适合评价中药注射液过敏反应的动物模型研究
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摘要
中药注射剂是中药的创新剂型,与传统中药比较具有生物利用度较高,起效迅速等特点,能较好的发挥中药治疗急危重症的特点,尤其在心血管系统疾病、病毒性疾病和肿瘤的治疗中具有不可替代的作用。但是随着中药注射液研发和生产的规模不断扩大以及在临床的广泛应用,其安全性问题逐渐暴露出来。目前我国的43个中成药剂型中,注射剂这一种剂型引起的不良反应就占了75%,这种情况已经引起了国内外的广泛关注。但是长期以来,由于对中药注射液的不良反应重视不够,造成该方面的研究基础极为薄弱。中药注射液严重不良反应中尤以过敏反应的比例为最高,且反应类型多为速发型变态反应,但目前国内外均未建立针对中药致敏原的敏感动物模型和标准评价方法,严重影响了中药注射液的临床前安全性评价,使其在临床上的应用受到了很大的限制。
本研究以清开灵注射液、双黄连注射液及其中的疑似致敏成分黄芩苷、绿原酸作为致敏原,以天花粉蛋白为阳性对照,通过观察过敏反应发生率、过敏反应程度、组胺增长率、靶器官病变率和病变程度,对BN大鼠和豚鼠在评价中药注射液过敏反应中的适用性进行比较,筛选出适合评价中药注射液过敏反应的动物模型；并通过全身主动过敏试验(ASA)和被动皮肤过敏试验(PCA)对该模型的特异性进行研究；在此基础上,通过对致敏参数(致敏剂量、致敏次数、致敏途径、致敏时间间隔)和激发参数(攻击剂量、攻击途径、攻击间隔、攻击体积)的明确,建立一套适合评价中药注射液过敏反应的规范化研究方法；在建立的动物模型上,以清开灵注射液、双黄连注射液、鱼腥草注射液、穿琥宁注射液、灯盏花素注射液为受试样本,以青霉素为阳性对照,通过对肥大细胞脱颗粒、IgE抗体含量、血清中过敏介质和Th1/Th2型细胞因子含量的研究,对中药注射液过敏反应的机制进行探讨。本研究获得国家自然科学基金重大研究计划的资助。
1.BN大鼠和豚鼠对评价中药注射液过敏反应的适用性研究
本研究以清开灵注射液、双黄连注射液、黄芩苷和绿原酸为致敏原,直接对BN大鼠和豚鼠进行致敏,致敏途径为腹腔注射、致敏剂量为人临床的等倍剂量,致敏次数为3次,隔日1次,在末次致敏后的第14天进行攻击,攻击途径为一次性静脉推注,攻击体积为致敏体积的2倍。攻击后观察过敏反应症状,根据SFDA的全身致敏性评价标准来计算过敏反应率、过敏反应程度；采用ELISA法检测血清、肺组织和气管中组胺含量并计算组胺增长率;HE染色法光镜下观察肺组织和气管病理改变并计算靶器官病变率和病变程度,以此为依据对BN大鼠和豚鼠在评价中药注射液过敏反应中的适用性进行比较。
结果显示：以清开灵注射液、双黄连注射液、黄芩苷和绿原酸为受试样本,①BN大鼠各组的过敏反应发生率分别为55.56%、61.11%、75.00%和83.33%,而豚鼠各组的过敏反应发生率分别为22.22%、27.77%、16.67%和41.67%,BN大鼠对四种受试的中药注射液及药物成分的过敏率均明显高于豚鼠,两种动物比较均具有显著性差异。②四种受试样本致BN大鼠的过敏程度均比豚鼠严重,其中双黄连注射液组和黄岑苷组的BN大鼠过敏反应程度与豚鼠比较具有显著性差异。③BN大鼠血清、肺组织和气管中的组胺增长率均明显高于豚鼠,其中清开灵注射液的血清、肺组织、气管,双黄连注射液的血清和肺组织,黄芩苷的血清,绿原酸的肺组织中的组胺增长率与豚鼠比较均具有显著性差异。④四种受试样本BN大鼠各组的肺组织病变率分别为66.67%、72.22%、66.67%和41.67%,而豚鼠各组的肺部病变率分别为44.44%、44.44%、25.00%和8.33%,BN大鼠对四种受试样本的肺组织病变率均明显高于豚鼠,其中黄芩苷组的BN大鼠肺组织病变率与豚鼠比较具有显著性差异。BN大鼠各组的气管病变率与豚鼠各组比较没有显著性差异。⑤四种受试样本致BN大鼠肺组织的病理改变程度均比豚鼠严重,其中双黄连注射液组和黄芩苷组的BN大鼠肺组织病变程度与豚鼠比较具有显著性差异；四种受试样本致BN大鼠气管的病理改变程度与豚鼠比较均没有显著性差异。
试验结果证明：在评价中药注射液过敏反应中,BN大鼠的敏感性明显高于豚鼠,说明BN大鼠可能为评价中药注射液过敏反应的适宜动物模型。
2.中药注射液致BN大鼠过敏反应的特异性研究
2.1中药注射液致BN大鼠的全身主动过敏试验(ASA)
本实验以清开灵注射液、双黄连注射液、黄芩苷和绿原酸为受试样本,以天花粉蛋白为阳性对照,通过观察BN大鼠的过敏反应症状,Elisa法检测血清、肺组织和气管中组胺含量的变化,HE染色法光镜下观察心脏、肝脏、脾脏、肺、气管、胃和小肠的病理改变,并从中筛选出特异性的靶器官,来明确中药注射液致BN大鼠过敏反应的特异性。
结果显示：①BN大鼠对清开灵注射液、双黄连注射液、黄芩苷和绿原酸均产生了特异性的过敏反应的症状和表现。BN大鼠产生的过敏反应症状主要包括躁动、颤抖、搔鼻、喷嚏、呼吸急促等,多在攻击后10-20min内出现,15min以内缓解,严重时出现静卧、肌张力下降、体表温度下降、抽搐、晕厥等过敏性休克的症状,甚至出现死亡。②BN大鼠过敏反应模型的血清、肺组织和气管中的组胺含量均明显升高,与正常对照组比较均具有显著性差异,说明BN大鼠的过敏反应均引起明显的组胺释放,组胺释放到靶器官并释放入血。③在过敏反应的靶器官筛选中发现心、肝、脾、胃、小肠均没有出现明显的病理改变,而各组BN大鼠的肺组织和气管均出现了明显的炎症性病理改变,说明肺和气管为中药注射液过敏反应的主要靶器官。④阳性药对照组BN大鼠也出现了特异性的过敏反应的症状,主要包括静卧、排粪(大便稀溏)、呼吸困难、呼吸急促、步态不稳、肌张力下降、反应敏感度下降、喘息等症状,多在攻击后10min内出现,30min-1h后缓解；血清、肺组织和气管中均可见明显的组胺释放；在过敏反应的靶器官筛选中发现,BN大鼠肺组织和气管中出现了明显的炎症性病理改变,据综合判断,肺和气管为阳性对照(天花粉蛋白)致BN大鼠过敏反应的主要靶器官。
试验结果证明：BN大鼠对以清开灵注射液、双黄连注射液、黄芩苷和绿原酸为代表的中药注射液和药物成分能产生特异性的全身速发型过敏反应。2.2中药注射液致BN大鼠的被动皮肤过敏试验(PCA)
本实验以清开灵注射液、双黄连注射液、鱼腥草注射液、穿琥宁注射液和灯盏花素注射液为受试样本,以青霉素钠为阳性对照,对BN大鼠致敏后制备抗血清,以1:25和1:50两个稀释度的抗血清对Balb/c小鼠进行致敏和激发,通过观察伊文思蓝渗出和蓝斑反应来评价BN大鼠在中药注射液致皮肤被动过敏反应中的特异性。
结果显示,1:25和1:50稀释度的抗血清各组均出现了明显的伊文思蓝渗出,对于多数中药注射液,1:25稀释度组的伊文思蓝吸光度高于1:50稀释度组,说明1:25稀释度抗血清中IgE抗体的含量较高。1:25稀释度的抗血清攻击后,各药物组均有动物出现了蓝斑反应,蓝斑直径大于5mm,其中双黄连注射液小剂量组、鱼腥草注射液小剂量组和阳性药对照组均有3只动物出现了蓝斑,反应阳性率为75%,与阴性对照组比较具有显著性差异；而1:50稀释度的抗血清攻击后,只有双黄连注射液小剂量组和阳性药对照组出现了蓝斑反应,反应阳性率分别为25%和50%。
试验结果证明：清开灵注射液、双黄连注射液、鱼腥草注射液、穿琥宁注射液、灯盏花素注射液均能通过BN大鼠制备的抗血清,使Balb/c小鼠产生明显的皮肤被动过敏反应,证明BN大鼠为研究中药注射液皮肤被动过敏反应的适宜动物模型。
