基于PK-PD与代谢组学方法研究何首乌肝毒性的毒效物质与致毒机理

Reports about hepatotoxicity induced by Polygonum multiflorum have been increasing in past decades, and it is connected with some chemical components and use method of Polygonum multiflorum . Polygonum multiflorum was recorded in “Ben-Cao-Zheng-Yi” with the properties of leading to liver and kidney, nourishing true Yin, which is inconsistent with the current reports of adverse drug reactions. Our studies suggested that the most important biomarkers of liver function varied to three or five fold than the normal value and liver damage occurred in HE dyed sections when rats were administrated orally by 15 g/kg of Polygonum multiflorum EtOAc extracts for ten days. These results indicated that the raw material of Polygonum multiflorum should contain some toxic components or toxic chemical groups. Therefore, we are going to systematically isolate the components from the toxic part of Polygonum multiflorum EtOAc extracts as well as screening cytotoxicity in L02 and HepG2 cell lines. Then, we will conduct some researches on the PK-PD model and metabolomics of toxic components or parts with determining several biomarkers of liver function such as ALT, AST, ALP, and SOD. Finally, we will illustrate the pharmacodynamic basis of hepatotoxicity and its related mechanism. All researches will provide theory support and a new assessment method to the hepatotoxicity for the rational clinical usage of Polygonum multiflorum and some Chinese herbal medicines containing the same components.

国内外有关何首乌肝脏不良反应的报道不断增加，显示其肝损伤与化学成分及使用方法有关。《本草正义》中记载“首乌，专入肝肾，补养真阴”。可见临床ADR报道与本草记载的益肝作用不符。前期研究结果表明：何首乌EtOAc部位剂量为0.5 g/kg时，给药7 d后大鼠ALT、AST和ALP出现3-5倍改变，HE染色切片显示明显肝细胞损伤。提示何首乌药材中含有致肝毒性的化合物或组合物。因此，拟采用半制备HPLC对此部位的化学成分进行快速分离，并利用L02和HepG2细胞进行毒性筛选；同时以ALT、AST、ALP为毒效指标，对毒性成分在大鼠体内PK-PD过程和代谢组学进行研究，从成分（组）-毒性评价（细胞）-毒性验证（动物）-毒性机制4个方面明确何首乌肝毒性的毒效物质和致毒机理。研究结果将为何首乌及多种具有相似化学成分的中药临床合理安全用药提供理论依据，为中药的安全评价提供新的方法，具有良好的临床应用价值。

何首乌为蓼科植物Polygonum multiflorum Thunb.的块根， 《本草纲目》记载 “能养血益肝，固精益肾，健筋骨，乌髭发，为滋补良药” 。其历来为古今医家、饮食家所推崇，成为滋补肝肾的佳品。但近年来，有关何首乌不良反应报道逐渐增多，主要集中在肝脏不良反应上，显示何首乌及其制剂致肝脏损伤与药物的化学成分及使用方法有关。本课题主要对何首乌毒效部位的化学成分进行系统分离，并利用L02和HepG2细胞进行毒性筛选；同时以ALT、AST、ALP的含量为毒效指标，对毒性组分在体内PK-PD过程和代谢组学进行研究，以期明确何首乌肝毒性的毒效物质和致毒机理。.首先给予大鼠何首乌95%乙醇提取物（HSW-Ex）28天，结果显示高、中剂量组的有明显肝损伤， ASP、ALT和AST水平显著增加， SOD活性显著降低。低剂量组大鼠没有明显肝损伤。代谢组学分析发现，有10个代谢物可能与HSW-Ex诱导的肝损伤有关联。.其次从HSW-Ex中分离出20个化合物，包括大黄素及其糖苷类化合物8个，白藜芦醇及其糖苷类化合物6个，二苯乙烯苷类化合物6个。对20个化合物进行细胞毒性筛选，发现对LO2有明显毒性的化合物RES和EMO。毒性评价显示RES 可以抑制LO2细胞增殖，IC50 为22.69±5.91 μM。Hoechst 33258、DAPI/MDC染色可发现细胞皱缩、染色质浓缩、细胞核碎片、细胞核溶解等明显细胞凋亡特征，电镜下可观察到凋亡小体形成。流式分析显示RES阻滞LO2细胞周期的S期抑制增殖。West blot analysis分析显示RES可以上调Bax蛋白和下调Bcl-2 蛋白，同时激活凋亡蛋白caspase-3和 caspase-9 在LO2细胞中的高表达。.综上所述，何首乌的肝毒性物质基础为RES 和EMO，其一定剂量下均可诱导肝细胞损伤。