目的 建立并初步测试肠-肝体外模型，模拟对乙酰氨基酚（APAP）口服后的吸收过程和肝脏毒性。方法 使用人结肠癌细胞系Caco-2和HT29-MTX-E12在Transwell中构建肠道模型，检测单位面积跨膜电阻（TEER）；苏木精-伊红（HE）染色和阿尔辛蓝（AB）-过碘酸希夫（PAS）染色观察肠道结构；免疫荧光检测紧密连接相关闭合蛋白（Occludin）和闭锁小带蛋白-1（ZO-1）、转运体多药耐药蛋白1（MDR1）和多药耐药相关蛋白2（MRP2）、黏蛋白2（MUC2），验证模型的完整性和转运蛋白表达。将肠道模型与人肝癌细胞系HepG2共同培养建立肠-肝模型，连续培养9 d，期间每天测定TEER；试剂盒法测定单肝模型（单独培养HepG2细胞）、肠道模型和肠-肝模型总腺嘌呤核苷三磷酸（ATP）和乳酸脱氢酶（LDH）、天冬氨酸氨基转移酶（AST）、丙氨酸氨基转移酶（ALT）、白蛋白（ALB）分泌水平。CCK-8法检测APAP对Caco-2和HepG2细胞的毒性。在肠-肝模型中模拟口服APAP产生的肝脏毒性，共培养的第2天将肠-肝模型分为3组：对照组给予不含药培养基；肠道吸收组从肠室给予12 mmol·L^-1 APAP，肝室给予空白培养基，药物平衡后预期终浓度为3 mmol·L^-1;APAP组分别从肠室和肝室给予3 mmol·L^-1 APAP，高效液相色谱法测定肝室药物浓度；在给药48 h后测定TEER并使用Cell Titer Glo试剂盒测定细胞活力；试剂盒法检测AST、ALT和ALB分泌水平；对模型肝室进行活性氧（ROS）和线粒体膜电位（MMP）染色。结果 建立的肠道模型在第14天时屏障结构完整，Occludin和ZO-1呈现网状结构分布，转运蛋白MDR1和MRP2分布于单侧；第21天仍含有分泌黏蛋白的HT29-MTX-E12细胞。肠-肝静态模型中，肠道细胞的数量稳定，肝脏细胞的数量不断增长，在共培养前5 d肝脏功能和细胞活力维持情况良好，适用于毒性测试。CCK-8结果显示，APAP对HepG2细胞的毒性强于Caco-2细胞，选取3 mmol·L^-1作为APAP测试的终浓度。APAP作用48 h后，肠道吸收组肠道细胞活力和TEER均无显著下降趋势，药物在肝室的浓度呈缓慢上升，在48 h时达到最大浓度（2.90±0.05）mmol·L^-1；与对照组比较，肠道吸收组和APAP组细胞存活率均显著下降（P<0.01）,APAP引起肝脏毒性标志物AST、ALT和ROS信号的升高，造成肝脏功能标志物ALB和MMP的降低；与APAP组相比，肠道吸收组细胞存活率略有升高，标志物改变程度略低。结论 构建的肠-肝体外模型能够实现药物肝毒性测试，肠道模型的存在通过影响药物暴露降低APAP诱导的肝脏毒性。

Objectives To establish a static gut-liver in vitro model to simulate the absorption process and hepatotoxic effects after administration of acteminophen（APAP）. Methods Human colon cancer cell lines Caco-2 and HT29-MTX-E12 were used to construct an intestinal model in Transwell, and the transepithelial electrical resistance（TEER） per unit area was measured. Hematoxylin-eosin（HE） staining and alcian blue（AB）-periodic acid-Schiff（PAS） staining were used to observe the intestinal structure. Immunofluorescence was used to detect tight junction proteins Occludin and zonula occludens-1（ZO-1）, transporters multidrug resistance protein 1（MDR1） and multidrug resistance-associated protein 2（MRP2）, and mucin 2（MUC2） to verify the integrity of the model and the expression of transport proteins. The intestinal model was co-cultured with human hepatoma cell line HepG2 to establish an intestinal-liver model, which was continuously cultured for 9 d. TEER was measured daily during the culture period. The levels of total adenosine triphosphate（ATP）, lactate dehydrogenase（LDH）, aspartate aminotransferase（AST）, alanine aminotransferase（ALT）, and albumin（ALB） secreted by the single liver model（cultured HepG2 cells alone）, intestinal model, and intestinal-liver model were determined by kits. The cytotoxicity of APAP to Caco-2 and HepG2 cells was detected by CCK-8 assay. The liver toxicity caused by oral APAP was simulated in the intestinal-liver model. On the second day of co-culture, the intestinal-liver model was divided into three groups: the control group was given drug-free medium; the intestinal absorption group was given 12 mmol·L^-1 APAP from the intestinal chamber and blank medium from the liver chamber, with an expected final concentration of 3 mmol·L^-1 in the liver chamber after drug equilibration; the APAP group was given 3 mmol·L^-1 APAP from both the intestinal and liver chambers. The drug concentration in the liver chamber was determined by high-performance liquid chromatography. TEER was measured and cell viability was determined using the Cell Titer Glo kit 48 h after drug administration. AST, ALT, and ALB secretion levels were detected by kits. Reactive oxygen species（ROS） and mitochondrial membrane potential（MMP） staining were performed on the liver chamber of the model. Results The established intestinal model had an intact barrier structure on the 14 th day, with Occludin and ZO-1 presenting a reticular distribution, and MDR1 and MRP2 distributed on one side. HT29-MTX-E12 cells secreting mucin were still present on the 21 st day. In the static intestinal-liver model, the number of intestinal cells was stable, and the number of liver cells continued to increase. The liver function and cell viability were maintained well in the first 5 days of co-culture, making it suitable for toxicity testing. The CCK-8 results showed that APAP was more toxic to HepG2 cells than Caco-2 cells, and 3 mmol·L^-1 was selected as the final concentration for APAP testing. After 48 h of APAP treatment, there was no significant decrease in cell viability and TEER in the intestinal absorption group, and the drug concentration in the liver chamber increased slowly, reaching the maximum concentration of（2.90 ±0.05） mmol·L^-1 at 48 h. Compared with the control group, the cell survival rates in the intestinal absorption group and the APAP group were significantly decreased（P ＜0.01）, and APAP caused an increase in liver toxicity markers AST, ALT, and ROS signals, and a decrease in liver function markers ALB and MMP. Compared with the APAP group, the cell survival rate in the intestinal absorption group was slightly increased, and the degree of marker changes was slightly lower. Conclusion The intestinal-liver in vitro model can be used for drug hepatotoxicity testing, and the presence of the intestinal model reduces the liver toxicity induced by APAP by affecting drug exposure.

R965

国家“十四五”重点专项(2022YFF0711100); 国家重点实验室项目(2023SKLDRS0129); 北京市科技计划项目(Z231100007223001)