3.中药注射液致BN大鼠过敏反应动物模型的规范化研究
本研究以清开灵注射液、双黄连注射液、黄芩苷和绿原酸作为受试样本,以豚鼠作为对照,采用不同的致敏参数和激发参数：致敏剂量(人临床等倍剂量、人临床2倍剂量)、致敏途径(ip、iv),攻击次数(1次、2次)、攻击时间间隔(末次致敏后的第14天和第21天)来进行全身主动过敏试验,通过不同参数下对过敏反应发生率,组胺含量,肺组织、气管的病变率和病变程度的比较,来明确适用于BN大鼠过敏反应动物模型的规范化条件。
结果显示：①第1次攻击BN大鼠各组均出现了过敏反应,其过敏反应率基本上都高于第2次攻击,因而确定攻击的次数为1次,攻击的时间间隔应为末次致敏后的第14d。②iv小剂量组(致敏途径为静脉注射、致敏剂量为人临床等倍剂量)对各受试样本均产生了过敏反应,对清开灵注射液、双黄连注射液、黄芩苷和绿原酸的过敏反应发生率分别为50%、66.67%、75%和75%；iv小剂量组血清和组织中的组胺含量均明显升高；iv小剂量组BN大鼠对清开灵注射液、双黄连注射液、黄芩苷和绿原酸的肺组织病变率分别为50%、50%、75%和25%,与同组的过敏反应发生率具有较好的对应关系,因此确定致敏途径为静脉注射、致敏剂量为临床人的等倍剂量,而相应的攻击剂量为临床人的2倍剂量。③在相同的致敏和攻击条件下,BN大鼠组的过敏反应发生率、组胺释放、肺组织、气管的病变率和病变程度基本上都高于豚鼠组,说明在同等条件下,BN大鼠对中药注射液过敏反应的敏感性优于豚鼠,重复性验证了前面的试验结果。
试验结果证明：以BN大鼠为动物模型评价中药注射液的过敏反应,其致敏和攻击条件应规范化为：致敏剂量为人临床等倍剂量、致敏间隔为1日、致敏途径为静脉注射,攻击剂量为致敏剂量的2倍、攻击途径为静脉一次性注射、攻击次数为1次、攻击时间间隔为末次致敏后的第14天,这种方法在动物数量较少的情况下也较为适用。
4.中药注射液对BN大鼠过敏反应机制的研究
本研究以清开灵注射液、双黄连注射液、鱼腥草注射液、穿琥宁注射液和灯盏花素注射液为受试样本,以青霉素为阳性对照,对BN大鼠进行致敏和激发,通过观察BN大鼠的过敏反应率、肺和气管的病变率来评价中药注射液致BN大鼠的过敏反应情况,通过观察腹腔肥大细胞脱颗粒,血清中IgE抗体、过敏介质和细胞因子含量的变化,来揭示中药注射液的致敏反应机制。
结果显示：①各注射液组的BN大鼠均出现了明显的过敏反应症状,小剂量组过敏反应发生率均高于大剂量组,与对照组比较具有显著性差异；各注射液组的肺组织和气管均出现了明显的炎症性病理改变。②各注射液组肥大细胞阳性率均明显升高,其中清开灵注射液组、鱼腥草注射液组、穿琥宁注射液组与对照组比较具有显著性差异；阳性对照药青霉素组的肥大细胞阳性率明显升高,与对照组比较具有显著性差异,且其阳性率高于中药注射液各组。③中药注射液诱导过敏反应后,BN大鼠血清中IgE抗体含量明显升高,与对照组比较具有显著性差异。④中药注射液诱导过敏反应后,BN大鼠血清中组胺、5-羟色胺、前列腺素D2和白三烯B4的含量均明显升高,细胞因子IFN-γ和IL-4的含量均明显升高,大部分组的IFN-γ/IL-4比值出现了降低的趋势。
试验结果证明：在BN大鼠动物模型上,中药注射液可引起IgE抗体介导的速发型过敏反应,其过敏反应机制可能与以下环节相关：①促进肥大细胞脱颗粒后,释放组胺、5-羟色胺、前列腺素D2和白三烯B4等过敏介质；②诱导Th1/Th2细胞分泌的细胞因子向Th2偏移,从而促进IgE抗体合成。
综上所述,我们得出以下研究结论：
●与豚鼠比较,BN大鼠可能为评价中药注射液过敏反应的适宜动物模型；
●在BN大鼠动物模型上,能产生特异性的全身主动过敏反应和皮肤被动过敏反应；
●采用BN大鼠模型对中药致敏原进行评价时,其规范化参数为：致敏剂量为人临床等倍剂量、致敏次数为3次、致敏间隔为1日、致敏途径为静脉注射,攻击剂量为致敏剂量的2倍、攻击途径为静脉注射、攻击次数为1次、攻击时间间隔为末次致敏后的第14天；
●在BN大鼠动物模型上,中药注射液可引起IgE抗体介导的速发型过敏反应,其过敏反应机制可能与以下环节相关：①促进肥大细胞脱颗粒后,释放组胺、5-羟色胺、前列腺素D2和白三烯B4等过敏介质；②诱导Th1/Th2细胞分泌的细胞因子向Th2偏移,从而促进IgE抗体合成。
Traditional Chinese medicine injection is a novelty form of TCM, the form has many virtues just as higher bioavailability, more efficiency etc. For example, it often plays an important role in treating severely acute diseases.Especially in curing cardiovascular diseases, viral diseases and tumor, TCM injection plays an irreplaceable role. But with the development of traditional TCM injection, expanding production and widely used in clinical applications, the safety problems of traditional Chinese medicine injection was gradually discovered. At present, among the 43 traditional Chinese medicine formulations,75 percent of adverse reaction is induced by using TCM injection, this phenomenon has already attracted wide attention at home and abroad. But for a long time, less attention on adverse reaction of TCM injection results in poor fundamental research about TCM injection. The highest proportion of seriously adverse reaction of TCM injection is anaphy lactic reaction. And mostly reaction type of anaphylactic reaction is immediate allergic response. There isn't standard animal model and assessing methodology, which is an important reason resulting in absence of preclinical safety evaluation, and then TCM injection also was restricted in extensive clinical application.
