应用腘窝淋巴结试验研究注射用双黄连的致敏性
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摘要
中药注射剂(Traditional Chinese Medicine Injection, TCMI)突破了中药传统给药方式,是我国自主创新研制的独有中药剂型,具有生物利用度高、作用迅速等特点,克服了传统中医药不能用于治疗危急重症的弱点,在心脑血管疾病、呼吸系统疾病和肿瘤治疗中发挥重要、甚至是不可替代的作用,是中药现代化的发展方向之一。然而随着TCMI临床应用的日益广泛,其诱发不良反应的相关报道日益增多。速发型超敏反应(immediate hypersensitivity reaction, IHR)通常被认为是TCMI出现最多、危害较大的不良反应,严重时可引起患者发生过敏性休克而死亡。使得人们越来越质疑TCMI使用的安全性,进而诱发了对TCMI乃至整个中药产业的“信任危机”。如何全面、客观、科学评价TCMI存在的不良反应和风险,保证其临床用药的安全性,成为解决此次“信任危机”的重中之重。解决这个问题的关键则需要明确TCMI不良反应发生的原因和机制。TCMI通常含有3～7种原料药,多时甚至可达12种。由于成分复杂,引起致敏反应的原因和机制尚不清楚。尽管临床上使用的TCMI均是通过了严格的临床前药理毒理研究的产品,但由于使用的实验模型不能准确预测TCMI的致敏潜能,动物实验结果与临床不良反应之间缺乏相关性。现广泛应用的动物模型如,主动皮肤过敏试验(the active cutaneous anaphylaxis assay, ACA)、被动皮肤过敏试验(the passive cutaneous anaphylaxis assay, PCA)和主动全身过敏试验(the active systemic anaphylaxis assay, ASA),主要用于大分子蛋白质类抗原的致敏性检测。对小分子药物(low molecular weight compound, LMWC)而言,ASA、ACA和PCA反应阳性,提示LMWC具有致敏潜能；但PCA等反应阴性并不确定其没有致敏能力,因此容易出现假阴性结果。综上可知,限制TCMI机制研究的瓶颈是缺乏适合的实验模型,即应用新的模型探讨TCMI不良反应发生的原因和机制是解决TCMI安全性的首要问题。
腘窝淋巴结试验(popliteal lymph node assay, PLNA)是目前唯一可靠的LMWC致敏性筛选方法。它具有简单、省时、灵敏、特异性高,实验动物用量少、样品使用量低的特点。依据动物足趾部接受处理因素的方式不同,PLNA可以分为直接法PLNA(Direct PLNA or Primary PLNA,d-PLNA),间接法PLNA (Secondary PLNA,s-PLNA)、过继法PLNA (Adoptive PLNA)、报告抗原PLNA (Modified PLNA or Reporter Antigen PLNA, RA-PLNA)和佐剂PLNA (Adjuvant PLNA)。其中d-PLNA和RA-PLNA最常用于药物的致敏性研究。迄今为止,不同实验室已经应用PLNA检测了130余个LMWC,研究结果获得了很好的一致性和可重复性。但已有PLNA研究的样品均是成分单一的LMWC,PLNA可否用于成分复杂的TCMI的致敏性研究尚未见报道。此外,国内目前尚未建立起PLNA方法的标准体系,亦未见PLNA方法检测化学药物或中药致敏性研究的报道。而国外文献报道所用的实验动物种属如CBA小鼠、C57BL小鼠、F344大鼠、BN大鼠等,在国内价格昂贵且不容易获得,缺乏这些动物的饲养经验和实验背景资料。鉴于此,本研究拟结合我国实际情况,首先建立d-PLNA和RA-PLNA实验模型；在国内常用的实验动物种属中选择确定敏感动物,建立一套规范、简便、可行的PLNA实验操作程序；并利用建立的d-PLNA和RA-PLNA模型研究TCMI的潜在致敏性；建立一个适于TCMI致敏性试验研究的特异、灵敏的新方法,以期明确其不良反应的发生机制,确定引起过敏反应的物质(致敏原)；这将为TCMI的安全性评价提供新的思路和实验依据,促进我国药物致敏性实验研究与国际接轨,对于我国新药的研发有重大的经济和实用价值。
双黄连注射剂是最具代表性的清热解毒类TCMI之一,临床应用广泛,疗效肯定。2009年7月注射用双黄连(冻干)针剂被列入到2005年中华人民共和国药典增补本,这不仅是我国惟一进入药典的一种TCMI,也是首个采用指纹图谱控制产品质量的TCMI。但是和其他类型的TCMI一样,双黄连注射剂引起的临床不良反应病例日趋增多。根据文献报道,双黄连注射剂与鱼腥草注射液是不良反应报道最多的TCMI。双黄连注射剂引起的不良反应以Ⅰ型超敏反应为主,绝大多数表现为轻度的皮肤红斑、皮疹等；严重不良反应以全身性损害、呼吸系统损害为主,致死原因为过敏性休克或过敏样反应。国家药品不良反应监测中心已在2001年和2009年2次通报了双黄连注射剂的超敏反应,说明双黄连注射剂也同样面临被市场淘汰的危险。双黄连注射剂包括注射用双黄连灭菌粉末及双黄连注射液。双黄连注射液是最早开发成功的TCMI品种。注射用双黄连(Shuanghuanglian Injection, SHLI)是在双黄连注射液基础上发展起来的新剂型。SHLI由黄芩、金银花和连翘三种中药材精制而成,绿原酸含量较为丰富,而绿原酸被高度怀疑是一种半抗原。此外,SHLI不含任何辅料,所含的主要成分均为分子量小于1000u的小分子化合物(low molecular weight compound, LMWC)。故以SHLI为代表,通过d-PLNA和RA-PLNA研究其诱导超反应的发生机制,确定致敏原,对于保证SHLI乃至所有TCMI的临床用药安全具有重要的意义。
1胭窝淋巴结试验的模型构建
雌性成年SPF级雌性昆明小鼠、BALB/c小鼠、C57BL/6J小鼠、Wistar大鼠、BN大鼠随机分为空白对照组、阴性对照组和模型组。模型组右侧后肢足趾部皮下分别注射已知致敏物氯化汞(Mercury chloride, HgCl2)、双氯芬酸(Diclofenac sodium salt, DF)、D-盐酸青霉胺(D-Penicillamine, D-pen)、苯妥英钠(Diphenylhydantoin, DP)、链脲佐菌素(Streptozotocin, STZ),小鼠给药体积25μl或50μl；大鼠为50μl或100μl;苯巴比妥(Phenobarbital, PB)或溶媒为阴性对照,左侧不做处理。给药7 d,吸入CO2处死动物,迅速取出左、右侧胭窝淋巴结,放入置于冰上盛有PBS (含1%BSA)的培养皿中,去除脂肪组织,吸干水分后称重,制备单细胞悬液,计算淋巴结重量指数(weight index,WI)和细胞指数(cellullarity index, CI),比较胭窝淋巴结(popliteal lymph node,PLN)反应,WI>2或CI>5为阳性反应。报告抗原胭窝淋巴结试验时则将受试物分别与TNP-OVA或TNP-Ficoll一起注入右侧后肢足趾部皮下,给药7 d,处死动物,迅速取出PLN,制备单细胞悬液,Elispot检测AFCs数量,TNP特异性AFCs增多为阳性反应。结果表明：C57BL/6J小鼠、BALB/c小鼠和BN大鼠均对阳性化合物反应敏感,提示成功构建了d-PLNA动物模型。小鼠后肢每侧均只有一个胭窝淋巴结,位置固定,容易摘出,大鼠PLN存有变异,操作不如小鼠方便,建议C57BL/6J小鼠和BALB/c小鼠可作为PLNA的首选敏感动物。在此基础上,利用工具药D-pen成功构建了BALB/c小鼠RA-PLNA模型。应用这两种模型进行下面的SHLI实验研究。
2直接腘窝淋巴结试验研究注射用双黄连的致敏性
雌性C57BL/6J小鼠随机分为空白对照(Veh)组、PB组(1 mg／只)、HgCl2组(50μg／只)和D-pen组(2 mg／只)、SHLI亻氐剂量组(L组,1 mg／只)和高剂量组(H组,5 mg／只),每组10只。小鼠一侧足趾部皮下给予受试物,第7d天处死动物,摘取两侧PLN,称重,计算WI；每组将4只小鼠的PLN以4%中性多聚甲醛固定用于病理学检查,其余6只制备单细胞悬液,计算CI。比较SHLI和致敏物引起的PLN反应,探讨SHLI的致敏潜能。结果表明：H组WI、CI较Veh组升高(P<0.05),WI>2和CI>5。病理检查,PLN体积变大,生发中心明显,HEV横断面增多。结果发现高剂量SHLI可诱导C57BL/6J小鼠产生明显的PLN反应,提示它具有一定的致敏潜能。但诱导PLN阳性反应需要较高的剂量,表明可能是SHLI中的微量成分发挥作用,这提示我们下一步需要对SHLI含有的不同成分进行研究,以确定致敏原存在的范围。此外,d-PLNA并未能揭示SHLI诱导PLNA反应的确切机制,这有待于下一步的RA-PLNA研究证实。
3直接胭窝淋巴结试验研究药典工艺制备注射用双黄连及其精制品的致敏性
按照药典经典工艺制备SHLI及其中间提取物,并用大孔树脂对其进行精制,共获得10个样品。BALB/c小鼠随机分组,右侧后肢足趾部酒精消毒,用胰岛素注射器或1 ml注射器皮下注射已知致敏物D-pen、供试品、以溶媒为阴性对照。小鼠给药体积均为50μl；利用d-PLNA研究5 mg／只剂量时的反应；对5mg／只出现PLNA阳性反应的样品,降低剂量为2.5mg／只给药；阴性反应的样品则提高剂量为10mg／只进行给药,以研究剂量-效应关系。结果发现,按药典原工艺制备的SHLI在高剂量下获得阳性反应,精制品PLNA反应降低,提示现有SHLI的药典工艺标准需要改进。另外,精制品水溶性较好,即使提高剂量也不易诱导PLNA阳性反应,表明有害物质可能是水溶性较差的成分,提示我们研究SHLI的致敏成分需用新方法分离提取三味药材的成分。
4直接胭窝淋巴结试验研究新工艺注射用双黄连有效组分的致敏性
利用d-PLNA研究采用新工艺提取制备的16个样品的致敏潜能。BALB/c小鼠随机分组,右侧后肢足趾部酒精消毒,用胰岛素注射器皮下注射。给药体积均为50μl；给药剂量均为10 mg／只；难溶于0.9%生理盐水的组分,先以10μl／只的纯DMSO溶解,然后再用0.9%生理盐水稀释为相应的浓度后给药。对出现PLNA阳性反应的化合物,进一步降低至5 mg／只、2.5 mg／只或0.25mg／只的剂量给药,直至发现无反应剂量,以研究剂量-效应关系。7／16个组分在10 mg／只出现明显的PLNA反应,提示这些部位中可能存有有害成分。引起PLNA阳性反应的组分分布于三种药材的提取物中,其中来源于双花提取物的2种,连翘提取物3种,黄芩提取物2种,说明有害成分在三种原药材中均可能存在。降低剂量至2.5 mg／只时阳性反应消失。表明反应存在剂量-效应关系,可对这些成分进一步提纯、分离其中的有害成分。但为了排除非特异性炎症刺激导致的d-PLNA假阳性反应,尚需进一步通过机制研究才能最终确定这些成分的致敏性。
5报告抗原胭窝淋巴结试验研究注射用双黄连的致敏性
本研究将高、低剂量的双黄连分别与TNP-OVA或TNP-Ficoll一起注射给BALB/c小鼠,利用RA-PLNA结合流式细胞术检测了SHLI的致敏机制。研究结果发现高剂量SHLI和两种报告抗原一起注射可以升高WI或CI,但阳性反应的程度并没有比单独注射高剂量SHLI时显著增加,表明注射报告抗原对高剂量SHLI诱导的PLNA反应没有显著影响；高剂量SHLI和TNP-OVA一起注射时,可明显增加TNP特异性AFC的数量,但与TNP-Ficoll一起注射或低剂量SHLI与二者一起注射时则未能增加AFC的数量；表明高剂量的SHLI提供了共刺激辅助性信号,导致了炎症刺激,降低剂量时则致炎作用消失。流式细胞术检测也发现高剂量SHLI与TNP-OVA一起可升高PLN中巨噬细胞的数量,提示了SHLI可激活体内的巨噬细胞,后者释放的细胞因子对促进炎症的发展具有重要的作用；但是SHLI在体内不会形成新的抗原表位,不能激活特异性T细胞,表明SHLI本身不具有致敏潜能,不会诱发抗原抗体特异性的免疫应答反应。所以,SHLI的成分本身不能直接诱导BALB/c小鼠产生IgE介导的超敏反应,即不具有致敏性。这与我们实验室另一研究小组的结论相一致,他们认为SHLI导致的急性过敏反应主要为类过敏反应。
6注射用双黄连有效单体成分的致敏性研究
6.1直接腘窝淋巴结试验研究绿原酸、黄芩苷和连翘苷的致敏性
d-PLNA预测单一成分LMWC致敏性的特异性和准确性已被多家实验室证实,但对于其预测成份复杂TCMI致敏潜能的有效性尚未得到验证。因此,利用d-PLNA检测SHLI有效单体成份的致敏性,其结果应该更准确可靠。CGA、黄芩苷和连翘苷分别是SHLI三种原料药金银花、黄芩和连翘的主要成分。多数人认为他们是发挥药效作用的物质基础,但亦有些研究认为他们与注射用双黄连ADR有关。故本实验首先利用d-PLNA检测了CGA、黄芩苷和连翘苷的致敏潜能。实验结果发现,CGA、黄芩苷和连翘苷在1mg／只的剂量下,均未能诱导d-PLNA阳性反应,提示这些主要成分不具致敏性。
6.2报告抗原胭窝淋巴结试验研究绿原酸的致敏性
CGA广泛分布在各种植物中,人们经常接触到CGA,但现在还不清楚暴露于CGA是否可以诱导产生超敏反应。许多研究者高度怀疑CGA是一种半抗原,但是本研究d-PLNA实验结果不支持这种结论。为进一步检测CGA的致敏潜能,并验证d-PLNA检测CGA,黄芩苷和连翘苷实验结果是否准确可靠,本研究以CGA为代表,利用流式细胞术和RA-PLNA进一步研究了CGA的致敏性。研究结果表明,CGA对BALB/c小鼠PLN细胞表型、激活标志物没有影响。RA-PLNA检测发现,无论与TNP-OVA还是与TNP-Ficoll一起注射,CGA均不能诱导TNP特异性AFCs形成,这表明CGA对BALB/c小鼠不具有致敏潜能。结果证实了d-PLNA和RA-PLNA结果一致。提示移除TCMI中的CGA或许不会显著降低其不良反应的发生,这或许有助于消除一些研究者对绿原酸全身暴露时安全性的担忧。
综上,本研究在国内首次成功建立了d-PLNA和RA-PLNA模型,并将之用于SHLI的致敏性研究；确认BALB/c小鼠和C57BL/6J小鼠为PLNA的首选敏感动物；研究结果证实,d-PLNA和RA-PLNA可用于SHLI及其有效组分或单体成分的致敏性研究；大剂量SHLI对小鼠具有辅助性炎症刺激作用,但不能激活抗原特异性的T淋巴细胞,表明SHLI的成分本身不能直接刺激BALB/c小鼠产生IgE介导的超敏反应,即不具有致敏性,提示IgE介导的超敏反应不是SHLI相关不良反应的主要发生机制,非抗原-抗体反应机制在其不良反应的发生、发展中起主导作用；这从另一个方面支持了我们实验室另一研究小组的结论：他们认为类过敏反应是SHLI临床不良反应发生的主要类型,SHLI直接刺激嗜碱性粒细胞或肥大细胞释放组胺等活性物质是其的主要机制。
Traditional Chinese medicine injection (TCMI), as a unique Chinese Medical formulation invented by our country and a developmental direction of the modernization of traditional Chinese medicine, has broken down the limitations of Traditional Chinese Medicine regiments. With features like high bioavailability and fast-acting, TCMI has already overcome the weakness of traditional Chinese medicine (cannot be used to cure the severe diseases) and played an important or even irreplaceable role in the treatment of conditions ranging from cardiovascular and cerebrovascular diseases, respiratory diseases and cancer. However, with the wide application of TCMI, the prevalence of adverse reactions related to TCMI appears to have increased significantly in China over the past ten years. Immediate hypersensitivity reactions (IHR), generally being considered as the most frequent and severe adverse reactions induced by TCMI, may cause anaphylactic shock or even be life-threatening. Those safety concerns caused a reduced use of TCMI and increasingly questioned its rationality, and thus ultimately lead to the "crisis of confidence" to the whole traditional Chinese medicine industry. In order to solve this crisis, the adverse reactions and risks induced by TCMI must be comprehensively, objectively and scientifically evaluated and the safety of clinical use should be ensured. Among those, the key point which needs to be further elucidated is to understand the cause and mechanisms of adverse reactions induced by TCMI. TCMI usually contains 3 to 7, sometimes even up to 12, kinds of raw material medicines. Thus TCMI does contain complex mixtures with a number of unknown constituent. Moreover the constituent that induced the adverse reactions and what mechanisms to be involved are poorly understood yet. Notably, all TCMIs approved for sale in the market and used in clinical are products which have been performed pre-clinical safety evaluations well. The results from safety evaluations showed that TCMIs did not induce any adverse reactions in animals. For this reason TCMIs were regarded as safe drugs in human, In fact, since it remains difficult for regulatory safety studies to predict the immunosensitizing potential of TCMI, there is poorly correlation between animal experiments results and clinical adverse reactions. Traditionally the animal model used to predict hypersensitivity reaction are the active cutaneous anaphylaxis assay (ACA), the passive cutaneous anaphylaxis assay (PCA) and the active systemic anaphylaxis assay (ASA), which are mainly used to detect the sensitization of macromolecular protein antigen. As far as low molecular weight compound(LMWC) is concerned, ASA, ACA, and PCA's positive results suggests that LMWC has immunosensitizing potential, but it is not always true for the negative results because of the possibility of false negative results in the test. In brief, the lack of suitable experimental models is the bottleneck that restricts the study on mechanism. Therefore, there is a great need for new animal models to study adverse reactions and mechanisms caused by TCMI prior to crisis of TCMI to be resolved.
