薯蓣皂苷致肝毒性及其机制的初步研究(1)
[摘要]薯蓣皂苷有广泛的生物学作用，具有广阔的应用前景，但是目前有关薯蓣皂苷毒理及其机制方面研究较少。该文从细胞水平上检测薯蓣皂苷(dioscin)对肝脏的毒性，同时对薯蓣皂苷作用于HepG2细胞后CYP1A在mRNA、蛋白水平的变化进行了研究，对CYP1A转录调控子芳香烃受体(AhR)的表达也进行了检测，旨在探索薯蓣皂苷的肝毒性及其可能的机制。将0.5～32 μmol·L-1薯蓣皂苷作用于HepG2细胞12 h，利用CCK-8法检测细胞活力，乳酸脱氢酶(LDH)释放实验检测细胞膜损伤，采用倒置显微镜观察细胞形态变化，流式细胞仪检测活性氧(ROS)的生成量;实时荧光定量PCR检测CYP1A及其转录调控子AhR mRNA的表达;Western blot 法检测CYP1A1的蛋白表达。结果显示薯蓣皂苷0.5～32 μmol·L-1作用于HepG2细胞可以显著抑制细胞活性，与对照组相比，LDH释放量显著升高，ROS的生成量显著增大，细胞形态发生变化并坏死。Q-PCR检测结果显示，与对照组相比，CYP1A及芳香烃受体(AhR)mRNA表达升高;在蛋白水平上，薯蓣皂苷作用于细胞后CYP1A1蛋白表达升高，且AhR的拮抗剂白藜芦醇(Res)与薯蓣皂苷合用后能使CYP1A1的表达下调。以上实验结果表明大剂量薯蓣皂苷具有肝细胞毒作用，这可能与其能激活芳香烃受体(AhR)，诱导CYP1A表达进而产生毒性作用有关。
[关键词]薯蓣皂苷;HepG2细胞;毒性作用;CYP1A
Preliminary study on hepatotoxicity induced by dioscin and its possible mechanism
ZHANG Ya-xin， WANG Yu-guang*， MA Zeng-chun， TANG Xiang-lin， LIANG Qian-de， TAN Hong-ling，XIAO Cheng-rong， ZHAO Yong-hong， GAO Yue*
(Institute of Radiation Medicine， Academy of Military Medical Sciences， Beijing 100850， China)
[Abstract]Dioscin has a wide range of biological effects and broad application prospects. However the studies concerning the toxicology and mechanism of dioscin is small. This article is to study the hepatotoxicity of dioscin and the effect of dioscin treatment on expression of aryl hydrocarbon receptor (AhR) mRNA and CYP1A mRNA and protein in HepG2 cells in vitro. Dioscin 0.5-32 μmol·L-1 exposed to HepG2 cells for 12 h，cell viability was examined by CCK-8 assay and the release rate of lactate dehydrogenase (LDH) was to evaluate cell membrane damage. HepG2 cells morphologic changes were quantified by inverted Microscope，and the effect on production of reactive oxygen species(ROS) was detected by flow cytometry. The mRNA expression of CYP1A and AhR was evaluated by RT-RCR. The protein expression of CYP1A1 was detected by western blot. The cell viability was significantly inhibited after HepG2 cells were exposed to dioscin 0.5-32 μmol·L-1. Compared with the control，the LDH release rate and ROS were significantly increased. The expression of CYP1A and AhR mRNA was increased. The expression of CYP1A1 protein was increased after dioscin treatment，and resveratrol，an AhR antagonist，could downregulate the expression of CYP1A1. It follows that large doses dioscin has potential hepatotoxicity. The possible mechanism may be dioscin can active aryl hydrocarbon receptor (AhR) and induce the expression of CYP1A.
[Key words]dioscin; HepG2 cells; toxic actions; CYP1A
doi：10.4268/cjcmm20151413 (张亚欣 王宇光 马增春 汤响林 梁乾德 谭洪玲 肖成荣 赵永红 高月)
[关键词]薯蓣皂苷;HepG2细胞;毒性作用;CYP1A
Preliminary study on hepatotoxicity induced by dioscin and its possible mechanism
ZHANG Ya-xin， WANG Yu-guang*， MA Zeng-chun， TANG Xiang-lin， LIANG Qian-de， TAN Hong-ling，XIAO Cheng-rong， ZHAO Yong-hong， GAO Yue*
(Institute of Radiation Medicine， Academy of Military Medical Sciences， Beijing 100850， China)
[Abstract]Dioscin has a wide range of biological effects and broad application prospects. However the studies concerning the toxicology and mechanism of dioscin is small. This article is to study the hepatotoxicity of dioscin and the effect of dioscin treatment on expression of aryl hydrocarbon receptor (AhR) mRNA and CYP1A mRNA and protein in HepG2 cells in vitro. Dioscin 0.5-32 μmol·L-1 exposed to HepG2 cells for 12 h，cell viability was examined by CCK-8 assay and the release rate of lactate dehydrogenase (LDH) was to evaluate cell membrane damage. HepG2 cells morphologic changes were quantified by inverted Microscope，and the effect on production of reactive oxygen species(ROS) was detected by flow cytometry. The mRNA expression of CYP1A and AhR was evaluated by RT-RCR. The protein expression of CYP1A1 was detected by western blot. The cell viability was significantly inhibited after HepG2 cells were exposed to dioscin 0.5-32 μmol·L-1. Compared with the control，the LDH release rate and ROS were significantly increased. The expression of CYP1A and AhR mRNA was increased. The expression of CYP1A1 protein was increased after dioscin treatment，and resveratrol，an AhR antagonist，could downregulate the expression of CYP1A1. It follows that large doses dioscin has potential hepatotoxicity. The possible mechanism may be dioscin can active aryl hydrocarbon receptor (AhR) and induce the expression of CYP1A.
[Key words]dioscin; HepG2 cells; toxic actions; CYP1A
doi：10.4268/cjcmm20151413 (张亚欣 王宇光 马增春 汤响林 梁乾德 谭洪玲 肖成荣 赵永红 高月)