【作者】 刘婷；

【导师】 梁爱华；

【作者基本信息】 中国中医科学院 ， 中药学， 2010， 硕士

【摘要】 鱼腥草注射液的致敏性研究鱼腥草注射液为鲜鱼腥草经二次蒸馏后所制成的灭菌水溶液,主要含有挥发性成分,包括甲基正壬酮、癸酰乙醛、月桂醛等,其蒸馏液中含有约50多种成分。药理研究证明鱼腥草注射液有抗病原微生物,消炎、抗过敏、增强机体免疫功能、利尿、镇痛、镇咳、抗活性氧、止血等作用,临床上被广泛用于治疗呼吸道感染、泌尿系统感染、皮肤病、耳鼻喉科感染、妇科感染等疾病的治疗。但随着鱼腥草注射液在临床上的广泛应用,其不良反应的报道也屡见不鲜。在对鱼腥草注射液进行不良反应调查中发现,鱼腥草注射液所致的不良反应可累及机体多个器官系统,临床表现亦复杂多样,严重不良反应有呼吸困难,急性肺水肿,喉头水肿,心跳呼吸骤停和死亡,直接死亡原因为过敏性休克。那么鱼腥草注射液的致敏特点是什么,其致敏物质又是什么,导致超敏反应发生的机理是什么,有必要对此进行深入研究,以便寻找防治鱼腥草注射液不良反应的途径和方法,确实保障人民群众的用药安全。[目的]明确鱼腥草注射液的超敏反应特点,确定鱼腥草注射液的过敏反应物质,并初步探讨其过敏反应发生的机理。[方法]1.鱼腥草注射液的“过敏反应”特点研究1.1豚鼠和BN大鼠的主动全身过敏试验将豚鼠和BN大鼠均随机分为以下各组：①阴性对照组(C)；②阳性对照组(P)；③鱼腥草注射液组(YT)；④吐温80组(T)；⑤鱼腥草蒸馏液组(Y)。试验分为致敏和激发二个阶段。致敏：于试验的第1、3、5天,各组分别腹腔注射相应受试物：C组为生理盐水;P组为0.6%血清白蛋白(BSA)；YT组为含0.5%吐温的鱼腥草注射液；T组为含0.5%吐温的生理盐水；Y组为鱼腥草二次蒸馏液(用NaCl调节渗透压)。注射体积均为lml／只。激发：分别于末次致敏后14天和21天进行激发试验。激发时相应实验组iv给予的受试物为：C组iv生理盐水；P组iv 2.4%的牛血清白蛋白1 ml；YT组iv鱼腥草注射液；T组iv 1%吐温80；Y组iv鱼腥草二次蒸馏液。注射体积均为1ml／只。观察指标：致敏期间每日观察每只动物的症状。静脉注射激发后立刻至60分钟,按照我国《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》中的有关全身主动过敏试验的症状观察和全身过敏评价标准,记录每只动物的过敏反应症状,分析过敏反应强度。1.2大鼠的被动皮肤过敏试验致敏血清制备：将BN大鼠随机分成以下各组：①正常对照组(C)；②佐剂对照组(A)；③阳性对照组(P)；④鱼腥草注射液组(YT)；⑤吐温80组(T)；⑥鱼腥草蒸馏液组(Y)；每组6只动物。将鱼腥草注射液、0.5%吐温80和鱼腥草蒸馏液组分别与弗氏完全佐剂以1：1研匀,配制各组的致敏受试物。将上述混匀后的致敏受试物物分别于D1、D3、D5给BN大鼠腹腔注射,各1 ml。末次注射后21天,将动物用戊巴比妥钠麻醉(30 mg／kg,ip),从腹腔动脉取血,3000转／分离心15分钟,分离血清,冻存于-80℃备用。被动皮肤致敏：将SD大鼠随机分成与BN大鼠各组对应的6组,每组5只。将上述各组的致敏血清用生理盐水稀释成1：1、1：2、1：4、1：8、1：16、1：32。将SD大鼠沿脊柱两侧预先脱毛3×4cm2,分别在脱毛区的左上、右上、左中、右中、左下、右下部位皮内注射1：1、1：2、1：4、1：8、1：16、1：32的稀释血清0.1mL以被动致敏。激发和皮肤蓝色斑点观察：SD大鼠被动致敏48小时后,各组静脉注射与致敏剂量相同的激发抗原(均不含佐剂)加等量的0.8%伊文思兰染料共1mL,进行激发。30分钟后,麻醉处死各组动物,剪取背部皮肤,测量皮肤内层的斑点大小,直径大于5mm者判定为阳性；不规则斑点的直径为长径与短径之和的一半,计算皮肤过敏反应阳性率。同时将蓝斑剪下,剪碎后用冷丙酮浸泡,用分光光度计于波长610nm处测定伊文思蓝含量。1.3 Beagle犬类过敏试验将健康雄性Beagle犬随机分为2组：对照组(C)和鱼腥草注射液组(YT),每组5只。C组iv生理盐水,YT组iv鱼腥草注射液组,给药体积均为3.3mL／kg。2周后,再次注射给药一次。首次注射和再次注射给药后即刻至30 min内,观察动物的“过敏反应”症状,并进行反应程度评分。于给药后30min测定收缩压(Systolic blood pressure, SBP),舒张压(Diastolic blood pressure, DBP);肉眼观察呼吸频率。2.鱼腥草注射液的致敏物质研究2.1 Beagle犬类过敏试验将健康雄性Beagle犬随机分为4组：C组、T组、Y组和YT组；每组4只。给药前,先测定动物血压、心率和呼吸。