Popliteal lymph node assay (PLNA) is regarded as a unique reliable and promising screening methods to indicate immunosensitization by LMWC. It is simple, time-saving, sensitive, high specific and with less uses of laboratory animals and samples. Five different versions of the PLNA have been proposed according to the different local treatments factors:the direct, secondary, adoptive, reporter antigen (RA) and Adjuvant PLNA. The direct PLNA (d-PLNA) and RA-PLNA are the most commonly used in drug immunosensitizing studies. Up to now, more than 130 LMWCs have been detected by PLNA in different laboratories and the findings showed good correlation, consistency and repeatability. However, existing research results are all about single-component LMWCs. it has not been reported that whether PLNA could be used to study the immunosensitizing potential of complex TCMI components. In addition, the standard system of PLNA has not been established at present, and there is no research report on the immunosesentizhing potential of chemicals or Chinese medicine allergens detected by PLNA in China. Moreover, animal species used in experiment reported in the foreign literatures, such as CBA mice, C57BL mice, F344 rats, and BN rats, were so expensive in China and not easily obtained. What's more, both the experience of animal's husbandry and the information of experimental background are scarce. For these reasons and combining the actual situation in our country, the aims of this study were to establish d-PLNA and RA-PLNA experimental model firstly; choose and define sensitive and appropriate animals from widely used laboratory animal species in China to define; secondly, establish a standardized, simple and feasible PLNA Experimental Operation procedures; thirdly, use d-PLNA and RA-PLNA methods that have been established to evaluate the immunosensitizing potential of TCMI and its single-components. We are trying to establish a special and sensitive method to evaluate the immunosensitizing potential of TCMI, and to clarify its mechanism and determine the allergen, which will provide new ideas and experimental evidences for TCMI safety evaluation, promote our immunosensitizing studies on drugs with international standards and have great economic and practical values on new drugs' R&D.
Shuanghuanglian injection (SHLI) is a representative of heat-clearing and detoxifying TCMI with a wide range of applications and good efficacy in clinical use. SHLI (lyophilized) was included in The People's Republic of China Pharmacopoeia Supplement (2005) in July,2009. It is the only TCMI that was put into the Pharmacopoeia and the first one to use fingerprint atlas to control its quality. However, just like other types of TCMIs, the reports of clinical adverse reactions related to SHLI are increasing. Analysis of data from the existing literatures report confirmed that IHR induced by SHLI or Houttuynia injection accounted for the most adverse reactions of all TCMI and came in the forefront. The adverse reaction induced by SHLI is allergy-based and the common symptoms are mild skin etthema and rash, etc.; serious adverse reactions include systemic damage, respiratory damage as the main ones. The main causes to death are anaphylactic shock and anaphylactoid reactions. National Adverse Drug Reaction Monitoring Center reported the hypersensitivity reactions related to SHLI twice in 2001 and 2009. This means that SHLI is also facing the danger of being eliminated from the market. SHLI was the first successful development of TCMI and was made up of three kinds of Chinese herbal medicines, including skullcap, honeysuckle and forsythia. Chlorogenic acid (CGA), which was highly suspected as a hapten, was rich in SHLI. In addition, the injection does not contain any excipient and the main component is LMWC. Therefore, allergens can be determined by studying the mechanisms of SHLI-induced allergic reactions through d-PLNA and RA-PLNA. This has great significance for SHLI as well as all TCMI in clinical drug safety.
1 Construction of animal models for popliteal lymph node assay
Specific pathogen-free female adult KM mice, BABL/c mice, C57BL/6J mice, Wistar rats and BN rats were randomly divided into Group Veh, negetive control group and model group. Animals were injected subcutaneously on the right hind footpad with Mercury chloride (HgCl2), Diclofenac sodium salt (DF), D-Penicillamine (D-pen), Diphenylhydantoin (DP) and Streptozotocin (STZ) in saline or 20%DMSO/saline. A volume of 25μl or 50μl was used for mice while 50μl or 100μl for rats. Phenobarbital (PB) was used for negative control. Normal animal were only injected on the right hind footpad with saline. After being injected for seven days, mice were sacrificed by inhalating CO2. Popliteal lymph node (PLN) was extracted and placed on ice in PBS-BSA (1%). Weighted PLN after the rid of adherent adipose tissues and being dried. Took preparation of single-cell suspension and got the weight index (WI= weight PLN treated side/weight PLN untreated side) and cellullarity index (CI). WI>2 or CI>5 is considered as a positive response. During the study, do the hypodermic on the toes of right hind leg with test material and TNP-OVA or TNP-Ficoll together for 7 days. After the administration, executed the animals and extracted the PLN for single cell susponsion. Use the Elispot method for testing the number of AFCs. The increase of AFCs is considered as a positive reaction. The study results showed that the C57BL/6J mice, BALB/c mice as well as BN rats could structure the d-PLNA models successfully. We found that mice only have one PLN on each side. PLN of mouse is easy to be located and removed than rat. So BABL/c mice and C57BL/6J are the preferred animals for PLNA. Based on those, we chose BABL/c mice to structure the RA-PLNA models and used on the following two SHLI studies.
2 Assessment of Immunosensitizing Potential of Shuanghuanglian Injection Using Direct Popliteal Lymph Node Assay in C57BL/6J Mice
Sixty C57BL/6J mice were randomly divided into 6 groups:vehicle (Veh), PB (1 mg/mouse), HgCl2 (50μg/mouse), D-pen(2 mg/mouse), SHLI low dose (1 mg/mouse) and high dose(5 mg/mouse),10 mice/group. Articles were injected subcutaneously into one hind foot pad of SPF female mice. On day 8 after injection, animals were sacrificed, the left (untreated side) and right (treated side) PLN were isolated, weighed respectively, and then PLN mass index (MI) was calculated. PLNs of 4 mice in each group were fixed in 4%paraformaldehyde solution, routinely stained with HE for histopathologic examination. The others (n=6) were prepared into single-cell suspensions, and then PLN cells index (CI) was calculated. PLN changes after administration of SHLI were observed and compared. We founded that MI and CI of SHLI treated group increased with the average MI≥2 and the average CI≥5,and the difference was significant when compared with group Veh (P<0.05) in C57BL/6J mice. Pathological examination showed that the volume of the right PLNs treated with SHLI became larger than those in left. Germinal center of PLN lymph was evident and vice cortex HEV cross-section increased. The results suggested that SHLI could induce significant PLN response that suggests it has the immuosensitizing potential.
3 Assessment of Immunosensitizing Potential of Shuanghuanglian Injection and its Refined products Prepared according to Chinese Pharmacopoeia Using Direct Popliteal Lymph Node Assay
Test articles, including 10 different pharmaceutical samples, were obtained from SHLI and its extracts after being refined by macroporous resin (D101).Randomly grouped the BALB/c mice and sterilized the right hind toes by alcohol. Use the insulin syringe or 1 ml syringe for injecting the sensitizer (D-pen), test material and dissolvent as the negative control. The treated volume of mice was 50μl. We used d-PLNA method to study on the reactions of the dose of 5 mg/mouse at first. At this moment, cut down the dose to 2.5mg/mouse when the sample had the PLNA positive reaction. When the negative reaction occurred, raise the dose to 10 mg/mouse. Dose-responses relationship could be studied through these methods. We found that SHLI prepared according to Pharmacopoeia procedure obtained positive results at high-dose, the rest did not respond; the refined products'PLNA reactions reduced. This suggested that the Pharmacopoeia procedure of SHLI was badly needed an improvement. What's more, the refined products were good in water-solubility and even the increase of dose wouldn't lead to positive reactions. This suggested that the adverse reactions may be induced by poor water-soluble components, In this sense, in order to study on the sensitized components of SHLI, new production procedure should be used for the separation of three medicinal compositions.
4 Assessment of Immunosensitizing Potential of Shuanghuanglian Injection Prepared by new Production Procedure Using Direct Popliteal Lymph Node Assay
d-PLNA was used for the assessment of immunosensitizing potential of 16 samples separated by new production procedure. The BALB/c mice were randomly grouped and the right hind toes were sterilized by 70%alcohol. The insulin syringe was used for injecting. The treated volume of mice was 50μl. The dose was 10 mg/mouse. For those constituents that were insoluble in 0.9%saline, were dissolved by pure DMSO of 10μl per mouse, and then diluted to the corresponding concentration with 0.9%saline for administration. For those compounds with PLNA positive results, kept on further reduction from 5 mg/mouse,2.5 mg/mouse or 0.25 mg/mouse to 0 mg/mouse for administration to get the dose-effect relationship. We found that 7/16 samples induced PLNA positive reactions at high dose (10 mg/animal); those suggested that those samples maybe contain harmful components. PLNA positive reactions caused by those components were found in the extracts of three herbs, including 2 extracts from honeysuckle,3 extract from Forsythia, and 2 extracts from skullcap, indicating that there may exist harmful components in all the 3 medicinal plants. Low dose (2.5 mg/animal) failed to induce the positive PLN reactions. Non-specific inflammatory stimulate may lead to d-PLNA false positive results, so further study on mechanism should be done for defining the sensitization of these compositions.
