基于类器官3D培养的何首乌易感物质肝毒性评价

Three dimensional organoids-based evaluation for hepatotoxicity of the susceptible compound in Polygonum multiflorum Thunb.

采用液滴重叠法构建类器官3D培养模型,评价何首乌易感物质顺式二苯乙烯苷(cis-SG)的肝损伤作用。结果表明,相对于普通2D培养肝细胞模型,所构建的类器官3D培养模型的L02细胞和HepG2细胞白蛋白表达分别提高2.5和6.7倍;在第21天时,尿素生成水平分别提高8.3和15.5倍,且HepG2细胞构建的类器官模型显著优于L02细胞;相对于2D培养肝细胞模型,HepG2细胞构建的类器官模型的药物Ⅰ相和Ⅱ相代谢酶表达量显著上调,如CYP2C9、CYP3A4和CYP2D6表达上调分别为381.9、87.0和312.6倍,药物转运体相关基因也明显上调,提示所建立的类器官3D培养模型的肝脏合成和代谢功能显著优于普通2D培养肝细胞模型。肝毒性评价结果表明,相对于普通2D培养肝细胞模型,类器官3D培养模型可以更灵敏地评价对乙酰氨基酚等肝毒性阳性药物的毒性差异,且在类器官3D培养模型上重复给药评价的半数抑制浓度(IC_(50))值显著低于单次给药方式;何首乌易感物质cis-SG在普通2D培养肝细胞模型未检测到IC_(50),在类器官3D培养模型上单次给药方式的IC_(50)是肝毒性阳性药环孢霉素的1.9倍,而其光学异构体反式二苯乙烯苷(trans-SG)的IC_(50)比cis-SG高4.1倍,与前期整体动物水平的毒性强弱基本一致;cis-SG在类器官3D培养模型上多次给药方式的IC_(50)进一步降低,提示长期用药可能增大何首乌肝损伤风险。综上,何首乌易感物质cis-SG对类器官的肝毒性大于trans-SG;类器官3D培养模型可长期培养且保留更多的肝脏合成和代谢功能,适用于中药成分低浓度、长时间暴露产生肝毒性评价和机制研究。

In this study, the three dimensional（3D） organoid culture system was established by liquid overlay method, and applied as an effective model to evaluate the hepatic injury of susceptible compounds in Polygonum multiflorum Thunb. Compared with the ordinary two dimensional（2D） culture of liver cells, the albumin expression of L02 cells and HepG2 cells were increased by 2.5 and 6.7 times in the 3D organoid culture system, respectively. After the cultivation of 21 days, urea generation levels of 3D culture were increased by 8.3 and 15.5 times. More importantly, HepG2 cells were more suitable to development of organoids than L02 cells. The gene expressions of phase Ⅰ and Ⅱ drug metabolism enzymes of HepG2 cells cultured as 3D organoids were significantly increased than that in 2D culture, such as the fold changes of CYP2C9 was up to 381.9, CYP3A4 to 87.0, CYP2D6 to 312.6. In addition, drug transporter relative genes were also up-regulated. The results demonstrated that the liver synthesis and metabolic function of the 3D model were better than that of the 2D cultured hepatocytes. The results of hepatotoxicity evaluation showed this developed model can be used to assess the hepatotoxicity of acetaminophen and other positive control drugs, which were considered with defined hepatotoxicity. On the 3D culture model, the IC_（50） value of repeated drug dose administration was significantly lower than that of single dose administration. However, the IC_（50） of 2,3,5,4＇-tetrahydroxy-cis-stilbene-2-O-β-glucoside（cis-SG）, which is the susceptible compound in Polygonum multiflorum Thunb., could not be detected in 2D cultured model. With the treatment of a single dose administration in organ 3D culture model, the IC_（50） of cis-SG was 1.9 times than that of cyclosporine A, and the IC_（50） of 2,3,5,4＇-tetrahydroxy-trans-stilbene-2-O-β-glucoside（trans-SG） was 4.1 times than cis-SG. The hepatotoxicity results of cis-SG and trans-SG on the 3D cultures were similar to in vivo toxicity results obtained in previous work. On organ 3D culture model, the IC_（50） of cis-SG with repeat of administration decreased compared with that with single dose administration, suggesting that long-term medication may increase the risk of liver injury. In summary, the 3D organoid culture system can be used for a long period to preserve the capacity of liver synthesis and metabolism. The organoids were a model suitable for evaluation of mechanism of the drugs with low toxicity.