In this study, Qingkailing injeciton, Shuanghuanglian injeciton, baicalin and chlorogenic acid were used as allergen, trichosanthin was used to positive control, we have observed the BN rats'and guinea pigs'incidence rates, degree of anaphylactic reactin, histamine release and degree of pathological changes in target organs,compared the applicability of anaphylactic reaction in BN rats and guinea pigs, selected suitable animal model which was used to evaluating TCM injection's anaphylactic reaction. We also observed the specificity of the BN animal model by systemic active anaphylactic reaction (ASA) and passive cutaneous anaphylaxis (PCA). Proceeding to the next step, sensitization parameters(sensitization dose, sensitization times, sensitization pathways,sensitization time interval)and excitation parameters(excitation dose,excitation pathways,excitation time interval,excitation volume) was determined, the standard methods which were suitable to evaluate anaphylactic reaction of TCM injection was established.
We have observed degranulation of mast cells in abdominal cavity, antibody levels, inflammatory factors and Th1/Th2 cytokines contents in serum with the eatablished model, then we also attempt to unpuzzle the mechanism of the anaphylactic reaction induced by TCM injection with the eatablished animal model. In this research, Yuxingcao injection, Chuanhuning injection, Dengzhanhuasu injection and ingredients baicalin, chlorogenic acid was used as allergen, penicillin as posistive control. Our research was supported by national nature science foundation of China.
1. Applicability Research of BN rat and guinea pig in evaluating anaphylactic reaction of TCM injection
In this section, we researched the applicability of BN rat and guinea pig in evaluating anaphylactic reaction of TCM injection. Qingkailing injection, Shuanghuanglian injection, baicalin and chlorogenic acid as allergens, BN rats and guinea pigs were sensitized directly. The sensitization pathway was intraperitoneal injection, the dose was equivalent with human clinical dosage. The sensitization was carried out every other day, totally 3 times. The provocation was done in the 14th day after the last sensitization, the provocation method is disposal intravenous injection, The provocation volume is double sensitization value. We would observe symptoms of the anaphylactic reaction after the initiation, calculate the rates and degree of anaphylactic reaction on the basis of SFDA systemic sensitization evaluation criteria. The rates of histamine increase were calculated by histamine content in serum, lung tissues and tracheas detected by ELISA respectively. Pathological changes in target organs were observed by HE staining under light microscope and the rates and degree of pathological changes were calculated. According to analysis these experiment data and result, we could determine the applicability of BN rat and guinea pig in evaluating anaphylactic reaction of TCM injections.
The main testing results are listed blow:
BN rats and guinea pigs are both sensitized by all of four allergens (Qingkailing injection, Shuanghuanglian injection, ingredients baicalin and Chlorogenic acid), but the allergic rates were obviously different between the two animals. The allergic rates of BN rats for every sample are 55.56%,61.11%,75.00% and 83.33% respectively, meanwhile the allergic rates of guinea pigs for every sample were 22.22%,27.77%,16.67% and 41.67% respectively.
The allergic degree of BN rats induced by four tested samples is more serious than that of guinea pigs, especially induced by Shuanghuanglian injection and ingredient baicalin.
Increase rates of histamine in BN rats'serum, lung and trachea are significantly higher than that of guinea pigs. For instance, compared with guinea pigs, increase rates of histamine in BN rats'serum (induced by Qingkailing injection group, Shuanghuanglian injection, ingredients baicalin), lung (induced by Qingkailing injection group, Shuanghuanglian injection, chlorogenic acid), and trachea (induced by Qingkailing injection group) are notabely different.
Significant difference could be found when we compare the pathological changes rate of BN rats'lung tissue with that of guinea pigs'lungs. The pathological change rates of BN rats'lung were 66.7%,72.22%,66.67% and 41.67% respectively when the rats were sensitized by all of allergens, meanwhile the pathological change rates were 44.44%、44.44%、25.00% and 8.33% respectively when parallel tests were done in guinea pigs. But no obvious difference was found when we compare the pathological rates of BN rats'trachea with that of guinea pigs'
Pathological change degree of BN rats'lung tissue was more serious than that of guinea pigs'when the two animals were induced by all of four allergens, especially they were sensitized by Shuanghuanlian injecition and ingredients baicalin. We didn't found obvious difference when we research the pathological change degree of BN rats'trachea with that of guinea pigs'too.
According to analysis above mentioned, we can draw a conclusion that BN rat was more sensitive than guinea pig for evaluating anaphylactic reaction induced by TCM injection, and BN rats is a suitable candidate as animal model for evaluating anaphylactic reaction induced by TCM injection.
2. Specificity research of BN rats'anaphylactic reaction induced by TCM injection
2.1 Active systemic anaphylactic reaction of BN rats induced by TCM injection (ASA)
In this section, we focused on the specificity of BN rats'anaphylactic reaction induced by TCM injection. We observed the BN rats'anaphylactic reactions, determined the level of histamine in serum and tissues by Elisa method, observed the pathological changes of heart, liver, spleen, lung, trachea, stomach and small intestine by HE staining under light microscope and selected specific target organs. Qingkailing injection, Shuanghuanglian injection, ingredients baicalin and chlorogenic acid was used as allergen, trichosanthin was positive control in these experiments.
We can draw the conclusion by above-mentioned research, all of four tested samples(Qingkailing injection, Shuanghuanglian injection, ingredients baicalin and bhlorogenic acid) can induced BN rats'allergic symptom and phenomena, and we found the similar results in positive control. The phenomena were scratching, nose shortness of breath, sneezing and some other responses, and the reactions mostly appeared in 10-20min after initiation and eased after 15min initiated by tested samples. The serious symptoms were hypotonia, the skin temperature lower, convulsion, swoon, other reactions of anaphylactic shock, severe ones even died.
