药学学报, 2021, 56(3): 808-815
引用本文:
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.
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.

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
潘韵铮, 李庆菊, 张琦, 蒋宝平, 张良, 许立
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     
The compound cell model-based evaluation for idiosyncratic liver injury of Cis-SG and Trans-SG
PAN Yun-zheng, LI Qing-ju, ZHANG Qi, JIANG Bao-ping, ZHANG Liang, XU Li
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   
收稿日期: 2020-11-17
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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参考文献:
[1] Fontana RJ. Pathogenesis of idiosyncratic drug-induced liver injury and clinical perspectives[J]. Gastroenterology, 2014, 146:914-928.
[2] Cho T, Uetrecht J. How reactive metabolites induce an immune response that sometimes leads to an idiosyncratic drug reaction[J]. Chem Res Toxicol, 2016, 30:295-314.
[3] Matzinger P. Tolerance, danger, and the extended family[J]. Immunology, 1994, 12:991-1045.
[4] Groslambert M, Py BF. Spotlight on the NLRP3 inflammasome pathway[J]. J Inflamm Res, 2018, 11:359-374.
[5] Wang Z, Xu G, Zhan X, et al. Carbamazepine promotes specific stimuli-induced NLRP3 inflammasome activation and causes idiosyncratic liver injury in mice[J]. Arch Toxicol, 2019, 93:3585-3599.
[6] Wang Z, Xu G, Wang H, et al. Icariside Ⅱ, a main compound in Epimedii Folium, induces idiosyncratic hepatotoxicity by enhancing NLRP3 inflammasome activation[J]. Acta Pharm Sin B, 2020, 10:1619-1633.
[7] China Pharmacopoeia Committee. Chinese Pharmacopoeia (中国药典)[M]. Beijing:China Medical Science Press, 2010:164.
[8] Ma NH, Chen Y, Xu CSH, et al. Research progress of liver injury induced by Polygoni Mulitiflori Radix[J]. China J Chin Mater Med (中国中药杂志), 2020, 45:3594-3602.
[9] Li C, Niu M, Bai Z, et al. Screening for main components associated with the idiosyncratic hepatotoxicity of a tonic herb, Polygonum multiflorum[J]. Front Med, 2017, 11:253-265.
[10] Meng YK, Li CY, Li RY, et al. Cis-Stilbene glucoside in Polygonum multiflorum induces immunological idiosyncratic hepatotoxicity in LPS-treated rats by suppressing PPAR-gamma[J]. Acta Pharmacol Sin, 2017, 38:1340-1352.
[11] He L, Yin P, Meng Y, et al. Immunological synergistic mechanisms of trans-/cis-stilbene glycosides in Heshouwu-related idiosyncratic liver injury[J]. Sci Bull, 2017, 62:748-751.
[12] Li TT, Li RH, Liu ZX, et al. Three dimensional organoids-based evaluation for hepatotoxicity of the susceptible compound in Polygonum multiflorum Thunb[J]. Acta Pharm Sin (药学学报), 2017, 52:1048-1054.
[13] Yamashita YI, Imai K, Mima K, et al. Idiosyncratic drug-induced liver injury:a short review[J]. Hepatol Commun, 2017, 1:494-500.
[14] Su Y, Zhang Y, Chen M, et al. Lipopolysaccharide exposure augments isoniazide-induced liver injury[J]. J Appl Toxicol, 2014, 34:1436-1442.
[15] Metushi IG, Hayes MA, Uetrecht J. Treatment of PD-1(-/-) mice with amodiaquine and anti-CTLA4 leads to liver injury similar to idiosyncratic liver injury in patients[J]. Hepatology, 2015, 61:1332-1342.
[16] Yokoi T, Oda S. Models of idiosyncratic drug-induced liver injury[J]. Annu Rev Pharmacol Toxicol, 2021, 61:20. 1-20. 22.
[17] Uetrecht J. Mechanisms of idiosyncratic drug-induced liver injury[J]. Adv Pharmacol, 2019, 85:133-163.
[18] Bettigole SE, Glimcher LH. Endoplasmic reticulum stress in immunity[J]. Annu Rev Immunol, 2015, 33:107-138.
[19] He Y, Hara H, Nunez G. Mechanism and regulation of NLRP3 inflammasome activation[J]. Trends Biochem Sci, 2016, 41:1012-1021.
[20] Su Q, Li L, Sun Y, et al. Effects of the TLR4/Myd88/NF-kappaB signaling pathway on NLRP3 inflammasome in coronary microembolization-induced myocardial injury[J]. Cell Physiol Biochem, 2018, 47:1497-1508.
[21] Qiao Y, Wang P, Qi J, et al. TLR-induced NF-kappaB activation regulates NLRP3 expression in murine macrophages[J]. FEBS Lett, 2012, 586:1022-1026.
[22] Wang W, Hu D, Feng Y, et al. Paxillin mediates ATP-induced activation of P2X7 receptor and NLRP3 inflammasome[J]. BMC Biol, 2020, 18:182-188.
[23] Keyel PA, Roth R, Yokoyama WM, et al. Reduction of streptolysin O (SLO) pore-forming activity enhances inflammasome activation[J]. Toxins (Basel), 2013, 5:1105-1118.
[24] Dostert C, Guarda G, Romero JF, et al. Malarial hemozoin is a Nalp3 inflammasome activating danger signal[J]. PLoS One, 2009, 4:e6510.
[25] Wree A, Eguchi A, McGeough MD, et al. NLRP3 inflammasome activation results in hepatocyte pyroptosis, liver inflammation, and fibrosis in mice[J]. Hepatology, 2014, 59:898-910.
[26] Mridha AR, Wree A, Robertson AAB, et al. NLRP3 inflammasome blockade reduces liver inflammation and fibrosis in experimental NASH in mice[J]. J Hepatol, 2017, 66:1037-1046.
[27] Kato R, Uetrecht J. Supernatant from hepatocyte cultures with drugs that cause idiosyncratic liver injury activates macrophage inflammasomes[J]. Chem Res Toxicol, 2017, 30:1327-1332.
[28] Mak A, Kato R, Weston K, et al. Editor's highlight:an impaired immune tolerance animal model distinguishes the potential of troglitazone/pioglitazone and tolcapone/entacapone to cause IDILI[J]. Toxicol Sci, 2018, 161:412-420.
[29] Tu C, Ge FL, Guo YM, et al. Analysis of clinical characteristics and medication rationality of Polygonum multiflorum Thunb. and its preparation-related liver injury[J]. Chin J Pharmacovig (中国药物警戒), 2013, 10:219-222.
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