5 Evaluation of the Immunosensitizing Potential of Shuanghuanglian injection Using a Reporter Antigen Popliteal Lymph Node Assay in mice
The immunosensitizing mechanisms of SHLI were investigated using RA-PLNA in mice. The results showed that high-dose of SHLI combined with TNP-OVA (T cell dependent antigen) or TNP-Ficoll (T cell independent antigen) could induce PLN positive response (WI>2 or CI>5), but the intense of positive reaction were similar to those induced by high-dose of SHLI alone. Mixed with TNP-OVA, high-dose of SHLI could significantly increase the number of the TNP-specific antibody-forming cells (AFCs), but with TNP-Ficoll and while Low-dose of SHLI with 2 RA failed to do so. Those results suggested that high-dose-SHLI provided costimulatory signals which could induce inflammatory reaction and this effect would disappear at low dose. Moreover, it has been demonstrated via flow cytometry that high-dose-SHLI co-injected with TNP-OVA could activate macrophages in vivo. Activated macrophages could release cytokines which played an important role in induction of inflammation. However, the results did not support that SHLI activated neoantigen-specific T cells in BALB/c mice, which indicated that SHLI by itself do not have immunosesensitizing potential. Thus SHLI did not induced immune response resulting from specific antigen. As a result we postulated that adverse reactions of SHLI may be due to anaphylactoid reaction rather than IgE-mediated hypersensitivity reactions, which is congruent with conclusions obtained by other researchers in our laboratory.
6 Evaluation of the immunosensitizing potential of Monomer constituents in Shanghuanglian Injection
6.1 Evaluation of the immunosensitizing potential of Chlorogenic acid, Baicalin and Phillyrin Using Direct Popliteal Lymph Node Assay
The great value of d-PLNA has been proved by several labs in predicting the immunosensitizing potential of pure LMWC. However, the value of d-PLNA has not been adequately validated in predicting TCMI which contain complex mixtures with a number of unknown constituents. Therefore, it is more reliable to evaluate the monomer constituents contained in SHLI by using d-PLNA. CGA, Baicalin and phillyrin are main constituents of honeysuckle, skullcap and forsythia respectively, which are composed crude drug of SHLI. Most researchers thought that those monomer constituents are the basis of pharmacodynamics of SHLI. The others thought that they are related to ADR induced by SHLI. To determine whether the CGA, Baicalin and phillyrin are associated with ADR, we detected their immunosensitizing potential using d-PLNA firstly. The results found that a dose of 1 mg CGA, baicalin and phillyrin per animal failed to induce BALB/c mice d-PLNA positive reaction, suggesting that they do not have immunosensitizing potential.
6.2 Evaluation of the immunosensitizing potential of chlorogenic acid using a popliteal lymph node assay in BALB/c mice
It has yet to be established whether CGA, a common xenobiotic with potential exposure risk to humans, is associated with immune-mediated hypersensitivity reactions (HRs).Previous results from the d-PLNA demonstrated that CGA failed to induce positive response. To further determine whether CGA may possess an intrinsic capacity to stimulate or dysregulate immune responses, and if so, what mechanisms may be involved, we characterized the popliteal lymph node reaction induced by CGA in naive female BALB/c mice using both PLNA combined with flow cytomtry and RA-PLNA method. CGA was selected since many investigators postulated that it is a hapten. Another aim of present study is to validate the accuracy and the reliability of results using d-PLNA to predict the immunosensitizing potential of CGA, baicalin and phillyrin.Our results show that CGA failed to induce immunoreactivity following a single subcutaneous injection either alone or when combined with TNP-OVA or TNP-Ficoll. These results indicated that CGA lacks the intrinsic capacity to sensitize or stimulate immune responses in BALB/c mice. The results form RA-PLNA confirmed those from d-PLNA. Moreover, these results suggest that exposure to CGA may not represent a safety concern for humans and that removal of CGA from Traditional Chinese Medicine Injections may not significantly decrease the prevalence of HRs.
In conclusion, we successfully established models of d-PLNA and RA-PLNA, and confirmed BALB/c mice and C57 BL/6J mice as the preferred sensitive animals of both methods in our laboratory; d-PLNA and RA-PLNA are suitable methods to determine the immunosensitizing potential of SHLI and its constituents, which could also reveal the primary mechanism involved in adverse reaction related to SHLI. Our results showed that high-dose-SHLI could provide costimulatory signals to induce inflammatory reactions, but could not activate antigen-specific T-lymphocytes. Those results indicated that SHLI or its constituents do not have the immunosensitizing potential. Form the results, we concluded that SHLI by itself can not directly stimulate BALB/c mice to produce IgE-mediated hypersensitivity reactions. As a result we propose that ADR related to SHLI may be due to anaphylactoid reaction rather than IgE-mediated hypersensitivity reactions, which is congruent with conclusions obtained by other researchers in our laboratory. The release of other active mediators from basophils or mast cells directly stimulated by SHLI may also be the primary mechanism of ADR in human.
腘窝淋巴结试验(popliteal lymph node assay, PLNA)是目前唯一可靠的LMWC致敏性筛选方法。它具有简单、省时、灵敏、特异性高,实验动物用量少、样品使用量低的特点。依据动物足趾部接受处理因素的方式不同,PLNA可以分为直接法PLNA(Direct PLNA or Primary PLNA,d-PLNA),间接法PLNA (Secondary PLNA,s-PLNA)、过继法PLNA (Adoptive PLNA)、报告抗原PLNA (Modified PLNA or Reporter Antigen PLNA, RA-PLNA)和佐剂PLNA (Adjuvant PLNA)。其中d-PLNA和RA-PLNA最常用于药物的致敏性研究。迄今为止,不同实验室已经应用PLNA检测了130余个LMWC,研究结果获得了很好的一致性和可重复性。但已有PLNA研究的样品均是成分单一的LMWC,PLNA可否用于成分复杂的TCMI的致敏性研究尚未见报道。此外,国内目前尚未建立起PLNA方法的标准体系,亦未见PLNA方法检测化学药物或中药致敏性研究的报道。而国外文献报道所用的实验动物种属如CBA小鼠、C57BL小鼠、F344大鼠、BN大鼠等,在国内价格昂贵且不容易获得,缺乏这些动物的饲养经验和实验背景资料。鉴于此,本研究拟结合我国实际情况,首先建立d-PLNA和RA-PLNA实验模型；在国内常用的实验动物种属中选择确定敏感动物,建立一套规范、简便、可行的PLNA实验操作程序；并利用建立的d-PLNA和RA-PLNA模型研究TCMI的潜在致敏性；建立一个适于TCMI致敏性试验研究的特异、灵敏的新方法,以期明确其不良反应的发生机制,确定引起过敏反应的物质(致敏原)；这将为TCMI的安全性评价提供新的思路和实验依据,促进我国药物致敏性实验研究与国际接轨,对于我国新药的研发有重大的经济和实用价值。
双黄连注射剂是最具代表性的清热解毒类TCMI之一,临床应用广泛,疗效肯定。2009年7月注射用双黄连(冻干)针剂被列入到2005年中华人民共和国药典增补本,这不仅是我国惟一进入药典的一种TCMI,也是首个采用指纹图谱控制产品质量的TCMI。但是和其他类型的TCMI一样,双黄连注射剂引起的临床不良反应病例日趋增多。根据文献报道,双黄连注射剂与鱼腥草注射液是不良反应报道最多的TCMI。双黄连注射剂引起的不良反应以Ⅰ型超敏反应为主,绝大多数表现为轻度的皮肤红斑、皮疹等；严重不良反应以全身性损害、呼吸系统损害为主,致死原因为过敏性休克或过敏样反应。国家药品不良反应监测中心已在2001年和2009年2次通报了双黄连注射剂的超敏反应,说明双黄连注射剂也同样面临被市场淘汰的危险。双黄连注射剂包括注射用双黄连灭菌粉末及双黄连注射液。双黄连注射液是最早开发成功的TCMI品种。注射用双黄连(Shuanghuanglian Injection, SHLI)是在双黄连注射液基础上发展起来的新剂型。SHLI由黄芩、金银花和连翘三种中药材精制而成,绿原酸含量较为丰富,而绿原酸被高度怀疑是一种半抗原。此外,SHLI不含任何辅料,所含的主要成分均为分子量小于1000u的小分子化合物(low molecular weight compound, LMWC)。故以SHLI为代表,通过d-PLNA和RA-PLNA研究其诱导超反应的发生机制,确定致敏原,对于保证SHLI乃至所有TCMI的临床用药安全具有重要的意义。
1胭窝淋巴结试验的模型构建
雌性成年SPF级雌性昆明小鼠、BALB/c小鼠、C57BL/6J小鼠、Wistar大鼠、BN大鼠随机分为空白对照组、阴性对照组和模型组。模型组右侧后肢足趾部皮下分别注射已知致敏物氯化汞(Mercury chloride, HgCl2)、双氯芬酸(Diclofenac sodium salt, DF)、D-盐酸青霉胺(D-Penicillamine, D-pen)、苯妥英钠(Diphenylhydantoin, DP)、链脲佐菌素(Streptozotocin, STZ),小鼠给药体积25μl或50μl；大鼠为50μl或100μl;苯巴比妥(Phenobarbital, PB)或溶媒为阴性对照,左侧不做处理。给药7 d,吸入CO2处死动物,迅速取出左、右侧胭窝淋巴结,放入置于冰上盛有PBS (含1%BSA)的培养皿中,去除脂肪组织,吸干水分后称重,制备单细胞悬液,计算淋巴结重量指数(weight index,WI)和细胞指数(cellullarity index, CI),比较胭窝淋巴结(popliteal lymph node,PLN)反应,WI>2或CI>5为阳性反应。报告抗原胭窝淋巴结试验时则将受试物分别与TNP-OVA或TNP-Ficoll一起注入右侧后肢足趾部皮下,给药7 d,处死动物,迅速取出PLN,制备单细胞悬液,Elispot检测AFCs数量,TNP特异性AFCs增多为阳性反应。结果表明：C57BL/6J小鼠、BALB/c小鼠和BN大鼠均对阳性化合物反应敏感,提示成功构建了d-PLNA动物模型。小鼠后肢每侧均只有一个胭窝淋巴结,位置固定,容易摘出,大鼠PLN存有变异,操作不如小鼠方便,建议C57BL/6J小鼠和BALB/c小鼠可作为PLNA的首选敏感动物。在此基础上,利用工具药D-pen成功构建了BALB/c小鼠RA-PLNA模型。应用这两种模型进行下面的SHLI实验研究。
2直接腘窝淋巴结试验研究注射用双黄连的致敏性
雌性C57BL/6J小鼠随机分为空白对照(Veh)组、PB组(1 mg／只)、HgCl2组(50μg／只)和D-pen组(2 mg／只)、SHLI亻氐剂量组(L组,1 mg／只)和高剂量组(H组,5 mg／只),每组10只。小鼠一侧足趾部皮下给予受试物,第7d天处死动物,摘取两侧PLN,称重,计算WI；每组将4只小鼠的PLN以4%中性多聚甲醛固定用于病理学检查,其余6只制备单细胞悬液,计算CI。比较SHLI和致敏物引起的PLN反应,探讨SHLI的致敏潜能。结果表明：H组WI、CI较Veh组升高(P<0.05),WI>2和CI>5。病理检查,PLN体积变大,生发中心明显,HEV横断面增多。结果发现高剂量SHLI可诱导C57BL/6J小鼠产生明显的PLN反应,提示它具有一定的致敏潜能。但诱导PLN阳性反应需要较高的剂量,表明可能是SHLI中的微量成分发挥作用,这提示我们下一步需要对SHLI含有的不同成分进行研究,以确定致敏原存在的范围。此外,d-PLNA并未能揭示SHLI诱导PLNA反应的确切机制,这有待于下一步的RA-PLNA研究证实。
3直接胭窝淋巴结试验研究药典工艺制备注射用双黄连及其精制品的致敏性
按照药典经典工艺制备SHLI及其中间提取物,并用大孔树脂对其进行精制,共获得10个样品。BALB/c小鼠随机分组,右侧后肢足趾部酒精消毒,用胰岛素注射器或1 ml注射器皮下注射已知致敏物D-pen、供试品、以溶媒为阴性对照。小鼠给药体积均为50μl；利用d-PLNA研究5 mg／只剂量时的反应；对5mg／只出现PLNA阳性反应的样品,降低剂量为2.5mg／只给药；阴性反应的样品则提高剂量为10mg／只进行给药,以研究剂量-效应关系。结果发现,按药典原工艺制备的SHLI在高剂量下获得阳性反应,精制品PLNA反应降低,提示现有SHLI的药典工艺标准需要改进。另外,精制品水溶性较好,即使提高剂量也不易诱导PLNA阳性反应,表明有害物质可能是水溶性较差的成分,提示我们研究SHLI的致敏成分需用新方法分离提取三味药材的成分。
4直接胭窝淋巴结试验研究新工艺注射用双黄连有效组分的致敏性
利用d-PLNA研究采用新工艺提取制备的16个样品的致敏潜能。BALB/c小鼠随机分组,右侧后肢足趾部酒精消毒,用胰岛素注射器皮下注射。给药体积均为50μl；给药剂量均为10 mg／只；难溶于0.9%生理盐水的组分,先以10μl／只的纯DMSO溶解,然后再用0.9%生理盐水稀释为相应的浓度后给药。对出现PLNA阳性反应的化合物,进一步降低至5 mg／只、2.5 mg／只或0.25mg／只的剂量给药,直至发现无反应剂量,以研究剂量-效应关系。7／16个组分在10 mg／只出现明显的PLNA反应,提示这些部位中可能存有有害成分。引起PLNA阳性反应的组分分布于三种药材的提取物中,其中来源于双花提取物的2种,连翘提取物3种,黄芩提取物2种,说明有害成分在三种原药材中均可能存在。降低剂量至2.5 mg／只时阳性反应消失。表明反应存在剂量-效应关系,可对这些成分进一步提纯、分离其中的有害成分。但为了排除非特异性炎症刺激导致的d-PLNA假阳性反应,尚需进一步通过机制研究才能最终确定这些成分的致敏性。
5报告抗原胭窝淋巴结试验研究注射用双黄连的致敏性
本研究将高、低剂量的双黄连分别与TNP-OVA或TNP-Ficoll一起注射给BALB/c小鼠,利用RA-PLNA结合流式细胞术检测了SHLI的致敏机制。研究结果发现高剂量SHLI和两种报告抗原一起注射可以升高WI或CI,但阳性反应的程度并没有比单独注射高剂量SHLI时显著增加,表明注射报告抗原对高剂量SHLI诱导的PLNA反应没有显著影响；高剂量SHLI和TNP-OVA一起注射时,可明显增加TNP特异性AFC的数量,但与TNP-Ficoll一起注射或低剂量SHLI与二者一起注射时则未能增加AFC的数量；表明高剂量的SHLI提供了共刺激辅助性信号,导致了炎症刺激,降低剂量时则致炎作用消失。流式细胞术检测也发现高剂量SHLI与TNP-OVA一起可升高PLN中巨噬细胞的数量,提示了SHLI可激活体内的巨噬细胞,后者释放的细胞因子对促进炎症的发展具有重要的作用；但是SHLI在体内不会形成新的抗原表位,不能激活特异性T细胞,表明SHLI本身不具有致敏潜能,不会诱发抗原抗体特异性的免疫应答反应。所以,SHLI的成分本身不能直接诱导BALB/c小鼠产生IgE介导的超敏反应,即不具有致敏性。这与我们实验室另一研究小组的结论相一致,他们认为SHLI导致的急性过敏反应主要为类过敏反应。
6注射用双黄连有效单体成分的致敏性研究
6.1直接腘窝淋巴结试验研究绿原酸、黄芩苷和连翘苷的致敏性
d-PLNA预测单一成分LMWC致敏性的特异性和准确性已被多家实验室证实,但对于其预测成份复杂TCMI致敏潜能的有效性尚未得到验证。因此,利用d-PLNA检测SHLI有效单体成份的致敏性,其结果应该更准确可靠。CGA、黄芩苷和连翘苷分别是SHLI三种原料药金银花、黄芩和连翘的主要成分。多数人认为他们是发挥药效作用的物质基础,但亦有些研究认为他们与注射用双黄连ADR有关。故本实验首先利用d-PLNA检测了CGA、黄芩苷和连翘苷的致敏潜能。实验结果发现,CGA、黄芩苷和连翘苷在1mg／只的剂量下,均未能诱导d-PLNA阳性反应,提示这些主要成分不具致敏性。
