报告抗原腘窝淋巴结试验评价注射用双黄连的致敏性

中国药物警戒 ›› 2017, Vol. 14 ›› Issue (1): 4-6.

报告抗原腘窝淋巴结试验评价注射用双黄连的致敏性

刘兆华¹, 姚景春², 孙蓉³, 郭岩乳¹, 曾繁光¹

1山东大学药学院,山东济南 250012；
2.鲁南制药集团股份有限公司,山东临沂 273400；
3.山东省中医研究院,山东济南 250014

收稿日期:2017-02-16 修回日期:2017-02-16 出版日期:2017-01-20 发布日期:2017-02-16
作者简介:刘兆华,男,博士,高级工程师,药物免疫毒性和毒性病理学。
基金资助:
山东省自然科学基金（ZR2012HM056）; 山东省自主创新及成果转化专项（2014ZZCX02104）; 山东省泰山学者工程专项经费(Ns201511107)

Assessment of Immunosensitizing Potential of Shuanghuanglian for Injection Using Reporter Antigen Popliteal Lymph Node Assay

LIU Zhao-hua¹, YAO Jing-chun², SUN Rong³, GUO Yan-ru¹, ZENG Fan-guang¹

1School of Pharmaceutical Sciences of Shandong University, Shandong Jinan 250012, China;
2 Lunan Pharmaceutical Group Co. Ltd, Shandong Linyi 273400, China;
3 Shandong Academy of Chinese Medicine, Shandong Jinan 250014, China

Received:2017-02-16 Revised:2017-02-16 Online:2017-01-20 Published:2017-02-16

摘要/Abstract

摘要： 目的利用报告抗原腘窝淋巴结试验（reporter antigen popliteal lymph node assay,RA-PLNA）评价注射用双黄连（Shuanghuanglian for injection,SHLI）的致敏性,并探讨其发生机制。方法SPF级7~8周雌性BALB/c小鼠,按体重随机分为生理盐水空白对照组（Veh）、D-盐酸青霉胺阳性对照组（D-pen,1 mg/只）、SHLI高剂量组（H,5 mg/只）和低剂量组（L,1 mg/只）,共4组,6只/组。将受试物分别与TNP-OVA（10 μg/只）混合,右侧足趾部皮下注射给予小鼠,终体积50 μL/只。药后7天处死动物,摘取两侧腘窝淋巴结（popliteal lymph node,PLN）,称重,计算重量指数（weight index,WI）;制备单细胞悬液,计算细胞指数（cells index,CI）,比较各组的RA-PLNA 反应。ELISPOT检测PLN中的TNP特异性抗体分泌细胞（AFC）的数量和类型,流式细胞术检测PLN中淋巴细胞和巨噬细胞数量。与TNP-Ficoll混合时,另取小鼠,方法同上。结果分别与两种报告抗原一起注射后,高剂量的SHLI诱导明显的RA-PLNA阳性反应（组平均WI≥2或CI≥5）;ELISPOT检测发现,仅高剂量SHLI和TNP-OVA一起注射明显增加TNP特异性AFC的数量,且分泌各类抗体的AFC均明显升高（P <0.05或P <0.01）;流式细胞术检测发现高剂量SHLI与TNP-OVA一起可升高PLN中巨噬细胞的数量（P <0.05）。结论在BALB/c小鼠体内SHLI不会形成新的抗原表位,也不会活化特异性T细胞,提示SHLI本身可能不具有致敏潜能。