We have found that content of histamine increased in the allergic BN rats'blood, lung and trachea significantly, so we believe the histamine was released from target organ and transfer into blood because of allergic reaction induced by the using TCM injection and ingredients in TCM injection. When we search target organ, we find there were no obvious pathological changes in heart, liver, spleen, stomach, small intestine, but there were pathological changes in lung and trachea. The symptom is obvious inflammatory pathological changes. It showed that lung and trachea were the main target organs in evaluating the allergic response induced by TCM injection.
We find similar phenomenon in positive control, these reactions mostly appeared in 10min after initiation and eased after 30min-lh. Obvious histamine release was found in serum, lung and trachea. Obvious inflammatory pathological change was found in BN rats'lung and trachea when we searched the target organ of allergic reaction. So we can draw the conclusion:lung and trachea are main target organs induced by trichosanthin.
The results demonstrated that BN rats'systemic rapid allergic response can be induced by four tested samples above mentioned.
2.2 Passive cutaneous anaphylaxis of BN rats induced by TCM injection (PCA)
In this section, we researched passive cutaneous anaphylaxis of BN rats induced by TCM injection(PCA experiment). Qingkailing injection,Shuanghuanglian injection,Yuxingcao injection,Chuanhuning injection,Dengzhanhuasu injection were used as allergens. Penicillin sodium was positive control. The antiserum was yielded after the BN rats were sensitized, then the Balb/c mice were sensitized by diluted antiserum (dilution factor:1:25 and 1:50). We would evaluate the specificity of passive cutaneous anaphylaxis of BN rats induced by TCM injection by observing the skin inner spot and Evans blue permeance.
The results showed that we could observe Evans blue permeance after the Balb/c mice were sensitized by all of antiserums. For mostly test samples, Evans blue absorption is higher when the dilution factor is 1:25 than the factor is 1:50, so we supposed the content of IgE in the antiserum whose dilute fator is 1:25 is higher than that in the antiserum whose dilute fator is 1:50.
We could observe skin inner spot in some Balb/c mice when the laboratory animals were sensitized by antiserum (dilution factor:1:25) which were yielding by all of five allergens above mentioned. Diameter of the spot is larger than 5 centimeters. Three Balb/c mice appeard skin inner spot when the allergens are Shuanghuanglian injection (low dose), Yuxiangcao injection and positive control respectively, this phenomenon was obviously different to negative control.But we didn't find remarkably difference when the dilution factor is 1:50. Only Shuanghuanglian injection (low dosage) and positive controls induced inner skin spot, and the rates of positive response were 25% and 50% respectively. That was similar to negative controls.
These results demonstrated that the antiserums can be yielded when the BN rats are sensitized by all of five allergens above mentioned. We can find obviously passive cutaneous anaphylaxis reaction too. So we considered BN rat is a suitable animal model to evaluate passive cutaneous anaphylaxis induced by TCM injection.
3. Standard research on anaphylactic reaction of BN rat animal model induced by TCM injection
In this section, we attempted to determine the standard parameters when the BN rats were used as animal model to evaluate anaphylactic reaction induced by TCM injection. Qingkailing injection, Shuanghuanglian injection, ingredients baicalin and chlorogenic acid were used as allergen, and the guinea pigs were used as control. The sensitizing and provocation parameters which were researched are listed blow: allergic dose (equivalent dose with clinic dosage or double), sensitized method (ip, iv), provocation times (one or two), provocation moment (the 14th day after last sensitization or 21st). According to analysis experimental results above mentioned, we could selected proper parameters when the BN rats were used as animal model to anaphylactic reaction induced by TCM injection.
The results showed that BN rats appeared allergic phenomena which were provocated only one time, the incidence carried out sensitization after first provocated was higher than second provocation. So we consider the provocation number of times is one, and the suitable moment is 14th day after the last sensitization.
All of iv low dosage tested groups (intravenous injection, equivalent dose with clinic dosage) appeared allergic reaction, the rates of anaphylaxis reaction were 50%, 66.67%,75% and 75% respectively. The content of histamine in blood of this group increased obviously. Pathological changes rate in BN rats'tissue which was sensitized by the four allergens were 50%,50%,75% and 25% respectively, the proportion is coincidence with rate of anaphylaxis reaction mostly. So we consider that the sensitization method is intravenous injection, the dose is equivalent with clinic dosage, and the provocation dose is double.
Under the same testing conditions, we found the anaphylactic reaction is more distinct in every aspect researched, such as the rate of anaphylactic reaction, histamine releasing, the rate of pathological changes and the degree of pathological changes. So we can draw a conclusion, the sensibility of BN rats induced by TCM injection is better than guinea pigs.
According to the testing results, we can consider that BN rat was a suitable animal model when we evaluated allergic reaction induced by TCM injection. The suitable allergic and provocation parameters are listed blow:the dose is equivalent with clinic dosage, the sensitization number of time is three and the sensitization is carried out anothor day, provocation number of times is one and provocation moment is the 14th day. These methods can also be used for less tested animals.
4. The research on mechanism of anaphylactic reaction induced by TCM injection
In this section, BN rats were sensitized and provacted by TCM injection, then the rates of anaphylactic reaction and pathological changes in lung and trachea were observed.To probe into the mechanism of anaphylactic reaction of TCM injection, we detected mast cells degranulation in abdominal cavity, the levels of IgE antibody, inflammatory factor and cytokines in serum. In these experiments, Qingkailing injection, Shuanghuanglian injection, ingredients baicalin and chlorogenic acid was used as allergen and penicillin sodium as positive control.
The main testing results are listed blow:
There were significant symptoms in BN rats sensitized by all the five TCM injections and positive control.The rate of anaphylactic reaction induced by the same injection in low dose group was higher that in high dose group.Compared with negative control, there was significant difference. Obvious inflammatory pathological changes were observed in lung and trachea tissues of BN rats sensitized by all the five TCM injections and positive control.The positive rates of mast cells degranulation increased obviously in every sample group. Compared with negtive control,significant difference was showed in some samples group,including Qingkailing injection group,Yuxingcao injection group and Chuanhuning injection group.In positive control (penicillin sodium) group,the positive rate of mast cells degranulation was higher that in every tested sample group. Compared with negtive control, significant difference was showed.After sensitized by TCM injection, the level of IgE antibody in serum of BN rats increased obviously.Compared with the negtive control, there was remarkably difference. After sensitized by TCM injection,both the levels of histamine,5-HT, PGD2, LTB4 and the content of cytokines IFN-y and IL-4 in serum of BN rats increased significantly,on the contrary,in mostly group the ratio of IFN-γ/IL-4 decreased to some extent.
The results demonstrated that TCM injection could induce immediate hypersensitivity mediated by IgE antibody with established BN rat animal model.The possible mechanism of this immediate hypersensitivity including:①TCM injections induced mast cell degranulation and then release histamine,5-HT, PGD2, LTB4.②TCM injections induced Thl/Th2 released cytokines shifting to Th2 and then enhance IgE synthesis.