6.2报告抗原胭窝淋巴结试验研究绿原酸的致敏性
CGA广泛分布在各种植物中,人们经常接触到CGA,但现在还不清楚暴露于CGA是否可以诱导产生超敏反应。许多研究者高度怀疑CGA是一种半抗原,但是本研究d-PLNA实验结果不支持这种结论。为进一步检测CGA的致敏潜能,并验证d-PLNA检测CGA,黄芩苷和连翘苷实验结果是否准确可靠,本研究以CGA为代表,利用流式细胞术和RA-PLNA进一步研究了CGA的致敏性。研究结果表明,CGA对BALB/c小鼠PLN细胞表型、激活标志物没有影响。RA-PLNA检测发现,无论与TNP-OVA还是与TNP-Ficoll一起注射,CGA均不能诱导TNP特异性AFCs形成,这表明CGA对BALB/c小鼠不具有致敏潜能。结果证实了d-PLNA和RA-PLNA结果一致。提示移除TCMI中的CGA或许不会显著降低其不良反应的发生,这或许有助于消除一些研究者对绿原酸全身暴露时安全性的担忧。
综上,本研究在国内首次成功建立了d-PLNA和RA-PLNA模型,并将之用于SHLI的致敏性研究；确认BALB/c小鼠和C57BL/6J小鼠为PLNA的首选敏感动物；研究结果证实,d-PLNA和RA-PLNA可用于SHLI及其有效组分或单体成分的致敏性研究；大剂量SHLI对小鼠具有辅助性炎症刺激作用,但不能激活抗原特异性的T淋巴细胞,表明SHLI的成分本身不能直接刺激BALB/c小鼠产生IgE介导的超敏反应,即不具有致敏性,提示IgE介导的超敏反应不是SHLI相关不良反应的主要发生机制,非抗原-抗体反应机制在其不良反应的发生、发展中起主导作用；这从另一个方面支持了我们实验室另一研究小组的结论：他们认为类过敏反应是SHLI临床不良反应发生的主要类型,SHLI直接刺激嗜碱性粒细胞或肥大细胞释放组胺等活性物质是其的主要机制。
Traditional Chinese medicine injection (TCMI), as a unique Chinese Medical formulation invented by our country and a developmental direction of the modernization of traditional Chinese medicine, has broken down the limitations of Traditional Chinese Medicine regiments. With features like high bioavailability and fast-acting, TCMI has already overcome the weakness of traditional Chinese medicine (cannot be used to cure the severe diseases) and played an important or even irreplaceable role in the treatment of conditions ranging from cardiovascular and cerebrovascular diseases, respiratory diseases and cancer. However, with the wide application of TCMI, the prevalence of adverse reactions related to TCMI appears to have increased significantly in China over the past ten years. Immediate hypersensitivity reactions (IHR), generally being considered as the most frequent and severe adverse reactions induced by TCMI, may cause anaphylactic shock or even be life-threatening. Those safety concerns caused a reduced use of TCMI and increasingly questioned its rationality, and thus ultimately lead to the "crisis of confidence" to the whole traditional Chinese medicine industry. In order to solve this crisis, the adverse reactions and risks induced by TCMI must be comprehensively, objectively and scientifically evaluated and the safety of clinical use should be ensured. Among those, the key point which needs to be further elucidated is to understand the cause and mechanisms of adverse reactions induced by TCMI. TCMI usually contains 3 to 7, sometimes even up to 12, kinds of raw material medicines. Thus TCMI does contain complex mixtures with a number of unknown constituent. Moreover the constituent that induced the adverse reactions and what mechanisms to be involved are poorly understood yet. Notably, all TCMIs approved for sale in the market and used in clinical are products which have been performed pre-clinical safety evaluations well. The results from safety evaluations showed that TCMIs did not induce any adverse reactions in animals. For this reason TCMIs were regarded as safe drugs in human, In fact, since it remains difficult for regulatory safety studies to predict the immunosensitizing potential of TCMI, there is poorly correlation between animal experiments results and clinical adverse reactions. Traditionally the animal model used to predict hypersensitivity reaction are the active cutaneous anaphylaxis assay (ACA), the passive cutaneous anaphylaxis assay (PCA) and the active systemic anaphylaxis assay (ASA), which are mainly used to detect the sensitization of macromolecular protein antigen. As far as low molecular weight compound(LMWC) is concerned, ASA, ACA, and PCA's positive results suggests that LMWC has immunosensitizing potential, but it is not always true for the negative results because of the possibility of false negative results in the test. In brief, the lack of suitable experimental models is the bottleneck that restricts the study on mechanism. Therefore, there is a great need for new animal models to study adverse reactions and mechanisms caused by TCMI prior to crisis of TCMI to be resolved.
Popliteal lymph node assay (PLNA) is regarded as a unique reliable and promising screening methods to indicate immunosensitization by LMWC. It is simple, time-saving, sensitive, high specific and with less uses of laboratory animals and samples. Five different versions of the PLNA have been proposed according to the different local treatments factors:the direct, secondary, adoptive, reporter antigen (RA) and Adjuvant PLNA. The direct PLNA (d-PLNA) and RA-PLNA are the most commonly used in drug immunosensitizing studies. Up to now, more than 130 LMWCs have been detected by PLNA in different laboratories and the findings showed good correlation, consistency and repeatability. However, existing research results are all about single-component LMWCs. it has not been reported that whether PLNA could be used to study the immunosensitizing potential of complex TCMI components. In addition, the standard system of PLNA has not been established at present, and there is no research report on the immunosesentizhing potential of chemicals or Chinese medicine allergens detected by PLNA in China. Moreover, animal species used in experiment reported in the foreign literatures, such as CBA mice, C57BL mice, F344 rats, and BN rats, were so expensive in China and not easily obtained. What's more, both the experience of animal's husbandry and the information of experimental background are scarce. For these reasons and combining the actual situation in our country, the aims of this study were to establish d-PLNA and RA-PLNA experimental model firstly; choose and define sensitive and appropriate animals from widely used laboratory animal species in China to define; secondly, establish a standardized, simple and feasible PLNA Experimental Operation procedures; thirdly, use d-PLNA and RA-PLNA methods that have been established to evaluate the immunosensitizing potential of TCMI and its single-components. We are trying to establish a special and sensitive method to evaluate the immunosensitizing potential of TCMI, and to clarify its mechanism and determine the allergen, which will provide new ideas and experimental evidences for TCMI safety evaluation, promote our immunosensitizing studies on drugs with international standards and have great economic and practical values on new drugs' R&D.
Shuanghuanglian injection (SHLI) is a representative of heat-clearing and detoxifying TCMI with a wide range of applications and good efficacy in clinical use. SHLI (lyophilized) was included in The People's Republic of China Pharmacopoeia Supplement (2005) in July,2009. It is the only TCMI that was put into the Pharmacopoeia and the first one to use fingerprint atlas to control its quality. However, just like other types of TCMIs, the reports of clinical adverse reactions related to SHLI are increasing. Analysis of data from the existing literatures report confirmed that IHR induced by SHLI or Houttuynia injection accounted for the most adverse reactions of all TCMI and came in the forefront. The adverse reaction induced by SHLI is allergy-based and the common symptoms are mild skin etthema and rash, etc.; serious adverse reactions include systemic damage, respiratory damage as the main ones. The main causes to death are anaphylactic shock and anaphylactoid reactions. National Adverse Drug Reaction Monitoring Center reported the hypersensitivity reactions related to SHLI twice in 2001 and 2009. This means that SHLI is also facing the danger of being eliminated from the market. SHLI was the first successful development of TCMI and was made up of three kinds of Chinese herbal medicines, including skullcap, honeysuckle and forsythia. Chlorogenic acid (CGA), which was highly suspected as a hapten, was rich in SHLI. In addition, the injection does not contain any excipient and the main component is LMWC. Therefore, allergens can be determined by studying the mechanisms of SHLI-induced allergic reactions through d-PLNA and RA-PLNA. This has great significance for SHLI as well as all TCMI in clinical drug safety.
1 Construction of animal models for popliteal lymph node assay
Specific pathogen-free female adult KM mice, BABL/c mice, C57BL/6J mice, Wistar rats and BN rats were randomly divided into Group Veh, negetive control group and model group. Animals were injected subcutaneously on the right hind footpad with Mercury chloride (HgCl2), Diclofenac sodium salt (DF), D-Penicillamine (D-pen), Diphenylhydantoin (DP) and Streptozotocin (STZ) in saline or 20%DMSO/saline. A volume of 25μl or 50μl was used for mice while 50μl or 100μl for rats. Phenobarbital (PB) was used for negative control. Normal animal were only injected on the right hind footpad with saline. After being injected for seven days, mice were sacrificed by inhalating CO2. Popliteal lymph node (PLN) was extracted and placed on ice in PBS-BSA (1%). Weighted PLN after the rid of adherent adipose tissues and being dried. Took preparation of single-cell suspension and got the weight index (WI= weight PLN treated side/weight PLN untreated side) and cellullarity index (CI). WI>2 or CI>5 is considered as a positive response. During the study, do the hypodermic on the toes of right hind leg with test material and TNP-OVA or TNP-Ficoll together for 7 days. After the administration, executed the animals and extracted the PLN for single cell susponsion. Use the Elispot method for testing the number of AFCs. The increase of AFCs is considered as a positive reaction. The study results showed that the C57BL/6J mice, BALB/c mice as well as BN rats could structure the d-PLNA models successfully. We found that mice only have one PLN on each side. PLN of mouse is easy to be located and removed than rat. So BABL/c mice and C57BL/6J are the preferred animals for PLNA. Based on those, we chose BABL/c mice to structure the RA-PLNA models and used on the following two SHLI studies.