关键词: 报告抗原腘窝淋巴结试验, 注射用双黄连, 超敏反应

Abstract: ObjectiveTo explore mechanism of adverse drug reactions induced by Shuanghuanglian for injection (SHLI) and further evaluate the immunosensitizing potential of SHLI using reporter antigen popliteal lymph node assay (RA-PLNA) in BALB/c mice. MethodsSPF female BALB/c mice (7~8 weeks old at the onset of the experiment), were randomly assigned into control (Veh), 1 mg D-Penicillamine hydrochloride (D-pen), 1 mg (L) and 5 mg (H) SHLI group. There are 4 groups and each group has 6 animals. Articles, respectively mixed with 10 μg Trinitropheny-Ovabumin (TNP-OVA), were injected subcutaneously into right hind foot pad of mice in 50 μL volume. On day 7 after injection, animals were sacrificed, the untreated and treated lateral popliteal lymph node (PLN) were isolated, weighed and prepared into single-cell suspensions respectively, and then PLN weight index (WI) and cells index (CI) were calculated. RA-PLNA responses were observed. Trinitrophenyl-specific antibody forming cell (AFC) and the number of lymphocyte and macrophages in PLN were determined by Enzyme-linked immunospot assay (Elispot) and flow cytometry, respectively. When SHLI mixed with TNP-Ficoll using some another mice, the method is the same as TNP-OVA. Results5 mg SHLI mixed with TNP-OVA or TNP-Ficoll induced positive reaction in RA-PLNA. However, only when 5 mg SHLI co-injected with TNP-OVA, TNP-specific AFC were observed in Elispot assay. Simultaneously, the number of macrophages in PLN increased in flow cytometry. Moreover, whether 5 mg SHLI with TNP-Ficoll or 1 mg SHLI mixed with TNP-OVA or TNP-Ficoll failed to induce TNP-specific reaction. ConclusionSHLI lacked the intrinsic capacity to sensitize or stimulate immune responses in BALB/c mice. This may imply that ADR induced by SHLI in clinic was mainly a non-IgE-mediated anaphylactoid reaction rather than IgE-mediated hypersensitivity reactions.

Key words: reporter antigen popliteal lymph node assay, Shuanghuanglian for injection, hypersensitivity reactions.

中图分类号:

R994

刘兆华, 姚景春, 孙蓉, 郭岩乳, 曾繁光. 报告抗原腘窝淋巴结试验评价注射用双黄连的致敏性[J]. 中国药物警戒, 2017, 14(1): 4-6.

LIU Zhao-hua, YAO Jing-chun, SUN Rong, GUO Yan-ru, ZENG Fan-guang. Assessment of Immunosensitizing Potential of Shuanghuanglian for Injection Using Reporter Antigen Popliteal Lymph Node Assay[J]. Chinese Journal of Pharmacovigilance, 2017, 14(1): 4-6.