All sum up, we can draw some conclusion:BN rat is possible more suitable animal model for estimating the anaphylactic reaction of traditional Chinese medicine injection than guinea pig. With BN rat animal model, active systemic anaphylaxis and passive cutaneous anaphylaxis can be found. When we forcast the allergen using BN rat animal model, the standard parameters is:the dose is equivalent with clinic dosage, the sensitization number of time is three and the sensitization is carried out another day, provocation number of times is one and provocation moment is the 14th day. The possible mechanism of this immediate hypersensitivity including:①TCM injections induced mast cell degranulation and then release histamine,5-HT, PGD2, LTB4.②TCM injections induced Thl/Th2 released cytokines shifting to Th2 and then enhance IgE synthesis.
本研究以清开灵注射液、双黄连注射液及其中的疑似致敏成分黄芩苷、绿原酸作为致敏原,以天花粉蛋白为阳性对照,通过观察过敏反应发生率、过敏反应程度、组胺增长率、靶器官病变率和病变程度,对BN大鼠和豚鼠在评价中药注射液过敏反应中的适用性进行比较,筛选出适合评价中药注射液过敏反应的动物模型；并通过全身主动过敏试验(ASA)和被动皮肤过敏试验(PCA)对该模型的特异性进行研究；在此基础上,通过对致敏参数(致敏剂量、致敏次数、致敏途径、致敏时间间隔)和激发参数(攻击剂量、攻击途径、攻击间隔、攻击体积)的明确,建立一套适合评价中药注射液过敏反应的规范化研究方法；在建立的动物模型上,以清开灵注射液、双黄连注射液、鱼腥草注射液、穿琥宁注射液、灯盏花素注射液为受试样本,以青霉素为阳性对照,通过对肥大细胞脱颗粒、IgE抗体含量、血清中过敏介质和Th1/Th2型细胞因子含量的研究,对中药注射液过敏反应的机制进行探讨。本研究获得国家自然科学基金重大研究计划的资助。
1.BN大鼠和豚鼠对评价中药注射液过敏反应的适用性研究
本研究以清开灵注射液、双黄连注射液、黄芩苷和绿原酸为致敏原,直接对BN大鼠和豚鼠进行致敏,致敏途径为腹腔注射、致敏剂量为人临床的等倍剂量,致敏次数为3次,隔日1次,在末次致敏后的第14天进行攻击,攻击途径为一次性静脉推注,攻击体积为致敏体积的2倍。攻击后观察过敏反应症状,根据SFDA的全身致敏性评价标准来计算过敏反应率、过敏反应程度；采用ELISA法检测血清、肺组织和气管中组胺含量并计算组胺增长率;HE染色法光镜下观察肺组织和气管病理改变并计算靶器官病变率和病变程度,以此为依据对BN大鼠和豚鼠在评价中药注射液过敏反应中的适用性进行比较。
结果显示：以清开灵注射液、双黄连注射液、黄芩苷和绿原酸为受试样本,①BN大鼠各组的过敏反应发生率分别为55.56%、61.11%、75.00%和83.33%,而豚鼠各组的过敏反应发生率分别为22.22%、27.77%、16.67%和41.67%,BN大鼠对四种受试的中药注射液及药物成分的过敏率均明显高于豚鼠,两种动物比较均具有显著性差异。②四种受试样本致BN大鼠的过敏程度均比豚鼠严重,其中双黄连注射液组和黄岑苷组的BN大鼠过敏反应程度与豚鼠比较具有显著性差异。③BN大鼠血清、肺组织和气管中的组胺增长率均明显高于豚鼠,其中清开灵注射液的血清、肺组织、气管,双黄连注射液的血清和肺组织,黄芩苷的血清,绿原酸的肺组织中的组胺增长率与豚鼠比较均具有显著性差异。④四种受试样本BN大鼠各组的肺组织病变率分别为66.67%、72.22%、66.67%和41.67%,而豚鼠各组的肺部病变率分别为44.44%、44.44%、25.00%和8.33%,BN大鼠对四种受试样本的肺组织病变率均明显高于豚鼠,其中黄芩苷组的BN大鼠肺组织病变率与豚鼠比较具有显著性差异。BN大鼠各组的气管病变率与豚鼠各组比较没有显著性差异。⑤四种受试样本致BN大鼠肺组织的病理改变程度均比豚鼠严重,其中双黄连注射液组和黄芩苷组的BN大鼠肺组织病变程度与豚鼠比较具有显著性差异；四种受试样本致BN大鼠气管的病理改变程度与豚鼠比较均没有显著性差异。
试验结果证明：在评价中药注射液过敏反应中,BN大鼠的敏感性明显高于豚鼠,说明BN大鼠可能为评价中药注射液过敏反应的适宜动物模型。
2.中药注射液致BN大鼠过敏反应的特异性研究
2.1中药注射液致BN大鼠的全身主动过敏试验(ASA)
本实验以清开灵注射液、双黄连注射液、黄芩苷和绿原酸为受试样本,以天花粉蛋白为阳性对照,通过观察BN大鼠的过敏反应症状,Elisa法检测血清、肺组织和气管中组胺含量的变化,HE染色法光镜下观察心脏、肝脏、脾脏、肺、气管、胃和小肠的病理改变,并从中筛选出特异性的靶器官,来明确中药注射液致BN大鼠过敏反应的特异性。
结果显示：①BN大鼠对清开灵注射液、双黄连注射液、黄芩苷和绿原酸均产生了特异性的过敏反应的症状和表现。BN大鼠产生的过敏反应症状主要包括躁动、颤抖、搔鼻、喷嚏、呼吸急促等,多在攻击后10-20min内出现,15min以内缓解,严重时出现静卧、肌张力下降、体表温度下降、抽搐、晕厥等过敏性休克的症状,甚至出现死亡。②BN大鼠过敏反应模型的血清、肺组织和气管中的组胺含量均明显升高,与正常对照组比较均具有显著性差异,说明BN大鼠的过敏反应均引起明显的组胺释放,组胺释放到靶器官并释放入血。③在过敏反应的靶器官筛选中发现心、肝、脾、胃、小肠均没有出现明显的病理改变,而各组BN大鼠的肺组织和气管均出现了明显的炎症性病理改变,说明肺和气管为中药注射液过敏反应的主要靶器官。④阳性药对照组BN大鼠也出现了特异性的过敏反应的症状,主要包括静卧、排粪(大便稀溏)、呼吸困难、呼吸急促、步态不稳、肌张力下降、反应敏感度下降、喘息等症状,多在攻击后10min内出现,30min-1h后缓解；血清、肺组织和气管中均可见明显的组胺释放；在过敏反应的靶器官筛选中发现,BN大鼠肺组织和气管中出现了明显的炎症性病理改变,据综合判断,肺和气管为阳性对照(天花粉蛋白)致BN大鼠过敏反应的主要靶器官。
试验结果证明：BN大鼠对以清开灵注射液、双黄连注射液、黄芩苷和绿原酸为代表的中药注射液和药物成分能产生特异性的全身速发型过敏反应。2.2中药注射液致BN大鼠的被动皮肤过敏试验(PCA)
本实验以清开灵注射液、双黄连注射液、鱼腥草注射液、穿琥宁注射液和灯盏花素注射液为受试样本,以青霉素钠为阳性对照,对BN大鼠致敏后制备抗血清,以1:25和1:50两个稀释度的抗血清对Balb/c小鼠进行致敏和激发,通过观察伊文思蓝渗出和蓝斑反应来评价BN大鼠在中药注射液致皮肤被动过敏反应中的特异性。
