目的 使用一种在微米尺度空间对流体进行操控，以模拟体内微环境为主要特征的微流控肝器官芯片技术，评价雷公藤提取物（Tripterygium wilfordii extract，TWE）和雷公藤多苷片提取物（Tripterygium Glycosides Tablet extract，TGE）的肝脏毒性，比较微流控-精密肝切片、静态-精密肝切片、微流控-HepaRG细胞培养体系的差异性。方法 将新鲜的大鼠肝脏进行精密切片处理后，分别置于静态/微流控培养体系中进行培养，微流控-HepaRG细胞培养体系则是将HepaRG细胞接种到已预先接种人脐静脉内皮细胞（human umbilical vein endothelial cells，HUVECs）的培养小室中加药培养；TWE终质量浓度为0.30、0.60、1.20 mg/mL（以生药量计），TGE终质量浓度为31.25、62.50、125.00 μg/mL，共孵育24 h后进行肝脏损伤标志物的测定及形态学观察。结果 在不额外添加药物的正常培养情况下，微流控/静态精密肝切片培养上清中丙氨酸氨基转移酶（alanine aminotransferase，ALT）、乳酸脱氢酶（lactate dehydrogenase，LDH）、总胆汁酸（total biliary acid，TBA）的表达均明显高于微流控-HepaRG细胞培养体系。与TWE和TGE共培养后，微流控-精密肝切片培养体系中，高剂量的TWE和TGE均可导致培养上清中ALT、LDH和谷氨酰转移酶（gamma glutamyl transferase，GGT）活性的显著升高，TWE还可以显著升高总胆红素（total bilirubin，TBil）的含量，并显示出一定的量-毒关系；而在静态-精密肝切片培养体系中，仅中剂量的TGE可导致ALT和LDH活性的显著升高，且未见剂量相关性；在微流控-HepaRG细胞培养体系中，仅GGT的含量显著升高。组织病理学检查结果显示，TWE和TGE低、中、高剂量对共培养体系中的HUVECs形态均未见明显影响，说明受试药不影响共培养体系的血管仿生结构；TWE和TGE在微流控培养体系或静态培养体系下，低、中、高剂量均会对精密肝切片造成不同程度的损伤，肝细胞出现不同程度肿胀，弥漫性肝细胞核溶解、消失，肝细胞索结构松散，但仅在微流控-精密肝切片培养体系下，形态学的改变呈现出一定的剂量相关性；在微流控-HepaRG细胞培养体系中，仅高剂量的TWE和TGE可导致细胞形态的明显改变。结论 在微流控-精密肝切片培养体系下，TWE和TGE所造成的肝损伤特点一致，具有明显的量-毒关系，表明微流控肝器官芯片技术可以更灵敏、更准确地反映出受试物对肝脏的损伤情况，且有可能成为临床前中药肝毒性评价的新的技术手段。

Objective To manipulate fluids in micron-scale space to simulate the in vivo micro-environment as the main feature by using a microfluidic liver-on-a-chip technology, so as to evaluate the hepatotoxicity of Tripterygium wilfordii extract (TWE) and Tripterygium Glycosides Tablet (雷公藤多苷片) extract (TGE), and compare the difference between the microfluidic precision-cut liver slices model and the microfluidic HepaRG cell model and the static precision-cut liver slices model. Methods Fresh rat livers were subjected to precision-cut liver slice and cultured in a static/microfluidic culture model, HepaRG cells were inoculated into a culture chamber pre-inoculated with human umbilical vein endothelial cells (HUVECs) and then incubated with drugs. TWE final concentrations were 0.30, 0.60, 1.20 mg/mL (calculated by the amount of crude drug), and TGE final concentrations were 31.25, 62.50, 125.00 μg/mL, respectively. After 24 h, the determination of liver injury markers and morphological observation were done. Results Under normal culture conditions, the expression levels of alanine aminotransferase (ALT), lactate dehydrogenase (LDH), and total biliary acid (TBA) in the supernatant of precision-cut liver slice culture were significantly higher than single HepaRG cell culture model when using two culture vectors. After co-culture with TWE and TGE, in the microfluidic precision-cut liver slice model, high-dose TWE and TGE could significantly increase the activities of ALT, LDH and gamma glutamyl transferase (GGT) in the culture supernatant, while TWE could also significantly increase the content of total bilirubin (TBil), and showed a certain dose-toxicity relationship; Only medium-dose TGE could significantly increase the levels of ALT and LDH in the static precision-cut liver slice model, with no dose-dependent effect; In the microfluidic HepaRG cell model, only the GGT content increased significantly. Histopathological examination showed that low-, medium-and high-dose of TWE and TGE had no significant effect on the morphology of HUVECs in the co-culture model, indicating that the bionic structure of blood vessels in the co-culture model was not affected by the experimental drugs; The low-, medium-and high-dose of TWE and TGE made different degrees of damage to precision-cut liver slices in two microfluidics culture model and static culture model. The liver cells appear different degrees of swelling, diffuse dissolution and disappearance of liver nuclei, and loose structure of liver cells. However, only in microfluidic precision-cut liver slice model, morphological changes showed a certain dose correlation; In the HepaRG cell culture model, only high-dose of TWE and TGE made significant changes in cell morphology. Conclusion In the microfluidic precision-cut liver slice culture model, the liver injury characteristics caused by TWE and TGE are consistent, with a clear dose-toxicity relationship. The microfluidic liver-on-a-chip technology can more sensitively and accurately reflect the damage of the test substance to the liver, which can become a new technology for preclinical liver toxicity evaluation of traditional Chinese medicine.

R285.5

北京市自然科学基金资助项目（7232301）；中国中医科学院科技创新工程项目（CI2021A04804）；中央级公益性科研院所基本科研业务费专项资金资助（ZZ14-YQ-025，ZXKT22018，ZXKT21009）