参考文献

[1] 张玉生. 双黄连注射剂药物不良反应的探讨和研究[D], 济南：山东大学,2009.
[2] 顾媛媛, 李殿明, 石朗,等. 注射用双黄连安全性问题研究近况[J]. 中国药房, 2015, (15):2156-2157.
[3] Simons F E R, Frew A J, Ansotegui I J, et al. Risk assessment in anaphylaxis: current and future approaches[J]. J Allergy Clin Immunol,2007, 120(1): S2-24.
[4] FDA. Guidance for Industry Immunotoxicology Evaluation of Investigational New Drugs[EB/OL].(2002-10-01)[2016-11-17].http://www.fda.gov/RegulatoryInformation/Guidances/default.htm.
[5] CFDA.中药、天然药物免疫毒性（过敏性、光变态反应）研究技术指导原则[EB/OL].( 2007-08-23) [2016-11-17]. http://www.cde.org.cn/zdyz.do?method=initValue&frameStr=0.
[6] CFDA.化学药物刺激性、过敏性和溶血性研究技术指导原则[EB/OL]. ( 2007-08-23) [2016-11-17]. http://www.cde.org.cn/zdyz.do?method=initValue&frameStr=0.
[7] Tuschl H, Landsteiner H T, Kovac R. Application of the popliteal lymph node assay in immunotoxicity testing: complementation of the direct popliteal lymph node assay with flow cytometric analyses[J]. Toxicology, 2002, 172(1):35-48.
[8] 刘兆华, 程芳, 周庚寅,等. C57BL/6J小鼠腘窝淋巴结试验评价注射用双黄连的致敏性研究[J]. 中国中西医结合杂志,2010, 30(1):64-67.
[9] Aida T, Kimura T, Ishikawa N, et al. Evaluation of allergenic potential of low-molecular compounds by mouse popliteal lymph node assay[J].J Toxicol Sci, 1998, 23(5):425-432.
[10] Løvik M, Alberg T, Nygaard U C, et al. Popliteal lymph node (PLN) assay to study adjuvant effects on respiratory allergy[J]. Methods,2007, 41(1):72-79.
[11] Ravel G, Descotes J. Popliteal lymph node assay: facts and perspectives[J]. J Appl Toxicol, 2005, 25(6):451-458.
[12] Gutting B W, Schomaker S J, Kaplan A H, et al. A comparison of the direct and reporter antigen popliteal lymph node assay for the detection of immunomodulation by low molecular weight compounds[J].Toxicol Sci, 1999, 51(1): 71-79.
[13] Shinkai K, Nakamura K, Tsutsui N, et al. Mouse popliteal lymph node assay for assessment of allergic and autoimmunity-inducing potentials of low-molecular-weight drugs[J]. J Toxicol Sci, 1999, 24(2):95-102.
[14] Carey J B, Allshire A, van Pelt F N. Immune modulation by cadmium and lead in the acute reporter antigen-popliteal lymph node assay[J]. Toxicol Sci, 2006, 91(1): 113-122.
[15] Nierkens S, Pieters R. The Reporter Antigen Popliteal Lymph Node Assay[S/OL]. (2006-09-01)[2016-11-17]. http://www.currentprotocols.com/WileyCDA/CPUnit/refId-tx1812.html
[16] 郝园, 孔翔瑜, 吴泰相. 277 篇 1486 例清开灵注射液不良反应/不良事件系统评价[J]. 中国循证医学杂志, 2010, 10(2): 162-175.
[17] 程芳. 注射用双黄连类过敏反应及其机制研究[D]. 济南：山东大学,2009.
[18] Uetrecht J. Idiosyncratic drug reactions: past, present, and future[J].Chem Res Toxicol, 2008, 21(1): 84-92.
[19] Pieters R, Albers R. Screening tests for autoimmune-related immunotoxicity[J]. Environ Health Perspect, 1999, 107( Suppl 5): 673-677.
[20] Pieters R. Detection of autoimmunity by pharmaceuticals[J]. Methods,2007, 41(1): 112-117.
[21] Gutting B W, Updyke L W, Amacher D E. Amacher, Investigating the TNP-OVA and direct popliteal lymph node assays for the detection of immunostimulation by drugs associated with anaphylaxis in humans[J]. J Appl Toxicol, 2002, 22(3): 177-183.
[22] Gutting B W, Updyke L W, Amacher D E. Diclofenac activates T cells in the direct popliteal lymph node assay and selectively induces IgG(1) and IgE against co-injected TNP-OVA[J]. Toxicol Lett, 2002, 131(3): 167-180.
[23] Albers R, Broeders A, Van d P A, et al. The use of reporter antigens in the popliteal lymph node assay to assess immunomodulation by chemicals[J]. Toxicol Appl Pharmacol, 1997, 143(1): 102-109.
[24] 张晶, 周富荣. 中药注射剂质量标准及有关问题评述[J]. 中药新药与临床药理, 2001, 12(002): 67-73.
[25] Han J, Yan Y, Li C, et al. Involvement of Histamine and RhoA/ROCK in Penicillin Immediate Hypersensitivity Reactions[J].Scientific Reports, 2016, 6:33192.
[26] 仇士东, 刘兆华,刘兆平. 双黄连注射剂导致类过敏反应的特点[J]. 中国药理学与毒理学杂志, 2013, 27(5): 818-824.