结果显示,1:25和1:50稀释度的抗血清各组均出现了明显的伊文思蓝渗出,对于多数中药注射液,1:25稀释度组的伊文思蓝吸光度高于1:50稀释度组,说明1:25稀释度抗血清中IgE抗体的含量较高。1:25稀释度的抗血清攻击后,各药物组均有动物出现了蓝斑反应,蓝斑直径大于5mm,其中双黄连注射液小剂量组、鱼腥草注射液小剂量组和阳性药对照组均有3只动物出现了蓝斑,反应阳性率为75%,与阴性对照组比较具有显著性差异；而1:50稀释度的抗血清攻击后,只有双黄连注射液小剂量组和阳性药对照组出现了蓝斑反应,反应阳性率分别为25%和50%。
试验结果证明：清开灵注射液、双黄连注射液、鱼腥草注射液、穿琥宁注射液、灯盏花素注射液均能通过BN大鼠制备的抗血清,使Balb/c小鼠产生明显的皮肤被动过敏反应,证明BN大鼠为研究中药注射液皮肤被动过敏反应的适宜动物模型。
3.中药注射液致BN大鼠过敏反应动物模型的规范化研究
本研究以清开灵注射液、双黄连注射液、黄芩苷和绿原酸作为受试样本,以豚鼠作为对照,采用不同的致敏参数和激发参数：致敏剂量(人临床等倍剂量、人临床2倍剂量)、致敏途径(ip、iv),攻击次数(1次、2次)、攻击时间间隔(末次致敏后的第14天和第21天)来进行全身主动过敏试验,通过不同参数下对过敏反应发生率,组胺含量,肺组织、气管的病变率和病变程度的比较,来明确适用于BN大鼠过敏反应动物模型的规范化条件。
结果显示：①第1次攻击BN大鼠各组均出现了过敏反应,其过敏反应率基本上都高于第2次攻击,因而确定攻击的次数为1次,攻击的时间间隔应为末次致敏后的第14d。②iv小剂量组(致敏途径为静脉注射、致敏剂量为人临床等倍剂量)对各受试样本均产生了过敏反应,对清开灵注射液、双黄连注射液、黄芩苷和绿原酸的过敏反应发生率分别为50%、66.67%、75%和75%；iv小剂量组血清和组织中的组胺含量均明显升高；iv小剂量组BN大鼠对清开灵注射液、双黄连注射液、黄芩苷和绿原酸的肺组织病变率分别为50%、50%、75%和25%,与同组的过敏反应发生率具有较好的对应关系,因此确定致敏途径为静脉注射、致敏剂量为临床人的等倍剂量,而相应的攻击剂量为临床人的2倍剂量。③在相同的致敏和攻击条件下,BN大鼠组的过敏反应发生率、组胺释放、肺组织、气管的病变率和病变程度基本上都高于豚鼠组,说明在同等条件下,BN大鼠对中药注射液过敏反应的敏感性优于豚鼠,重复性验证了前面的试验结果。
试验结果证明：以BN大鼠为动物模型评价中药注射液的过敏反应,其致敏和攻击条件应规范化为：致敏剂量为人临床等倍剂量、致敏间隔为1日、致敏途径为静脉注射,攻击剂量为致敏剂量的2倍、攻击途径为静脉一次性注射、攻击次数为1次、攻击时间间隔为末次致敏后的第14天,这种方法在动物数量较少的情况下也较为适用。
4.中药注射液对BN大鼠过敏反应机制的研究
本研究以清开灵注射液、双黄连注射液、鱼腥草注射液、穿琥宁注射液和灯盏花素注射液为受试样本,以青霉素为阳性对照,对BN大鼠进行致敏和激发,通过观察BN大鼠的过敏反应率、肺和气管的病变率来评价中药注射液致BN大鼠的过敏反应情况,通过观察腹腔肥大细胞脱颗粒,血清中IgE抗体、过敏介质和细胞因子含量的变化,来揭示中药注射液的致敏反应机制。
结果显示：①各注射液组的BN大鼠均出现了明显的过敏反应症状,小剂量组过敏反应发生率均高于大剂量组,与对照组比较具有显著性差异；各注射液组的肺组织和气管均出现了明显的炎症性病理改变。②各注射液组肥大细胞阳性率均明显升高,其中清开灵注射液组、鱼腥草注射液组、穿琥宁注射液组与对照组比较具有显著性差异；阳性对照药青霉素组的肥大细胞阳性率明显升高,与对照组比较具有显著性差异,且其阳性率高于中药注射液各组。③中药注射液诱导过敏反应后,BN大鼠血清中IgE抗体含量明显升高,与对照组比较具有显著性差异。④中药注射液诱导过敏反应后,BN大鼠血清中组胺、5-羟色胺、前列腺素D2和白三烯B4的含量均明显升高,细胞因子IFN-γ和IL-4的含量均明显升高,大部分组的IFN-γ/IL-4比值出现了降低的趋势。
试验结果证明：在BN大鼠动物模型上,中药注射液可引起IgE抗体介导的速发型过敏反应,其过敏反应机制可能与以下环节相关：①促进肥大细胞脱颗粒后,释放组胺、5-羟色胺、前列腺素D2和白三烯B4等过敏介质；②诱导Th1/Th2细胞分泌的细胞因子向Th2偏移,从而促进IgE抗体合成。
综上所述,我们得出以下研究结论：
●与豚鼠比较,BN大鼠可能为评价中药注射液过敏反应的适宜动物模型；
●在BN大鼠动物模型上,能产生特异性的全身主动过敏反应和皮肤被动过敏反应；
●采用BN大鼠模型对中药致敏原进行评价时,其规范化参数为：致敏剂量为人临床等倍剂量、致敏次数为3次、致敏间隔为1日、致敏途径为静脉注射,攻击剂量为致敏剂量的2倍、攻击途径为静脉注射、攻击次数为1次、攻击时间间隔为末次致敏后的第14天；
●在BN大鼠动物模型上,中药注射液可引起IgE抗体介导的速发型过敏反应,其过敏反应机制可能与以下环节相关：①促进肥大细胞脱颗粒后,释放组胺、5-羟色胺、前列腺素D2和白三烯B4等过敏介质；②诱导Th1/Th2细胞分泌的细胞因子向Th2偏移,从而促进IgE抗体合成。
Traditional Chinese medicine injection is a novelty form of TCM, the form has many virtues just as higher bioavailability, more efficiency etc. For example, it often plays an important role in treating severely acute diseases.Especially in curing cardiovascular diseases, viral diseases and tumor, TCM injection plays an irreplaceable role. But with the development of traditional TCM injection, expanding production and widely used in clinical applications, the safety problems of traditional Chinese medicine injection was gradually discovered. At present, among the 43 traditional Chinese medicine formulations,75 percent of adverse reaction is induced by using TCM injection, this phenomenon has already attracted wide attention at home and abroad. But for a long time, less attention on adverse reaction of TCM injection results in poor fundamental research about TCM injection. The highest proportion of seriously adverse reaction of TCM injection is anaphy lactic reaction. And mostly reaction type of anaphylactic reaction is immediate allergic response. There isn't standard animal model and assessing methodology, which is an important reason resulting in absence of preclinical safety evaluation, and then TCM injection also was restricted in extensive clinical application.