2 Assessment of Immunosensitizing Potential of Shuanghuanglian Injection Using Direct Popliteal Lymph Node Assay in C57BL/6J Mice
Sixty C57BL/6J mice were randomly divided into 6 groups:vehicle (Veh), PB (1 mg/mouse), HgCl2 (50μg/mouse), D-pen(2 mg/mouse), SHLI low dose (1 mg/mouse) and high dose(5 mg/mouse),10 mice/group. Articles were injected subcutaneously into one hind foot pad of SPF female mice. On day 8 after injection, animals were sacrificed, the left (untreated side) and right (treated side) PLN were isolated, weighed respectively, and then PLN mass index (MI) was calculated. PLNs of 4 mice in each group were fixed in 4%paraformaldehyde solution, routinely stained with HE for histopathologic examination. The others (n=6) were prepared into single-cell suspensions, and then PLN cells index (CI) was calculated. PLN changes after administration of SHLI were observed and compared. We founded that MI and CI of SHLI treated group increased with the average MI≥2 and the average CI≥5,and the difference was significant when compared with group Veh (P<0.05) in C57BL/6J mice. Pathological examination showed that the volume of the right PLNs treated with SHLI became larger than those in left. Germinal center of PLN lymph was evident and vice cortex HEV cross-section increased. The results suggested that SHLI could induce significant PLN response that suggests it has the immuosensitizing potential.
3 Assessment of Immunosensitizing Potential of Shuanghuanglian Injection and its Refined products Prepared according to Chinese Pharmacopoeia Using Direct Popliteal Lymph Node Assay
Test articles, including 10 different pharmaceutical samples, were obtained from SHLI and its extracts after being refined by macroporous resin (D101).Randomly grouped the BALB/c mice and sterilized the right hind toes by alcohol. Use the insulin syringe or 1 ml syringe for injecting the sensitizer (D-pen), test material and dissolvent as the negative control. The treated volume of mice was 50μl. We used d-PLNA method to study on the reactions of the dose of 5 mg/mouse at first. At this moment, cut down the dose to 2.5mg/mouse when the sample had the PLNA positive reaction. When the negative reaction occurred, raise the dose to 10 mg/mouse. Dose-responses relationship could be studied through these methods. We found that SHLI prepared according to Pharmacopoeia procedure obtained positive results at high-dose, the rest did not respond; the refined products'PLNA reactions reduced. This suggested that the Pharmacopoeia procedure of SHLI was badly needed an improvement. What's more, the refined products were good in water-solubility and even the increase of dose wouldn't lead to positive reactions. This suggested that the adverse reactions may be induced by poor water-soluble components, In this sense, in order to study on the sensitized components of SHLI, new production procedure should be used for the separation of three medicinal compositions.
4 Assessment of Immunosensitizing Potential of Shuanghuanglian Injection Prepared by new Production Procedure Using Direct Popliteal Lymph Node Assay
d-PLNA was used for the assessment of immunosensitizing potential of 16 samples separated by new production procedure. The BALB/c mice were randomly grouped and the right hind toes were sterilized by 70%alcohol. The insulin syringe was used for injecting. The treated volume of mice was 50μl. The dose was 10 mg/mouse. For those constituents that were insoluble in 0.9%saline, were dissolved by pure DMSO of 10μl per mouse, and then diluted to the corresponding concentration with 0.9%saline for administration. For those compounds with PLNA positive results, kept on further reduction from 5 mg/mouse,2.5 mg/mouse or 0.25 mg/mouse to 0 mg/mouse for administration to get the dose-effect relationship. We found that 7/16 samples induced PLNA positive reactions at high dose (10 mg/animal); those suggested that those samples maybe contain harmful components. PLNA positive reactions caused by those components were found in the extracts of three herbs, including 2 extracts from honeysuckle,3 extract from Forsythia, and 2 extracts from skullcap, indicating that there may exist harmful components in all the 3 medicinal plants. Low dose (2.5 mg/animal) failed to induce the positive PLN reactions. Non-specific inflammatory stimulate may lead to d-PLNA false positive results, so further study on mechanism should be done for defining the sensitization of these compositions.
5 Evaluation of the Immunosensitizing Potential of Shuanghuanglian injection Using a Reporter Antigen Popliteal Lymph Node Assay in mice
The immunosensitizing mechanisms of SHLI were investigated using RA-PLNA in mice. The results showed that high-dose of SHLI combined with TNP-OVA (T cell dependent antigen) or TNP-Ficoll (T cell independent antigen) could induce PLN positive response (WI>2 or CI>5), but the intense of positive reaction were similar to those induced by high-dose of SHLI alone. Mixed with TNP-OVA, high-dose of SHLI could significantly increase the number of the TNP-specific antibody-forming cells (AFCs), but with TNP-Ficoll and while Low-dose of SHLI with 2 RA failed to do so. Those results suggested that high-dose-SHLI provided costimulatory signals which could induce inflammatory reaction and this effect would disappear at low dose. Moreover, it has been demonstrated via flow cytometry that high-dose-SHLI co-injected with TNP-OVA could activate macrophages in vivo. Activated macrophages could release cytokines which played an important role in induction of inflammation. However, the results did not support that SHLI activated neoantigen-specific T cells in BALB/c mice, which indicated that SHLI by itself do not have immunosesensitizing potential. Thus SHLI did not induced immune response resulting from specific antigen. As a result we postulated that adverse reactions of SHLI may be due to anaphylactoid reaction rather than IgE-mediated hypersensitivity reactions, which is congruent with conclusions obtained by other researchers in our laboratory.
6 Evaluation of the immunosensitizing potential of Monomer constituents in Shanghuanglian Injection
6.1 Evaluation of the immunosensitizing potential of Chlorogenic acid, Baicalin and Phillyrin Using Direct Popliteal Lymph Node Assay
The great value of d-PLNA has been proved by several labs in predicting the immunosensitizing potential of pure LMWC. However, the value of d-PLNA has not been adequately validated in predicting TCMI which contain complex mixtures with a number of unknown constituents. Therefore, it is more reliable to evaluate the monomer constituents contained in SHLI by using d-PLNA. CGA, Baicalin and phillyrin are main constituents of honeysuckle, skullcap and forsythia respectively, which are composed crude drug of SHLI. Most researchers thought that those monomer constituents are the basis of pharmacodynamics of SHLI. The others thought that they are related to ADR induced by SHLI. To determine whether the CGA, Baicalin and phillyrin are associated with ADR, we detected their immunosensitizing potential using d-PLNA firstly. The results found that a dose of 1 mg CGA, baicalin and phillyrin per animal failed to induce BALB/c mice d-PLNA positive reaction, suggesting that they do not have immunosensitizing potential.
6.2 Evaluation of the immunosensitizing potential of chlorogenic acid using a popliteal lymph node assay in BALB/c mice
It has yet to be established whether CGA, a common xenobiotic with potential exposure risk to humans, is associated with immune-mediated hypersensitivity reactions (HRs).Previous results from the d-PLNA demonstrated that CGA failed to induce positive response. To further determine whether CGA may possess an intrinsic capacity to stimulate or dysregulate immune responses, and if so, what mechanisms may be involved, we characterized the popliteal lymph node reaction induced by CGA in naive female BALB/c mice using both PLNA combined with flow cytomtry and RA-PLNA method. CGA was selected since many investigators postulated that it is a hapten. Another aim of present study is to validate the accuracy and the reliability of results using d-PLNA to predict the immunosensitizing potential of CGA, baicalin and phillyrin.Our results show that CGA failed to induce immunoreactivity following a single subcutaneous injection either alone or when combined with TNP-OVA or TNP-Ficoll. These results indicated that CGA lacks the intrinsic capacity to sensitize or stimulate immune responses in BALB/c mice. The results form RA-PLNA confirmed those from d-PLNA. Moreover, these results suggest that exposure to CGA may not represent a safety concern for humans and that removal of CGA from Traditional Chinese Medicine Injections may not significantly decrease the prevalence of HRs.
In conclusion, we successfully established models of d-PLNA and RA-PLNA, and confirmed BALB/c mice and C57 BL/6J mice as the preferred sensitive animals of both methods in our laboratory; d-PLNA and RA-PLNA are suitable methods to determine the immunosensitizing potential of SHLI and its constituents, which could also reveal the primary mechanism involved in adverse reaction related to SHLI. Our results showed that high-dose-SHLI could provide costimulatory signals to induce inflammatory reactions, but could not activate antigen-specific T-lymphocytes. Those results indicated that SHLI or its constituents do not have the immunosensitizing potential. Form the results, we concluded that SHLI by itself can not directly stimulate BALB/c mice to produce IgE-mediated hypersensitivity reactions. As a result we propose that ADR related to SHLI may be due to anaphylactoid reaction rather than IgE-mediated hypersensitivity reactions, which is congruent with conclusions obtained by other researchers in our laboratory. The release of other active mediators from basophils or mast cells directly stimulated by SHLI may also be the primary mechanism of ADR in human.
引文
1.周超凡,徐植灵,林育华.从药物组成看中药注射剂[J].中国中药杂志,2006,31(011)：950-952.
2.张玉生.双黄连注射剂药物不良反应的探讨和研究[D].山东大学；2009.
3.王立军,周学琴.我院405例药品不良反应报告分析[J].中国药房,2007,18(002)：137-138.
4.李荔,杨静.我院456例药物不良反应分析[J].中国新药杂志,2005,14(005)：642-644.
5.温泽淮,庾慧,伍耀衡,金卓祥,杨志敏,林琳,刘旭生,黄穗平,林华,许卫华,赖世隆,梁伟雄.中药注射剂不良反应监测的初步报告[J].中药新药与临床药理,2003,14(004)：278-281.
6.张惠霞,陈建玉,宋成.3414例中药注射剂不良反应分析[J].中国药物警戒,2006,3(004)：232-235.
7.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]. Chin J Evid-based Med,2010,10(2):162-175.
8.程芳.注射用双黄连类过敏反应及其机制研究[D].山东大学；2009.
9. De Week, A. Immunopathological mechanisms and clinical aspects of allergic reactions to drugs [J]. Handb. Exp. Pharmacol.,1983.
10. Simons, F.E., Frew, A.J., Ansotegui,I.J., Bochner, B.S., Golden, D.B., Finkelman, F.D., Leung, D.Y., Lotvall, J., Marone, G, Metcalfe, D.D., Muller, U., Rosenwasser, L.J., Sampson, H.A., Schwartz,L.B., van Hage, M, Walls, A.F. Risk assessment in anaphylaxis:current and future approaches [J]. J Allergy Clin Immunol,2007,120(1 Suppl):S2-24.
11. FDA:Guidance for Industry Immunotoxicology Evaluation of Investigational New Drugs[S].2002,1-38. [cited:2010,3];Available from: http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/ Guidances/UCM079239.pdf.
12.食品药品监督管理局(SFDA):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[S].2005:1-28.[cited:2010,3]Available from: http://zq.gdda.gov.cn/publicfiles/business/cmsresources/gddec/cmsrsdocument/doc7 575.pdf
13.食品药品监督管理局(SFDA):化学药物刺激性、过敏性和溶血性研究技术指导原则[S].2005:1-30. [cited:2010,3];Available from: http://www.sda.gov.cn/gsz05106 /14.pdf
14. Tuschl, H., H.T. Landsteiner, and R. Kovac. 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.
15. Lovik, M., Alberg,T., Nygaard,U.C., Samuelsen, M., Groeng, E.C., Gaarder, P.I. Popliteal lymph node (PLN) assay to study adjuvant effects on respiratory allergy[J]. Methods,2007,41(1):72-79.
16. Pieters, R. Detection of autoimmunity by pharmaceuticals[J]. Methods,2007, 41(1):112-7.
17. Kamala, T. Hock immunization:a humane alternative to mouse footpad injections[J]. J Immunol Methods,2007,328(1-2):204-14.
18. Pieters, R. The popliteal lymph node assay in predictive testing for autoimmunity [J]. Toxicology Letters,2000,112:453-459.
19. Carey, J.B., A. Allshire, and F.N. van Pelt. Immune modulation by cadmium and lead in the acute reporter antigen-popliteal lymph node assay [J]. Toxicol Sci,2006, 91(1):113-22.
20. Gutting, B.W., Schomaker, S.J., Kaplan, A.H., Amacher, D.E. 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):71-9.
21. Nierkens, S., Pieters, R. The Reporter Antigen Popliteal Lymph Node Assay, in Curr. Protoc. Toxicol.2006. [cited:2010,3];Available from:http://mrw.interscience.wiley.com/ emrw/9780471140856/home.
22. Goyer, R.and W. Clarkson. Cassarett and Doull's. Toxicology:The basic science of poisons[M].2001,107(4).
23. Gad, S. Drug safety evaluation[M]. Wiley-Interscience;2002.
24. Kemp, S. Office approach to anaphylaxis:sooner better than later[J], The American Journal of Medicine,2007,120(8):664-668.
25. Ratajczak, H. Drug-induced hypersensitivity:role in drug development[J]. Toxicological reviews,2004,23(4):265-280.
26. Aida, T., Kimura, T., Ishikawa, N., Shinkai, K. Evaluation of allergenic potential of low-molecular compounds by mouse popliteal lymph node assay[J]. J Toxicol Sci,1998,23(5):425-32.
27. Aida, T., N. Ishikawa, and K. Shinkai. Sex differences in immune responses to cephalothin in guinea pigs [J]. J Toxicol Sci,1998,23(1):87-91.
28. Bloksma, N., Kubicka-Muranyi, M., Schuppe, H.C., Gleichmann, E., Gleichmann H. Predictive immunotoxicological test systems:suitability of the popliteal lymph node assay in mice and rats[J]. Crit Rev Toxicol,1995, 25(5):369-96.
