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药学学报, 2021, 56(3): 808-815 |
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引用本文： |
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潘韵铮, 李庆菊, 张琦, 蒋宝平, 张良, 许立. 基于复合细胞模型的顺式和反式二苯乙烯苷特异质肝损伤评价[J]. 药学学报, 2021, 56(3): 808-815. |
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PAN Yun-zheng, LI Qing-ju, ZHANG Qi, JIANG Bao-ping, ZHANG Liang, XU Li. The compound cell model-based evaluation for idiosyncratic liver injury of Cis-SG and Trans-SG[J]. Acta Pharmaceutica Sinica, 2021, 56(3): 808-815. |
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| 基于复合细胞模型的顺式和反式二苯乙烯苷特异质肝损伤评价 |
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| 潘韵铮, 李庆菊, 张琦, 蒋宝平, 张良, 许立 |
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| 南京中医药大学药学院, 江苏省中药药效与安全性评价重点实验室, 江苏 南京 210023 |
| 摘要: |
| 本研究从免疫炎症角度建立一种体外评价特异质型药物性肝损伤（idiosyncratic drug-induced liver injury，IDILI）的复合细胞模型，并应用此模型对顺式二苯乙烯苷（2，3，5，4'-tetrahydroxy-cis-stilbene-2-O-β-glucoside，Cis-SG）和反式二苯乙烯苷（2，3，5，4'-tetrahydroxy-trans-stilbene-2-O-β-glucoside，Trans-SG）的IDILI风险进行评价。CellTiter-Glo® 3D Cell Viability Assay法测定Cis-SG和Trans-SG对三维（three-dimension，3D）培养下HepG2细胞活力的影响；MTT法测定Cis-SG和Trans-SG对THP-1巨噬细胞活力的影响，确定低、中、高给药剂量。分别给予THP-1巨噬细胞1、5和25 μmol·L-1的Cis-SG和Trans-SG或其经3D HepG2细胞孵育的上清液，酶联免疫吸附分析（enzyme linked immunosorbent assay，ELISA）法检测THP-1巨噬细胞上清液中白细胞介素（interleukin，IL）-1β的水平；免疫印迹（Western blot）法和逆转录聚合酶链反应（reverse transcription-polymerase chain reaction，RT-PCR）分别检测THP-1巨噬细胞中凋亡相关斑点样蛋白（apoptosis-associated speck-like protein，ASC）、Nod样受体蛋白3（Nod-like receptor protein 3，NLRP3）炎症小体、天冬氨酸特异性半胱氨酸蛋白酶-1（cysteinyl aspartate specific proteinase-1，caspase-1）和IL-1β的表达。结果显示，1、5和25 μmol·L-1的Cis-SG和Trans-SG对THP-1巨噬细胞分泌IL-1β的水平无明显影响，而1、5和25 μmol·L-1的Cis-SG和25 μmol·L-1的Trans-SG经肝细胞孵育后的上清液能显著提高THP-1巨噬细胞分泌IL-1β的水平，并能明显提高ASC、NLRP3、caspase-1和IL-1β蛋白和mRNA的表达。综上，本研究建立的体外IDILI复合细胞评价模型在测试Cis-SG和Trans-SG上成功应用，此模型有助于在体外初步评价和筛选具有IDILI风险的药物，为药物的特异质肝毒性预测与解决提供方法。 |
| 关键词:
特异质型药物性肝损伤
何首乌
二苯乙烯苷
NLRP3炎症小体
评价模型
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| The compound cell model-based evaluation for idiosyncratic liver injury of Cis-SG and Trans-SG |
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| PAN Yun-zheng, LI Qing-ju, ZHANG Qi, JIANG Bao-ping, ZHANG Liang, XU Li |
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| Jiangsu Key Laboratory for Pharmacology and Safety Evaluation of Chinese Materia Medica, School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210023, China |
| Abstract: |
| In this study, a composite cell model for evaluation of idiosyncratic drug-induced liver injury (IDILI) was established in vitro from the perspective of immune inflammation. And this model was used to evaluate the risk of IDILI for 2,3,5,4'-tetrahydroxy-cis-stilbene-2-O-β-glucoside (Cis-SG) and 2,3,5,4'-tetrahydroxy-trans-stilbene-2-O-β-glucoside (Trans-SG). To determine the low, medium, and high dosage of Cis-SG and Trans-SG, CellTiter-Glo® 3D Cell Viability Assay was used to detect the effects of Cis-SG and Trans-SG on cell viability of HepG2 cells in three dimensional (3D) culture, and MTT assay was used to detect the effects of Cis-SG and Trans-SG on cell viability of THP-1 derived macrophages. THP-1 derived macrophages were incubated by Cis-SG and Trans-SG directly or supernatants from HepG2 cells incubated with them. Enzyme linked immunosorbent assay (ELISA) was used to detect the levels of interleukin-1β (IL-1β) in the supernatants of the THP-1 derived macrophages. Western blot and reverse transcription-polymerase chain reaction (RT-PCR) were used to determine the expression of apoptosis-associated speck-like protein (ASC), Nod-like receptor protein 3 (NLRP3), cysteinyl aspartate specific proteinase-1 (caspase-1), and IL-1β in THP-1 derived macrophages. The results showed that there was no effect on the secretion of IL-1β in THP-1 derived macrophages incubated by Cis-SG and Trans-SG directly. However, the secretion of IL-1β, the protein and mRNA expression of ASC, NLRP3, caspase-1, and IL-1β significantly increased in THP-1 derived macrophages incubated by supernatants from HepG2 cells incubated with 1, 5, and 25 μmol·L-1 Cis-SG or 25 μmol·L-1 Trans-SG. In summary, the composite cell model for evaluation of IDILI established in vitro has been successfully applied in testing Cis-SG and Trans-SG. This composite cell model is helpful to evaluate and screen drugs with IDILI risk in vitro preliminarily, which provides methods for predicting and solving the idiosyncratic liver toxicity of drugs. |
| Key words:
idiosyncratic drug-induced liver injury
Polygonum multiflorum Thunb.
stibene glucoside
NLRP3 inflammasome
evaluation model
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| 收稿日期: 2020-11-17
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| DOI: 10.16438/j.0513-4870.2020-1741 |
| 基金项目: 江苏省中药药效与安全性评价重点实验室资助项目（JKLPSE201810）；江苏省高校中药学优势学科建设工程资助项目（PAPD）. |
通讯作者: 许立,Tel:13851572203,E-mail:xuli64@163.com
Email: xuli64@163.com |
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| 潘韵铮 在本刊中的所有文章 |
| 李庆菊 在本刊中的所有文章 |
| 张琦 在本刊中的所有文章 |
| 蒋宝平 在本刊中的所有文章 |
| 张良 在本刊中的所有文章 |
| 许立 在本刊中的所有文章 |
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