In this study, Qingkailing injeciton, Shuanghuanglian injeciton, baicalin and chlorogenic acid were used as allergen, trichosanthin was used to positive control, we have observed the BN rats'and guinea pigs'incidence rates, degree of anaphylactic reactin, histamine release and degree of pathological changes in target organs,compared the applicability of anaphylactic reaction in BN rats and guinea pigs, selected suitable animal model which was used to evaluating TCM injection's anaphylactic reaction. We also observed the specificity of the BN animal model by systemic active anaphylactic reaction (ASA) and passive cutaneous anaphylaxis (PCA). Proceeding to the next step, sensitization parameters(sensitization dose, sensitization times, sensitization pathways,sensitization time interval)and excitation parameters(excitation dose,excitation pathways,excitation time interval,excitation volume) was determined, the standard methods which were suitable to evaluate anaphylactic reaction of TCM injection was established.
We have observed degranulation of mast cells in abdominal cavity, antibody levels, inflammatory factors and Th1/Th2 cytokines contents in serum with the eatablished model, then we also attempt to unpuzzle the mechanism of the anaphylactic reaction induced by TCM injection with the eatablished animal model. In this research, Yuxingcao injection, Chuanhuning injection, Dengzhanhuasu injection and ingredients baicalin, chlorogenic acid was used as allergen, penicillin as posistive control. Our research was supported by national nature science foundation of China.
1. Applicability Research of BN rat and guinea pig in evaluating anaphylactic reaction of TCM injection
In this section, we researched the applicability of BN rat and guinea pig in evaluating anaphylactic reaction of TCM injection. Qingkailing injection, Shuanghuanglian injection, baicalin and chlorogenic acid as allergens, BN rats and guinea pigs were sensitized directly. The sensitization pathway was intraperitoneal injection, the dose was equivalent with human clinical dosage. The sensitization was carried out every other day, totally 3 times. The provocation was done in the 14th day after the last sensitization, the provocation method is disposal intravenous injection, The provocation volume is double sensitization value. We would observe symptoms of the anaphylactic reaction after the initiation, calculate the rates and degree of anaphylactic reaction on the basis of SFDA systemic sensitization evaluation criteria. The rates of histamine increase were calculated by histamine content in serum, lung tissues and tracheas detected by ELISA respectively. Pathological changes in target organs were observed by HE staining under light microscope and the rates and degree of pathological changes were calculated. According to analysis these experiment data and result, we could determine the applicability of BN rat and guinea pig in evaluating anaphylactic reaction of TCM injections.
The main testing results are listed blow:
BN rats and guinea pigs are both sensitized by all of four allergens (Qingkailing injection, Shuanghuanglian injection, ingredients baicalin and Chlorogenic acid), but the allergic rates were obviously different between the two animals. The allergic rates of BN rats for every sample are 55.56%,61.11%,75.00% and 83.33% respectively, meanwhile the allergic rates of guinea pigs for every sample were 22.22%,27.77%,16.67% and 41.67% respectively.
The allergic degree of BN rats induced by four tested samples is more serious than that of guinea pigs, especially induced by Shuanghuanglian injection and ingredient baicalin.
Increase rates of histamine in BN rats'serum, lung and trachea are significantly higher than that of guinea pigs. For instance, compared with guinea pigs, increase rates of histamine in BN rats'serum (induced by Qingkailing injection group, Shuanghuanglian injection, ingredients baicalin), lung (induced by Qingkailing injection group, Shuanghuanglian injection, chlorogenic acid), and trachea (induced by Qingkailing injection group) are notabely different.
Significant difference could be found when we compare the pathological changes rate of BN rats'lung tissue with that of guinea pigs'lungs. The pathological change rates of BN rats'lung were 66.7%,72.22%,66.67% and 41.67% respectively when the rats were sensitized by all of allergens, meanwhile the pathological change rates were 44.44%、44.44%、25.00% and 8.33% respectively when parallel tests were done in guinea pigs. But no obvious difference was found when we compare the pathological rates of BN rats'trachea with that of guinea pigs'
Pathological change degree of BN rats'lung tissue was more serious than that of guinea pigs'when the two animals were induced by all of four allergens, especially they were sensitized by Shuanghuanlian injecition and ingredients baicalin. We didn't found obvious difference when we research the pathological change degree of BN rats'trachea with that of guinea pigs'too.
According to analysis above mentioned, we can draw a conclusion that BN rat was more sensitive than guinea pig for evaluating anaphylactic reaction induced by TCM injection, and BN rats is a suitable candidate as animal model for evaluating anaphylactic reaction induced by TCM injection.
2. Specificity research of BN rats'anaphylactic reaction induced by TCM injection
2.1 Active systemic anaphylactic reaction of BN rats induced by TCM injection (ASA)
In this section, we focused on the specificity of BN rats'anaphylactic reaction induced by TCM injection. We observed the BN rats'anaphylactic reactions, determined the level of histamine in serum and tissues by Elisa method, observed the pathological changes of heart, liver, spleen, lung, trachea, stomach and small intestine by HE staining under light microscope and selected specific target organs. Qingkailing injection, Shuanghuanglian injection, ingredients baicalin and chlorogenic acid was used as allergen, trichosanthin was positive control in these experiments.
We can draw the conclusion by above-mentioned research, all of four tested samples(Qingkailing injection, Shuanghuanglian injection, ingredients baicalin and bhlorogenic acid) can induced BN rats'allergic symptom and phenomena, and we found the similar results in positive control. The phenomena were scratching, nose shortness of breath, sneezing and some other responses, and the reactions mostly appeared in 10-20min after initiation and eased after 15min initiated by tested samples. The serious symptoms were hypotonia, the skin temperature lower, convulsion, swoon, other reactions of anaphylactic shock, severe ones even died.
We have found that content of histamine increased in the allergic BN rats'blood, lung and trachea significantly, so we believe the histamine was released from target organ and transfer into blood because of allergic reaction induced by the using TCM injection and ingredients in TCM injection. When we search target organ, we find there were no obvious pathological changes in heart, liver, spleen, stomach, small intestine, but there were pathological changes in lung and trachea. The symptom is obvious inflammatory pathological changes. It showed that lung and trachea were the main target organs in evaluating the allergic response induced by TCM injection.
We find similar phenomenon in positive control, these reactions mostly appeared in 10min after initiation and eased after 30min-lh. Obvious histamine release was found in serum, lung and trachea. Obvious inflammatory pathological change was found in BN rats'lung and trachea when we searched the target organ of allergic reaction. So we can draw the conclusion:lung and trachea are main target organs induced by trichosanthin.