29. Spanhaak, S.The ICH S8 immunotoxicity guidance. Immune function assessment and toxicological pathology:Autonomous or synergistic methods to predict immunotoxicity?[J]. Exp Toxicol Pathol.,2006,57(5-6):373-376.
30. van Loveren, H., Cockshott A, Gebel T, Gundert-Remy U, de Jong WH, Matheson J, McGarry H, Musset L, Selgrade MK, Vickers C. Skin sensitization in chemical risk assessment:Report of a WHO/IPCS international workshop focusing on dose-response assessmen[J]. Regul Toxicol Pharmacol.,2008,50(2):155-162.
31. Gerberick, G.F., Ryan, C.A., Dearman, R.J., Kimber, I. Local lymph node assay (LLNA) for detection of sensitization capacity of chemicals[J]. Methods,2007, 41(1):54-60.
32. Goebel, C, Griem P, Sachs B, Bloksma N, Gleichmann E. The popliteal lymph node assay in mice:screening of drugs and other chemicals for immunotoxic hazard [J]. Inflamm Res,1996,45 Suppl 2:S85-90.
33. Pichler, W.J. Immune mechanism of drug hypersensitivity [J]. Immunol Allergy Clin North Am.,2004,24(3):373-97.
34. Gleichmann, H. Studies on the mechanism of drug sensitization: T-cell-dependent popliteal lymph node reaction to diphenylhydantoin[J]. Clin Immunol Immunopathol, 1981,18(2):203-11.
35. Ravel, G, Descotes, J. Popliteal lymph node assay:facts and perspectives [J]. J Appl Toxicol,2005,25(6):451-8.
36. Gutting, B.W., Bouzahzah, F., Kong,P.L., Updyke LW, Amacher, D.E., Craft, J. Oxazolone and diclofenac-induced popliteal lymph node assay reactions are attenuated in mice orally pretreated with the respective compound:potential role for the induction of regulatory mechanisms following enteric administration[J]. Toxicol Appl Pharmacol,2003,189(2):120-33.
37. Nierkens, S.andR. Pieters. Murine models of drug hypersensitivity[J]. Curr Opin Allergy Clin Immunol,2005,5(4):331-5.
38. Pieters, R.and Albers, R. Screening tests for autoimmune-related immunotoxicity[J]. Environ Health Perspect,1999,107 Suppl 5:673-7.
39. Uetrecht, J. Idiosyncratic drug reactions:current understanding[J]. Annu Rev Pharmacol Toxicol,2007,47:513-39.
40. Pieters, R. The popliteal lymph node assay:a tool for predicting drug allergies[J]. Toxicology,2001,158(1-2):65-9.
41. Roujeau, J.C. Immune mechanisms in drug allergy[J]. Allergol Int,2006, 55(1):27-33.
42. Cavani, A.and O. De Pita. The role of T cells in drug reaction[J]. Curr Allergy Asthma Rep,2006,6(1):20-4.
43. Parker, C.W. Allergic reactions in man[J]. Pharmacol Rev,1982,34(1):85-104.
44. Pichler, W.J. Immune mechanism of drug hypersensitivity[J]. Immunol Allergy Clin North Am,2004,24(3):373-97, ⅴ-ⅵ.
45. Schnyder, B., Mauri-Hellweg, D., Zanni, M., Bettens, F. Pichler, W.J. Direct, MHC-dependent presentation of the drug sulfamethoxazole to human alphabeta T cell clones[J]. J Clin Invest,1997,100(1):136-41.
46. Zanni, M.P., von Greyerz, S., Schnyder, B., Wendland, T., Pichler, W.J. Allele-unrestricted presentation of lidocaine by HLA-DR molecules to specific alphabeta+T cell clones[J]. Int Immunol,1998,10(4):507-15.
47. Naisbitt, D.J., Farrell,J., Wong,G, Depta, J.P., Dodd, C.C., Hopkins, J.E., Gibney, C.A., Chadwick, D.W., Pichler, W.J., Pirmohamed, M., Park, B.K. Characterization of drug-specific T cells in lamotrigine hypersensitivity[J]. J Allergy Clin Immunol,2003,111(6):1393-403.
48. Lehmann, P.V., Sercarz, E.E., Forsthuber,T., Dayan, C.M., Gammon,G. Determinant spreading and the dynamics of the autoimmune T-cell repertoire [J]. Immunol Today,1993,14(5):203-8.
49. Akbari, O., Stock,P., DeKruyff, R.H., Umetsu, D.T.Role of regulatory T cells in allergy and asthma[J]. Curr Opin Immunol,2003,15(6):627-33.
50. Nierkens, S., Aalbers, M., Bleumink, R., Boon, L., Pieters, R. Drug-induced type 1 and type 2 immune responses are characterized by distinct profiles of cell kinetics, cytokine production, and expression of co-stimulatory molecules in the popliteal lymph node assay[J]. J Immunotoxicol,2005,2(3):141-50.
51. Uetrecht, J. Idiosyncratic drug reactions:past, present, and future[J]. Chem Res Toxicol,2008,21(1):84-92.
52.孙炯.应用胭窝淋巴结实验评价干扰素的免疫毒性[J].中国药理学通报,2005,21(8).
53. Friedrich, K., Delgado, I.F., Santos, L.M., Paumgartten, F.J. Assessment of sensitization potential of monoterpenes using the rat popliteal lymph node assay [J]. Food Chem Toxicol.,2007,45(8):1516-1522.
54. Fernandez Cabezudo, M.J., Petroianu, G,. Al-Ramadi, B., Langer, R.D. Iosimenol, a new non-ionic dimeric contrast medium, does not induce immunoreactivity in the popliteal lymph node assay [J]. Br J Radiol,2007, 80(957):713-8.
55. Shinkai, K., Nakamura, K., Tsutsui, N., Kuninishi, Y., Iwaki, Y., Nishida, H., Suzuki, R., Vohr, H.W., Takahashi, M., Takahashi, K., Kamimura, Y, Maki, E. 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.
56. Albers, R., Broeders, A., van der Pijl, A., Seinen, W., Pieters, R. The use of reporter antigens in the popliteal lymph node assay to assess immunomodulation by chemicals[J]. Toxicol Appl Pharmacol,1997,143(1):102-9.
57. Gutting, B.W., Updyke, L.W., Amacher, D.E. 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):177-83.
58. 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):167-80.
59. Goud, S.N., Muthusamy, N., Subbarao, B. Differential responses of B cells from the spleen and lymph node to TNP-Ficoll[J]. J Immunol,1988,140(9):2925-30.
60. Nierkens, S., Nieuwenhuijsen, L., Thomas, M., Pieters, R.Evaluation of the use of reporter antigens in an auricular lymph node assay to assess the immunosensitizing potential of drugs[J]. Toxicol Sci,2004,79(1):90-7.
61. Carey, J., Allshire, A., van Pelt, F. Immune modulation by cadmium and lead in the acute reporter antigen-popliteal lymph node assay [J]. Toxicological Sciences,2006,91(1):113.
62. Lovik, M., H(?)gseth, A,K., Gaarder, P.I., Hagemann, R., Eide, I. Diesel exhaust particles and carbon black have adjuvant activity on the local lymph node response and systemic IgE production to ovalbumin[J]. Toxicology,1997,121(2):165-78.
63. Choquet-Kastylevsky, G., Descotes, J. Popliteal lymph node responses to acetone and ethanol differ from those induced by streptozotocin[J]. Arch Toxicol,2004,78(11):649-54.
64. Verdier, R, Virat, M., Descotes, J. Applicability of the popliteal lymph node assay in the Brown-Norway rat[J]. Immunopharmacol Immunotoxicol,1990, 12(4):669-77.
65. Choquet-Kastylevsky, G, Tedone, R., Ducluzeau, M.T., Kehren, J., Nicolas, J. F., Descotes, J. The popliteal lymph node response to streptozotocin is under type 1, MHC class-I restricted, CD8(+) T-cell control[J]. Toxicology,2000,146(1):73-82.
66. Krzystyniak, K., Brouland, J,P., Panaye, G., Patriarca, C., Verdier, F., Descotes, J., Revillard, J.P. Activation of CD4+and CD8+lymphocyte subsets by streptozotocin in murine popliteal lymph node (PLN) test[JJ. J Autoimmun,1992, 5(2):183-97.
67.袁洞君,何周康.双黄连注射剂在临床上的应用[J].中医药导报,2006,12(005)：101-102.
68.徐生波,魏中银,程百学,黄福祥,吕风成,郭祁伟.双黄连和利巴韦林联合治疗流行性乙型脑炎疗效观察[J].中国误诊学杂志,2006,6(009)：1691-1692.
69.徐开伦,梁传余,戚晓清,张剑,任贤灵,邓世明,罗通勇.内镜鼻窦术后应用双黄连液冲洗鼻腔的疗效观察[J].临床耳鼻咽喉科杂志,2005,19：23.
70.李秋,王珊,黄素芳.双黄连注射液治疗婴幼儿喘憋性肺炎疗效观察[J].时珍国医国药,2005,16(006)：569-570.
71.曾国萍,黎金玲.双黄连注射剂对急性上呼吸道感染的疗效观察[J].武警医学院学报,2008,17(010)：888-889.
72.黄智芬,黎汉忠,张作军,谭志强,陈闯,陈蔚.注射用双黄连(冻干)合注射用丹参(冻干)治疗老年晚期非小细胞肺癌并发肺部感染30例[J].河北中医,2009,31(003)：431-433.
73.旷飒飒,刘玲忠.LEEP刀联合双黄连粉针剂治疗重度宫颈糜烂疗效分析[J].医学临床研究,2009,26(007)：1288-1289.
74.刘剑英,高丽丽.注射用双黄连粉针剂药理作用概况[J].中外医疗,2009,28(001)：84-84.
75.赵海东.双黄连粉针和水针临床应用不良反应比较[J].现代医药卫生,2008(8)：1219-1219.
76.吴嘉瑞,张冰.双黄连注射剂不良反应文献的数据挖掘研究[J].中国药物警戒,2008,5(003)：139-143.
77.吴嘉瑞,张冰.双黄连注射剂不良反应文献病案的系统研究[J].中成药,2008,30(005)：640-643.
78.袁迎.清开灵,双黄连注射剂不良反应病例分析[J].陕西中医,2009,(007)：892-893.
79.梁进权,邹元平,邓响潮.中药注射剂不良反应的文献调查与分析[J].中国医院药学杂志,2003,23(008)：486-488.
80.郑加嘉,杨小娟.注射用双黄连2176例临床用药不良反应分析[J].中国实用医药,2006,1(001)：105-107
81.赖宇红,杨卫荣.中药注射剂变态反应研究亟待加强[J].中药新药与临床药理,2002,13(005)：324-326.
82.刘红.双黄连注射液的不良反应[J].中华医学实践杂志,2005,4(6)41-2.
83. Freedman, S., Krupey, J., Sehon,A. Chlorogenic acid:an allergen in green coffee bean[J]. Nature (Inglaterra),1961,192(4799):241-243.
84. Freedman, S., Siddiqi, A.I., Krupey, J.H., Sehon, A.H. Identification of a simple chemical compound (chlorogenic acid) as an allergen in plant materials causing human atopic disease[J]. Am J Med Sci.,1962,244:548.
85. Freedman, S., Shulman, R., Krupey, J. Loss of allergenicity of chlorogenic acid in the gastrointestinal tract[J]. J Allergy Clin Immunol.,1964,35(2):108-116.
86. Freedman, S., Shulman, R., Krupey, J., Sehon, A.H. Antigenic properties of chlorogenic acid[J]. J Allergy Clin Immunol.,1964,35(2):97-107.
87. Bariana, D., Krupey, J., Scarpati, L.M., Freedman, S.O., Sehon, A.H. Chlorogenic acid:further evidence for its antigenic and allergenic activity[J]. Nature,1965,207(5002):1155.
88.黄芳华.绿原酸及其中药注射剂的安全性问题状况分析[J].中国中药杂志,2008,33(022)：2716-2719.
89.李钦,张信岳,陈国神.含绿原酸的清热解毒类中药注射剂不良反应及其机理探讨[J].中国现代应用药学,2009：555-558.
90.童路.双黄连注射剂的不良反应与成分间的关系[J].中成药,1997,19(004)：47-48.
91.田锋奇.黄芩苷的致过敏作用机制[D].郑州大学；2007.
92.赵新先.中药注射剂学[M].广州：广东科技出版社；2000.
93.邓良,袁华,喻宗沅.绿原酸的研究进展[J].化学与生物工程,2005,22(007)：4-6.
94.张鞍灵,马琼.绿原酸及其类似特与生物活性[J].中草药,2001,32(002)：173-176.
95.张建春,张华,施瑛,胡浩.黄芩苷的研究近况[J].时珍国医国药,2005,16(003)：247-249.
96.罗飞,包旭,林大胜,杨华蓉,周莎,徐小平.绿原酸对动物的致敏性研究[J].华西药学杂志,2009,24(002)：181-183.
97.李瑾翡,黎旸,陈琪,程朝辉,谭舜.注射用双黄连的免疫毒性研究[J].中药新药与临床药理,2008,19(003)：172-174.
98. Bala, S., Weaver, J., Hastings, K.L. Clinical relevance of preclinical testing for allergic side effects[J]. Toxicology,2005,209(2):195-200
99. Weaver, J.L., Staten, D., Swann, J., Armstrong, G,. Bates, M., Hastings, K.L. Detection of systemic hypersensitivity to drugs using standard guinea pig assays[J]. Toxicology,2003,193(3):203-17.
