatocytes,the tail DNA% and tail distance of peripheral blood lymphocytes and hepatocytes were all increased significantly in ethyl methanesulfonate group(P<0.01). Tail DNA% of peripheral blood lymphocytes was increased significantly in EG high-dose group(P<0.01). There was no statistical significance in the micronucleus formation rate of bone marrow polychromatic erythrocytes and hepatocytes,the tail DNA% and tail distance of hepatocytes among EG groups(P>0.05);with the increase of dose,there was an increasing trend. CONCLUSIONS:The results of this study suggest that in 2D cell model, EG not lead to chromosome breakage and DNA damage, but the long-term administration and repeated administration in vivo of 3D cell model show that EG has a certain risk of DNA damage,so the evaluation results of 3D Hepa RG cell model are more similar to those of rats in vivo.