灵芝三萜对鹅膏毒肽所致肝损伤保护作用的分子机理研究

Poisonous mushrooms are the most major causation for the fatality due to food poisoning in China. It has been demonstrated that the amatoxins in Amanita species are responsible for the mortality. Ganoderma lucidum has been used as a remedy for clinical treatmen of Amanita poisoning in China in recent years and the results showed that the clinical efficacy was obvious, with significant decrease in mortality.Our previous studies indicated that G. lucidum had obvious protective effects against liver injury induced by amanitin in mice, and its main bioactive constituent is triterpenoid. the protective effects may be in part related to its antioxidant properties. The purpose of this study was to examine the molecular mechanism of Ganoderma triterpenoids protection against amanitin-induced liver injury in human hepatocytes cultures. Firstly,the effect of triterpenoid on RNA polymerase II activity and the synthesis of protein and mRNA in human hepatocytes treated with amanitin will be evaluated. Secondly,the effect of triterpenoids on amanitin-induced apoptosis and apoptosis markers(TNF-α、caspase-3、p53、Bcl-2 et al.) in human hepatocytes cultures will be determinated and then the molecular mechanism of amanitin-induced apoptosis will be demonstrated. Thirdly, the active monomeric Ganoderma triterpenoid will be screened according to the molecular index. The implement of this project will be contribute to further understand the molecular mechanism of Ganoderma as antidotes for the treatment of amanita poisoning.The implement of this progect will have grand value for the medicine development for treatment of mushroom poisoning.

蘑菇中毒是我国食物中毒中导致死亡的最主要因素，主要是由鹅膏菌中所含的鹅膏毒肽所致。近年来利用灵芝治疗鹅膏中毒在临床上取得了良好效果，我们的前期研究结果表明灵芝对α-鹅膏毒肽中毒小鼠所致肝损伤具有很好的保护作用，其主要作用成分为灵芝三萜，并从自由基清除和抗氧化活性方面阐述了部分机理。本申请项目拟在前期研究的基础上采用人肝细胞培养进一步开展灵芝三萜对鹅膏毒肽所致肝损伤保护作用的分子机制研究：（1）、探讨灵芝三萜对鹅膏毒肽处理后RNA聚合酶Ⅱ活性和mRNA与蛋白质合成的影响；（2）、探讨灵芝三萜对鹅膏毒肽所引起的细胞凋亡以及细胞凋亡关键因子（TNF-α、caspase-3、p53、Bcl-2等）的影响，阐述其作用途径;(3)、依据分子水平的指标筛选出具有活性的灵芝三萜单体化合物。该项目的实施有助于从分子水平上进一步明确灵芝治疗鹅膏中毒的机理，筛选出的活性灵芝三萜单体化合物可用于进一步的药物开发。

蘑菇中毒是我国食物中毒事件中导致死亡的最主要因素，在我国导致蘑菇中毒死亡的70%以上是由含鹅膏肽类毒素的鹅膏菌属中的剧毒种类所引起。鹅膏毒肽不仅能导致肝细胞坏死，也是一个强的细胞凋亡诱导剂，并且认为凋亡在鹅膏毒肽引起肝损害的发病机制中起着非常重要的作用。近年来利用灵芝治疗鹅膏中毒在临床上取得了良好效果，我们的前期研究结果表明灵芝水提液对α-鹅膏毒肽中毒小鼠所致肝损伤具有很好的保护作用，其主要活性成分为灵芝三萜。本申请项目在前期研究的基础上采用人肝细胞培养和小鼠实验从凋亡的角度进一步开展灵芝三萜对鹅膏毒肽所致肝损伤保护作用的分子机制研究。研究结果表明：1、利用α-鹅膏毒肽处理培养的人肝细胞，通过细胞凋亡形态、细胞周期、DNA损伤、凋亡相关酶学、细胞内活性氧变化等系列指标说明了鹅膏毒肽能导致人肝细胞的凋亡，证实了凋亡是鹅膏毒肽引起中毒、导致肝损伤的一个重要因素。2、通过α-鹅膏毒肽对人肝细胞线粒体膜电位、线粒体差异蛋白质组学、线粒体内的细胞色素C的释放的影响研究从机理上阐明了鹅膏毒肽是通过作用于细胞线粒体途径来引起凋亡的。3、评价了灵芝总三萜和5种灵芝三萜单体化合物的治疗效果，表明灵芝总三萜和灵芝三萜单体化合物对鹅膏毒肽所致肝损伤具有很好的保护作用，其中灵芝酸C2、灵芝酸B和灵芝酸F效果最好；4、利用DNA梯形法和凋亡关键因子分析了灵芝三萜单体化合物灵芝酸C2对鹅膏毒肽诱导肝细胞凋亡的抑制效果，表明灵芝酸C2能够抑制α-AMA引起的肝细胞凋亡。该项目所获得的研究结果有助于从分子水平上进一步明确灵芝治疗鹅膏中毒的机理，筛选出的活性灵芝三萜单体化合物对于进一步的药物开发具有重要科学价值。