100. Ju, C, Uetrecht,J.P. Mechanism of idiosyncratic drug reactions:reactive metabolite formation, protein binding and the regulation of the immune system[J]. Curr Drug Metab,2002,3(4):367-77.
101. Brouland, J.P., Verdier, F., Patriarca, C., Vial, T., Descotes, J.Morphology of popliteal lymph node responses in Brown-Norway rats[J]. J Toxicol Environ Health,1994,41(1):95-108.
102. De Bakker, J.M., Kammiil ler, M. E., Muller, E. S. M., Lam, A. W., Seinen, W. Bloksma, N. Kinetics and morphology of chemically induced popliteal lymph node reactions compared with antigen-, mitogen-, and graft-versus-host-reaction-induced responses[J]. Virchows Arch B Cell Pathol Incl Mol Pathol,1990,58(4):279-87.
103. Koch, E., Jaggy, H., Chatterjee,S.S.. Evidence for immunotoxic effects of crude Ginkgo biloba L. leaf extracts using the popliteal lymph node assay in the mouse[J]. Int J Immunopharmacol,2000,22(3):229-36.
104.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.
105.谢华,何韶衡.哮喘中IgE依赖性和非IgE依赖性肥大细胞激活的研究[J].汕头大学医学院学报,2003,16(001)：12-15.
106. Findlay, S., Dvorak, A.M., Kagey-Sobotka, A., Lichtenstein, L.M. Hyperosmolar triggering of histamine release from human basophils[J]. J Clin Invest.,1981,67(6):1604-13.
107.王玉俊,杨卫华,万年红,张志萍.荨麻疹患者血清变应原特异性免疫球蛋白E检测分析[J].临床荟萃,2005,20(022)：1310-1311.
108. de Weck, A.L., Gamboa, P. M., Esparza, R., Sanz, M. L. Hypersensitivity to aspirin and other nonsteroidal anti-inflammatory drugs (NSAIDs)[J]. Curr Pharm Des,2006,12(26):3347-58.
109. Szebeni, J. Complement activation-related pseudoallergy:a new class of drug-induced acute immune toxicity[J]. Toxicology,2005,216(2-3):106-21.
110. Zebeni, J. Complement activation-related pseudoallergy caused by amphiphilic drug carriers:the role of lipoproteins[J]. Curr Drug Deliv,2005,2(4):443-9.
111.徐国良,张卓辉,张增珠,张启云.双黄连注射剂过敏症动物模型的研究[J].中国临床药理学与治疗学,2008,13(002)：154-157.
112. Chen, X.F.,, Lin, J.M., Zheng, L.P. The Effect of Shuanghuanglian Injection on Elevating of Blood Serum Histamine in Mice [J]. J Fujian College Tradit Chine Med,2005.
113吴友苹,徐潘生,卢觅佳,宣尧仙.鱼腥草注射液Ⅰ型过敏反应试验[J].时珍国医国药,2009,20(6).
114 J. zhang, Y.-K.L., L.-L. Li, W. Hao, W.-J Yan. sensitization of beagle dogs to Houttuynia cordata distillation mixed with three solubilizers [J]. Chin J New Drugs,2008,17(17):1494-1498
115. Ezendam, J., Staedtler, F., Pennings, J., Vandebriel, R.J., Pieters, R., Harleman, J.H., Vos, J.G. Toxicogenomics of subchronic hexachlorobenzene exposure in Brown Norway rats [J]. Environ Health Perspect,2004,112(7):782.
116.吴梅,闫慧,张春梅.黄芩苷作为一种抗生物制剂的过敏反应的研究[J].微生物学免疫学进展,2007,4(4).
117.杜国安,李烈,卢素国.常用中药注射液静脉滴注临床不良反应分析[J].时珍国医国药,2006,4(9).
118. Ko, R. A US perspective on the adverse reactions from traditional Chinese medicines[J]. J CHIN MED ASSOCIATION,2004,67(3):109-116.
119. Gong, J., F.T. Liu, and S.S. Chen. Polyphenolic antioxidants enhance IgE production[J]. Immunol Invest,2004,33(3):295-307.
120. Kang, J., Liu, Y., Meng, X. X., Song, L., Jiang, M.,Wang,Y.D. Interactions of human serum albumin with chlorogenic acid and ferulic acid[J]. Biochimica Et BiophysicaActa-General Subjects,2004,1674(2):205-214.
2.张玉生.双黄连注射剂药物不良反应的探讨和研究[D].山东大学；2009.
3.王立军,周学琴.我院405例药品不良反应报告分析[J].中国药房,2007,18(002)：137-138.
4.李荔,杨静.我院456例药物不良反应分析[J].中国新药杂志,2005,14(005)：642-644.
5.温泽淮,庾慧,伍耀衡,金卓祥,杨志敏,林琳,刘旭生,黄穗平,林华,许卫华,赖世隆,梁伟雄.中药注射剂不良反应监测的初步报告[J].中药新药与临床药理,2003,14(004)：278-281.
6.张惠霞,陈建玉,宋成.3414例中药注射剂不良反应分析[J].中国药物警戒,2006,3(004)：232-235.
7.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]. Chin J Evid-based Med,2010,10(2):162-175.
8.程芳.注射用双黄连类过敏反应及其机制研究[D].山东大学；2009.
9. De Week, A. Immunopathological mechanisms and clinical aspects of allergic reactions to drugs [J]. Handb. Exp. Pharmacol.,1983.
10. Simons, F.E., Frew, A.J., Ansotegui,I.J., Bochner, B.S., Golden, D.B., Finkelman, F.D., Leung, D.Y., Lotvall, J., Marone, G, Metcalfe, D.D., Muller, U., Rosenwasser, L.J., Sampson, H.A., Schwartz,L.B., van Hage, M, Walls, A.F. Risk assessment in anaphylaxis:current and future approaches [J]. J Allergy Clin Immunol,2007,120(1 Suppl):S2-24.
11. FDA:Guidance for Industry Immunotoxicology Evaluation of Investigational New Drugs[S].2002,1-38. [cited:2010,3];Available from: http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/ Guidances/UCM079239.pdf.
12.食品药品监督管理局(SFDA):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[S].2005:1-28.[cited:2010,3]Available from: http://zq.gdda.gov.cn/publicfiles/business/cmsresources/gddec/cmsrsdocument/doc7 575.pdf
13.食品药品监督管理局(SFDA):化学药物刺激性、过敏性和溶血性研究技术指导原则[S].2005:1-30. [cited:2010,3];Available from: http://www.sda.gov.cn/gsz05106 /14.pdf
14. Tuschl, H., H.T. Landsteiner, and R. Kovac. 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.
15. Lovik, M., Alberg,T., Nygaard,U.C., Samuelsen, M., Groeng, E.C., Gaarder, P.I. Popliteal lymph node (PLN) assay to study adjuvant effects on respiratory allergy[J]. Methods,2007,41(1):72-79.
16. Pieters, R. Detection of autoimmunity by pharmaceuticals[J]. Methods,2007, 41(1):112-7.
17. Kamala, T. Hock immunization:a humane alternative to mouse footpad injections[J]. J Immunol Methods,2007,328(1-2):204-14.
18. Pieters, R. The popliteal lymph node assay in predictive testing for autoimmunity [J]. Toxicology Letters,2000,112:453-459.
19. Carey, J.B., A. Allshire, and F.N. van Pelt. Immune modulation by cadmium and lead in the acute reporter antigen-popliteal lymph node assay [J]. Toxicol Sci,2006, 91(1):113-22.
20. Gutting, B.W., Schomaker, S.J., Kaplan, A.H., Amacher, D.E. 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):71-9.
21. Nierkens, S., Pieters, R. The Reporter Antigen Popliteal Lymph Node Assay, in Curr. Protoc. Toxicol.2006. [cited:2010,3];Available from:http://mrw.interscience.wiley.com/ emrw/9780471140856/home.
22. Goyer, R.and W. Clarkson. Cassarett and Doull's. Toxicology:The basic science of poisons[M].2001,107(4).
23. Gad, S. Drug safety evaluation[M]. Wiley-Interscience;2002.
24. Kemp, S. Office approach to anaphylaxis:sooner better than later[J], The American Journal of Medicine,2007,120(8):664-668.
25. Ratajczak, H. Drug-induced hypersensitivity:role in drug development[J]. Toxicological reviews,2004,23(4):265-280.
26. Aida, T., Kimura, T., Ishikawa, N., Shinkai, K. Evaluation of allergenic potential of low-molecular compounds by mouse popliteal lymph node assay[J]. J Toxicol Sci,1998,23(5):425-32.
27. Aida, T., N. Ishikawa, and K. Shinkai. Sex differences in immune responses to cephalothin in guinea pigs [J]. J Toxicol Sci,1998,23(1):87-91.
28. Bloksma, N., Kubicka-Muranyi, M., Schuppe, H.C., Gleichmann, E., Gleichmann H. Predictive immunotoxicological test systems:suitability of the popliteal lymph node assay in mice and rats[J]. Crit Rev Toxicol,1995, 25(5):369-96.
29. Spanhaak, S.The ICH S8 immunotoxicity guidance. Immune function assessment and toxicological pathology:Autonomous or synergistic methods to predict immunotoxicity?[J]. Exp Toxicol Pathol.,2006,57(5-6):373-376.
30. van Loveren, H., Cockshott A, Gebel T, Gundert-Remy U, de Jong WH, Matheson J, McGarry H, Musset L, Selgrade MK, Vickers C. Skin sensitization in chemical risk assessment:Report of a WHO/IPCS international workshop focusing on dose-response assessmen[J]. Regul Toxicol Pharmacol.,2008,50(2):155-162.
31. Gerberick, G.F., Ryan, C.A., Dearman, R.J., Kimber, I. Local lymph node assay (LLNA) for detection of sensitization capacity of chemicals[J]. Methods,2007, 41(1):54-60.
32. Goebel, C, Griem P, Sachs B, Bloksma N, Gleichmann E. The popliteal lymph node assay in mice:screening of drugs and other chemicals for immunotoxic hazard [J]. Inflamm Res,1996,45 Suppl 2:S85-90.
33. Pichler, W.J. Immune mechanism of drug hypersensitivity [J]. Immunol Allergy Clin North Am.,2004,24(3):373-97.
34. Gleichmann, H. Studies on the mechanism of drug sensitization: T-cell-dependent popliteal lymph node reaction to diphenylhydantoin[J]. Clin Immunol Immunopathol, 1981,18(2):203-11.
35. Ravel, G, Descotes, J. Popliteal lymph node assay:facts and perspectives [J]. J Appl Toxicol,2005,25(6):451-8.
36. Gutting, B.W., Bouzahzah, F., Kong,P.L., Updyke LW, Amacher, D.E., Craft, J. Oxazolone and diclofenac-induced popliteal lymph node assay reactions are attenuated in mice orally pretreated with the respective compound:potential role for the induction of regulatory mechanisms following enteric administration[J]. Toxicol Appl Pharmacol,2003,189(2):120-33.
37. Nierkens, S.andR. Pieters. Murine models of drug hypersensitivity[J]. Curr Opin Allergy Clin Immunol,2005,5(4):331-5.
38. Pieters, R.and Albers, R. Screening tests for autoimmune-related immunotoxicity[J]. Environ Health Perspect,1999,107 Suppl 5:673-7.
39. Uetrecht, J. Idiosyncratic drug reactions:current understanding[J]. Annu Rev Pharmacol Toxicol,2007,47:513-39.
40. Pieters, R. The popliteal lymph node assay:a tool for predicting drug allergies[J]. Toxicology,2001,158(1-2):65-9.
41. Roujeau, J.C. Immune mechanisms in drug allergy[J]. Allergol Int,2006, 55(1):27-33.
42. Cavani, A.and O. De Pita. The role of T cells in drug reaction[J]. Curr Allergy Asthma Rep,2006,6(1):20-4.
43. Parker, C.W. Allergic reactions in man[J]. Pharmacol Rev,1982,34(1):85-104.
44. Pichler, W.J. Immune mechanism of drug hypersensitivity[J]. Immunol Allergy Clin North Am,2004,24(3):373-97, ⅴ-ⅵ.
45. Schnyder, B., Mauri-Hellweg, D., Zanni, M., Bettens, F. Pichler, W.J. Direct, MHC-dependent presentation of the drug sulfamethoxazole to human alphabeta T cell clones[J]. J Clin Invest,1997,100(1):136-41.
46. Zanni, M.P., von Greyerz, S., Schnyder, B., Wendland, T., Pichler, W.J. Allele-unrestricted presentation of lidocaine by HLA-DR molecules to specific alphabeta+T cell clones[J]. Int Immunol,1998,10(4):507-15.
47. Naisbitt, D.J., Farrell,J., Wong,G, Depta, J.P., Dodd, C.C., Hopkins, J.E., Gibney, C.A., Chadwick, D.W., Pichler, W.J., Pirmohamed, M., Park, B.K. Characterization of drug-specific T cells in lamotrigine hypersensitivity[J]. J Allergy Clin Immunol,2003,111(6):1393-403.
48. Lehmann, P.V., Sercarz, E.E., Forsthuber,T., Dayan, C.M., Gammon,G. Determinant spreading and the dynamics of the autoimmune T-cell repertoire [J]. Immunol Today,1993,14(5):203-8.