The results demonstrated that BN rats'systemic rapid allergic response can be induced by four tested samples above mentioned.
2.2 Passive cutaneous anaphylaxis of BN rats induced by TCM injection (PCA)
In this section, we researched passive cutaneous anaphylaxis of BN rats induced by TCM injection(PCA experiment). Qingkailing injection,Shuanghuanglian injection,Yuxingcao injection,Chuanhuning injection,Dengzhanhuasu injection were used as allergens. Penicillin sodium was positive control. The antiserum was yielded after the BN rats were sensitized, then the Balb/c mice were sensitized by diluted antiserum (dilution factor:1:25 and 1:50). We would evaluate the specificity of passive cutaneous anaphylaxis of BN rats induced by TCM injection by observing the skin inner spot and Evans blue permeance.
The results showed that we could observe Evans blue permeance after the Balb/c mice were sensitized by all of antiserums. For mostly test samples, Evans blue absorption is higher when the dilution factor is 1:25 than the factor is 1:50, so we supposed the content of IgE in the antiserum whose dilute fator is 1:25 is higher than that in the antiserum whose dilute fator is 1:50.
We could observe skin inner spot in some Balb/c mice when the laboratory animals were sensitized by antiserum (dilution factor:1:25) which were yielding by all of five allergens above mentioned. Diameter of the spot is larger than 5 centimeters. Three Balb/c mice appeard skin inner spot when the allergens are Shuanghuanglian injection (low dose), Yuxiangcao injection and positive control respectively, this phenomenon was obviously different to negative control.But we didn't find remarkably difference when the dilution factor is 1:50. Only Shuanghuanglian injection (low dosage) and positive controls induced inner skin spot, and the rates of positive response were 25% and 50% respectively. That was similar to negative controls.
These results demonstrated that the antiserums can be yielded when the BN rats are sensitized by all of five allergens above mentioned. We can find obviously passive cutaneous anaphylaxis reaction too. So we considered BN rat is a suitable animal model to evaluate passive cutaneous anaphylaxis induced by TCM injection.
3. Standard research on anaphylactic reaction of BN rat animal model induced by TCM injection
In this section, we attempted to determine the standard parameters when the BN rats were used as animal model to evaluate anaphylactic reaction induced by TCM injection. Qingkailing injection, Shuanghuanglian injection, ingredients baicalin and chlorogenic acid were used as allergen, and the guinea pigs were used as control. The sensitizing and provocation parameters which were researched are listed blow: allergic dose (equivalent dose with clinic dosage or double), sensitized method (ip, iv), provocation times (one or two), provocation moment (the 14th day after last sensitization or 21st). According to analysis experimental results above mentioned, we could selected proper parameters when the BN rats were used as animal model to anaphylactic reaction induced by TCM injection.
The results showed that BN rats appeared allergic phenomena which were provocated only one time, the incidence carried out sensitization after first provocated was higher than second provocation. So we consider the provocation number of times is one, and the suitable moment is 14th day after the last sensitization.
All of iv low dosage tested groups (intravenous injection, equivalent dose with clinic dosage) appeared allergic reaction, the rates of anaphylaxis reaction were 50%, 66.67%,75% and 75% respectively. The content of histamine in blood of this group increased obviously. Pathological changes rate in BN rats'tissue which was sensitized by the four allergens were 50%,50%,75% and 25% respectively, the proportion is coincidence with rate of anaphylaxis reaction mostly. So we consider that the sensitization method is intravenous injection, the dose is equivalent with clinic dosage, and the provocation dose is double.
Under the same testing conditions, we found the anaphylactic reaction is more distinct in every aspect researched, such as the rate of anaphylactic reaction, histamine releasing, the rate of pathological changes and the degree of pathological changes. So we can draw a conclusion, the sensibility of BN rats induced by TCM injection is better than guinea pigs.
According to the testing results, we can consider that BN rat was a suitable animal model when we evaluated allergic reaction induced by TCM injection. The suitable allergic and provocation parameters are listed blow:the dose is equivalent with clinic dosage, the sensitization number of time is three and the sensitization is carried out anothor day, provocation number of times is one and provocation moment is the 14th day. These methods can also be used for less tested animals.
4. The research on mechanism of anaphylactic reaction induced by TCM injection
In this section, BN rats were sensitized and provacted by TCM injection, then the rates of anaphylactic reaction and pathological changes in lung and trachea were observed.To probe into the mechanism of anaphylactic reaction of TCM injection, we detected mast cells degranulation in abdominal cavity, the levels of IgE antibody, inflammatory factor and cytokines in serum. In these experiments, Qingkailing injection, Shuanghuanglian injection, ingredients baicalin and chlorogenic acid was used as allergen and penicillin sodium as positive control.
The main testing results are listed blow:
There were significant symptoms in BN rats sensitized by all the five TCM injections and positive control.The rate of anaphylactic reaction induced by the same injection in low dose group was higher that in high dose group.Compared with negative control, there was significant difference. Obvious inflammatory pathological changes were observed in lung and trachea tissues of BN rats sensitized by all the five TCM injections and positive control.The positive rates of mast cells degranulation increased obviously in every sample group. Compared with negtive control,significant difference was showed in some samples group,including Qingkailing injection group,Yuxingcao injection group and Chuanhuning injection group.In positive control (penicillin sodium) group,the positive rate of mast cells degranulation was higher that in every tested sample group. Compared with negtive control, significant difference was showed.After sensitized by TCM injection, the level of IgE antibody in serum of BN rats increased obviously.Compared with the negtive control, there was remarkably difference. After sensitized by TCM injection,both the levels of histamine,5-HT, PGD2, LTB4 and the content of cytokines IFN-y and IL-4 in serum of BN rats increased significantly,on the contrary,in mostly group the ratio of IFN-γ/IL-4 decreased to some extent.
The results demonstrated that TCM injection could induce immediate hypersensitivity mediated by IgE antibody with established BN rat animal model.The possible mechanism of this immediate hypersensitivity including:①TCM injections induced mast cell degranulation and then release histamine,5-HT, PGD2, LTB4.②TCM injections induced Thl/Th2 released cytokines shifting to Th2 and then enhance IgE synthesis.
All sum up, we can draw some conclusion:BN rat is possible more suitable animal model for estimating the anaphylactic reaction of traditional Chinese medicine injection than guinea pig. With BN rat animal model, active systemic anaphylaxis and passive cutaneous anaphylaxis can be found. When we forcast the allergen using BN rat animal model, the standard parameters is:the dose is equivalent with clinic dosage, the sensitization number of time is three and the sensitization is carried out another day, provocation number of times is one and provocation moment is the 14th day. The possible mechanism of this immediate hypersensitivity including:①TCM injections induced mast cell degranulation and then release histamine,5-HT, PGD2, LTB4.②TCM injections induced Thl/Th2 released cytokines shifting to Th2 and then enhance IgE synthesis.
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