49. Akbari, O., Stock,P., DeKruyff, R.H., Umetsu, D.T.Role of regulatory T cells in allergy and asthma[J]. Curr Opin Immunol,2003,15(6):627-33.
50. Nierkens, S., Aalbers, M., Bleumink, R., Boon, L., Pieters, R. Drug-induced type 1 and type 2 immune responses are characterized by distinct profiles of cell kinetics, cytokine production, and expression of co-stimulatory molecules in the popliteal lymph node assay[J]. J Immunotoxicol,2005,2(3):141-50.
51. Uetrecht, J. Idiosyncratic drug reactions:past, present, and future[J]. Chem Res Toxicol,2008,21(1):84-92.
52.孙炯.应用胭窝淋巴结实验评价干扰素的免疫毒性[J].中国药理学通报,2005,21(8).
53. Friedrich, K., Delgado, I.F., Santos, L.M., Paumgartten, F.J. Assessment of sensitization potential of monoterpenes using the rat popliteal lymph node assay [J]. Food Chem Toxicol.,2007,45(8):1516-1522.
54. Fernandez Cabezudo, M.J., Petroianu, G,. Al-Ramadi, B., Langer, R.D. Iosimenol, a new non-ionic dimeric contrast medium, does not induce immunoreactivity in the popliteal lymph node assay [J]. Br J Radiol,2007, 80(957):713-8.
55. Shinkai, K., Nakamura, K., Tsutsui, N., Kuninishi, Y., Iwaki, Y., Nishida, H., Suzuki, R., Vohr, H.W., Takahashi, M., Takahashi, K., Kamimura, Y, Maki, E. 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.
56. Albers, R., Broeders, A., van der Pijl, A., Seinen, W., Pieters, R. The use of reporter antigens in the popliteal lymph node assay to assess immunomodulation by chemicals[J]. Toxicol Appl Pharmacol,1997,143(1):102-9.
57. Gutting, B.W., Updyke, L.W., Amacher, D.E. 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):177-83.
58. 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):167-80.
59. Goud, S.N., Muthusamy, N., Subbarao, B. Differential responses of B cells from the spleen and lymph node to TNP-Ficoll[J]. J Immunol,1988,140(9):2925-30.
60. Nierkens, S., Nieuwenhuijsen, L., Thomas, M., Pieters, R.Evaluation of the use of reporter antigens in an auricular lymph node assay to assess the immunosensitizing potential of drugs[J]. Toxicol Sci,2004,79(1):90-7.
61. Carey, J., Allshire, A., van Pelt, F. Immune modulation by cadmium and lead in the acute reporter antigen-popliteal lymph node assay [J]. Toxicological Sciences,2006,91(1):113.
62. Lovik, M., H(?)gseth, A,K., Gaarder, P.I., Hagemann, R., Eide, I. Diesel exhaust particles and carbon black have adjuvant activity on the local lymph node response and systemic IgE production to ovalbumin[J]. Toxicology,1997,121(2):165-78.
63. Choquet-Kastylevsky, G., Descotes, J. Popliteal lymph node responses to acetone and ethanol differ from those induced by streptozotocin[J]. Arch Toxicol,2004,78(11):649-54.
64. Verdier, R, Virat, M., Descotes, J. Applicability of the popliteal lymph node assay in the Brown-Norway rat[J]. Immunopharmacol Immunotoxicol,1990, 12(4):669-77.
65. Choquet-Kastylevsky, G, Tedone, R., Ducluzeau, M.T., Kehren, J., Nicolas, J. F., Descotes, J. The popliteal lymph node response to streptozotocin is under type 1, MHC class-I restricted, CD8(+) T-cell control[J]. Toxicology,2000,146(1):73-82.
66. Krzystyniak, K., Brouland, J,P., Panaye, G., Patriarca, C., Verdier, F., Descotes, J., Revillard, J.P. Activation of CD4+and CD8+lymphocyte subsets by streptozotocin in murine popliteal lymph node (PLN) test[JJ. J Autoimmun,1992, 5(2):183-97.
67.袁洞君,何周康.双黄连注射剂在临床上的应用[J].中医药导报,2006,12(005)：101-102.
68.徐生波,魏中银,程百学,黄福祥,吕风成,郭祁伟.双黄连和利巴韦林联合治疗流行性乙型脑炎疗效观察[J].中国误诊学杂志,2006,6(009)：1691-1692.
69.徐开伦,梁传余,戚晓清,张剑,任贤灵,邓世明,罗通勇.内镜鼻窦术后应用双黄连液冲洗鼻腔的疗效观察[J].临床耳鼻咽喉科杂志,2005,19：23.
70.李秋,王珊,黄素芳.双黄连注射液治疗婴幼儿喘憋性肺炎疗效观察[J].时珍国医国药,2005,16(006)：569-570.
71.曾国萍,黎金玲.双黄连注射剂对急性上呼吸道感染的疗效观察[J].武警医学院学报,2008,17(010)：888-889.
72.黄智芬,黎汉忠,张作军,谭志强,陈闯,陈蔚.注射用双黄连(冻干)合注射用丹参(冻干)治疗老年晚期非小细胞肺癌并发肺部感染30例[J].河北中医,2009,31(003)：431-433.
73.旷飒飒,刘玲忠.LEEP刀联合双黄连粉针剂治疗重度宫颈糜烂疗效分析[J].医学临床研究,2009,26(007)：1288-1289.
74.刘剑英,高丽丽.注射用双黄连粉针剂药理作用概况[J].中外医疗,2009,28(001)：84-84.
75.赵海东.双黄连粉针和水针临床应用不良反应比较[J].现代医药卫生,2008(8)：1219-1219.
76.吴嘉瑞,张冰.双黄连注射剂不良反应文献的数据挖掘研究[J].中国药物警戒,2008,5(003)：139-143.
77.吴嘉瑞,张冰.双黄连注射剂不良反应文献病案的系统研究[J].中成药,2008,30(005)：640-643.
78.袁迎.清开灵,双黄连注射剂不良反应病例分析[J].陕西中医,2009,(007)：892-893.
79.梁进权,邹元平,邓响潮.中药注射剂不良反应的文献调查与分析[J].中国医院药学杂志,2003,23(008)：486-488.
80.郑加嘉,杨小娟.注射用双黄连2176例临床用药不良反应分析[J].中国实用医药,2006,1(001)：105-107
81.赖宇红,杨卫荣.中药注射剂变态反应研究亟待加强[J].中药新药与临床药理,2002,13(005)：324-326.
82.刘红.双黄连注射液的不良反应[J].中华医学实践杂志,2005,4(6)41-2.
83. Freedman, S., Krupey, J., Sehon,A. Chlorogenic acid:an allergen in green coffee bean[J]. Nature (Inglaterra),1961,192(4799):241-243.
84. Freedman, S., Siddiqi, A.I., Krupey, J.H., Sehon, A.H. Identification of a simple chemical compound (chlorogenic acid) as an allergen in plant materials causing human atopic disease[J]. Am J Med Sci.,1962,244:548.
85. Freedman, S., Shulman, R., Krupey, J. Loss of allergenicity of chlorogenic acid in the gastrointestinal tract[J]. J Allergy Clin Immunol.,1964,35(2):108-116.
86. Freedman, S., Shulman, R., Krupey, J., Sehon, A.H. Antigenic properties of chlorogenic acid[J]. J Allergy Clin Immunol.,1964,35(2):97-107.
87. Bariana, D., Krupey, J., Scarpati, L.M., Freedman, S.O., Sehon, A.H. Chlorogenic acid:further evidence for its antigenic and allergenic activity[J]. Nature,1965,207(5002):1155.
88.黄芳华.绿原酸及其中药注射剂的安全性问题状况分析[J].中国中药杂志,2008,33(022)：2716-2719.
89.李钦,张信岳,陈国神.含绿原酸的清热解毒类中药注射剂不良反应及其机理探讨[J].中国现代应用药学,2009：555-558.
90.童路.双黄连注射剂的不良反应与成分间的关系[J].中成药,1997,19(004)：47-48.
91.田锋奇.黄芩苷的致过敏作用机制[D].郑州大学；2007.
92.赵新先.中药注射剂学[M].广州：广东科技出版社；2000.
93.邓良,袁华,喻宗沅.绿原酸的研究进展[J].化学与生物工程,2005,22(007)：4-6.
94.张鞍灵,马琼.绿原酸及其类似特与生物活性[J].中草药,2001,32(002)：173-176.
95.张建春,张华,施瑛,胡浩.黄芩苷的研究近况[J].时珍国医国药,2005,16(003)：247-249.
96.罗飞,包旭,林大胜,杨华蓉,周莎,徐小平.绿原酸对动物的致敏性研究[J].华西药学杂志,2009,24(002)：181-183.
97.李瑾翡,黎旸,陈琪,程朝辉,谭舜.注射用双黄连的免疫毒性研究[J].中药新药与临床药理,2008,19(003)：172-174.
98. Bala, S., Weaver, J., Hastings, K.L. Clinical relevance of preclinical testing for allergic side effects[J]. Toxicology,2005,209(2):195-200
99. Weaver, J.L., Staten, D., Swann, J., Armstrong, G,. Bates, M., Hastings, K.L. Detection of systemic hypersensitivity to drugs using standard guinea pig assays[J]. Toxicology,2003,193(3):203-17.
100. Ju, C, Uetrecht,J.P. Mechanism of idiosyncratic drug reactions:reactive metabolite formation, protein binding and the regulation of the immune system[J]. Curr Drug Metab,2002,3(4):367-77.
101. Brouland, J.P., Verdier, F., Patriarca, C., Vial, T., Descotes, J.Morphology of popliteal lymph node responses in Brown-Norway rats[J]. J Toxicol Environ Health,1994,41(1):95-108.
102. De Bakker, J.M., Kammiil ler, M. E., Muller, E. S. M., Lam, A. W., Seinen, W. Bloksma, N. Kinetics and morphology of chemically induced popliteal lymph node reactions compared with antigen-, mitogen-, and graft-versus-host-reaction-induced responses[J]. Virchows Arch B Cell Pathol Incl Mol Pathol,1990,58(4):279-87.
103. Koch, E., Jaggy, H., Chatterjee,S.S.. Evidence for immunotoxic effects of crude Ginkgo biloba L. leaf extracts using the popliteal lymph node assay in the mouse[J]. Int J Immunopharmacol,2000,22(3):229-36.
104.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.
105.谢华,何韶衡.哮喘中IgE依赖性和非IgE依赖性肥大细胞激活的研究[J].汕头大学医学院学报,2003,16(001)：12-15.
106. Findlay, S., Dvorak, A.M., Kagey-Sobotka, A., Lichtenstein, L.M. Hyperosmolar triggering of histamine release from human basophils[J]. J Clin Invest.,1981,67(6):1604-13.
107.王玉俊,杨卫华,万年红,张志萍.荨麻疹患者血清变应原特异性免疫球蛋白E检测分析[J].临床荟萃,2005,20(022)：1310-1311.
108. de Weck, A.L., Gamboa, P. M., Esparza, R., Sanz, M. L. Hypersensitivity to aspirin and other nonsteroidal anti-inflammatory drugs (NSAIDs)[J]. Curr Pharm Des,2006,12(26):3347-58.
109. Szebeni, J. Complement activation-related pseudoallergy:a new class of drug-induced acute immune toxicity[J]. Toxicology,2005,216(2-3):106-21.
110. Zebeni, J. Complement activation-related pseudoallergy caused by amphiphilic drug carriers:the role of lipoproteins[J]. Curr Drug Deliv,2005,2(4):443-9.
111.徐国良,张卓辉,张增珠,张启云.双黄连注射剂过敏症动物模型的研究[J].中国临床药理学与治疗学,2008,13(002)：154-157.
112. Chen, X.F.,, Lin, J.M., Zheng, L.P. The Effect of Shuanghuanglian Injection on Elevating of Blood Serum Histamine in Mice [J]. J Fujian College Tradit Chine Med,2005.
113吴友苹,徐潘生,卢觅佳,宣尧仙.鱼腥草注射液Ⅰ型过敏反应试验[J].时珍国医国药,2009,20(6).
114 J. zhang, Y.-K.L., L.-L. Li, W. Hao, W.-J Yan. sensitization of beagle dogs to Houttuynia cordata distillation mixed with three solubilizers [J]. Chin J New Drugs,2008,17(17):1494-1498
115. Ezendam, J., Staedtler, F., Pennings, J., Vandebriel, R.J., Pieters, R., Harleman, J.H., Vos, J.G. Toxicogenomics of subchronic hexachlorobenzene exposure in Brown Norway rats [J]. Environ Health Perspect,2004,112(7):782.
116.吴梅,闫慧,张春梅.黄芩苷作为一种抗生物制剂的过敏反应的研究[J].微生物学免疫学进展,2007,4(4).
117.杜国安,李烈,卢素国.常用中药注射液静脉滴注临床不良反应分析[J].时珍国医国药,2006,4(9).
118. Ko, R. A US perspective on the adverse reactions from traditional Chinese medicines[J]. J CHIN MED ASSOCIATION,2004,67(3):109-116.
119. Gong, J., F.T. Liu, and S.S. Chen. Polyphenolic antioxidants enhance IgE production[J]. Immunol Invest,2004,33(3):295-307.
120. Kang, J., Liu, Y., Meng, X. X., Song, L., Jiang, M.,Wang,Y.D. Interactions of human serum albumin with chlorogenic acid and ferulic acid[J]. Biochimica Et BiophysicaActa-General Subjects,2004,1674(2):205-214.