然后C组iv生理盐水；T组iv 0.5%吐温80；YT组iv鱼腥草注射液组；Y组iv鱼腥草蒸馏液；各组给药体积均为3 ml／kg,缓慢推注,2分钟推完。给药后即刻开始,记录动物的过敏反应症状及其出现的时间以及反应程度,并于给药后10min和30min,分别测定动物的血压、心率和呼吸。首次给药一个月后,再次采用上述同样的给药方法进行试验。2.2小鼠类过敏试验将ICR小鼠随机分为10组,每组10只动物：C组(iv生理盐水);Compound 48/80 (iv5 mg／kg)；Y组(iv鱼腥草蒸馏液)；T1组(iv 0.5%吐温80)；T2组(iv 2.5%吐温80)；T3组(iv 5%吐温80)；T4组(iv 10%吐温80)；YTl组(iv含0.5%吐温80鱼腥草注射液)；YT2组(iv含2.5%吐温80鱼腥草注射液)；YT3组(iv含5.0%吐温80鱼腥草注射液)；YT4组(iv含10%吐温80鱼腥草注射液)。以上所有组的受试物包括生理盐水均用0.4%伊文思蓝配制。各组小鼠分别静脉给予20ml／kg。给药后观察1h内动物的行为学变化,耳廓蓝染情况。小鼠于给药后1h剪下双耳,用3ml冷丙酮浸泡,用分光光度计检测耳廓的伊文思蓝渗出量。3.吐温80的致类过敏机理初步研究3.1吐温80对Beagle犬补体系统的作用在2.1试验中,于给药后10、30min取血,分离血浆,用半数溶血试验法和ELISA试剂盒测定血浆的总补体活性(CH50)；用ELISA试剂盒测定末端补体激活复合物(C5b-9)含量。3.2吐温80对SD大鼠的血浆组胺和补体活性的影响将SD大鼠分为：C组、0.5%T组、2.0%T组、YT组、阳性药compand48/80 0.625mg/kg组。给药组动物均iv给予相应的受试物,给药量10 mL／kg,1min内给药完毕。C组给予等体积生理盐水。分别于注射后10、20、30 min,取血,分离血清。用ELISA试剂盒测定总补体活性、补体激活片段C5b-9、组胺及类胰蛋白酶含量的影响。3.3吐温80对BN大鼠及豚鼠血清IgE含量的影响将BN大鼠、豚鼠分别随机分为：C组,佐剂组,0.2%吐温80组,4)佐剂+吐温组。于试验的第1、3、5天给动物腹腔注射0.2%吐温lml以致敏,于末次致敏后第7、14天,吐温组静脉注射含0.8%吐温生理盐水乳液1 ml／只进行激发刺激；而对照组静脉注射相同体积生理盐水。每次激发后1 h内,从眼眶静脉丛取血,分离血清,采用ELISA竞争试验法检测IgE含量。[结果]1.鱼腥草注射液的“过敏反应”特点1.1鱼腥草注射液不引起免疫介导的过敏反应豚鼠和BN大鼠全身过敏试验结果显示,T组、YT组和Y组无论是致敏(D1、3、5天腹腔注射)给药,还是二次激发给药后,所有动物均未见典型的过敏反应症状。而阳性药BSA组激发后出现典型的过敏反应,阳性率为100%,过敏反应程度为强阳性反应。结果提示,鱼腥草注射液组、吐温80以及鱼腥草蒸馏液均不引起免疫介导的主动全身过敏反应大鼠的被动皮肤过敏试验结果显示,只有阳性对照组(BSA组)的被动皮肤过敏反应为阳性,阳性率为100%。其他实验组包括T组、YT组和Y组均未能诱导被动皮肤过敏反应,结果均为阴性。结果提示,鱼腥草注射液组、吐温80以及鱼腥草蒸馏液均不引起免疫介导的被动皮肤过敏反应。以上二类试验结果表明,鱼腥草注射液的“过敏反应”可能不是由免疫介导的过敏反应。1.2鱼腥草注射液可引起的类过敏反应Beagle犬首次给药后,YT组的所有动物迅速(给药后约2-5min)出现口唇、耳部红肿、烦躁、甩头,前肢挠头、挠耳、蹭鼻、添舌,步态不稳,静卧,排尿,排便,恶心等类似过敏反应的症状,反应阳性率为100%,反应程度达强阳性标准。同时,该组动物的血压明显下降,呼吸频率增快。再次给药后的症状与单次给药相似,反应未见增强。C组的所有动物均未出现异常反应,血压正常。由于鱼腥草注射液首次静脉注射即可导致明显的反应,因此,认为不是免疫介导的过敏反应,而是类过敏反应。2.鱼腥草注射液的致敏物质是吐温80根据Beagle犬类过敏试验结果,鱼腥草蒸馏液无论是首次静脉注射或再次静脉注射给药,Beagle犬均未见任何过敏样反应的症状。表明鱼腥草注射液的过敏样反应与鱼腥草二次蒸馏液关系不大。而鱼腥草注射液(含0.5%吐温80)和0.5%吐温80组的所有Beagle犬均在首次注射和再次注射给药后表现为明显的过敏样反应症状,包括皮肤粘膜、消化道症状、呼吸循环以及神经系统症状,与临床报道的鱼腥草注射液“过敏反应”表现类似。结果表明,鱼腥草注射液的“过敏反应”与吐温80有关,吐温80是鱼腥草注射液中的主要致敏物质。小鼠静脉注射加有0.4%伊文思蓝为指示剂的0.5～10.0%吐温80、含0.5～10.0%吐温80鱼腥草注射液后,均可见耳廓血管通透性增高,耳廓呈不同程度蓝染,并呈现剂量依赖性。而鱼腥草蒸馏液不引起小鼠耳廓血管通透性增高。该结果也证明吐温80是鱼腥草注射液中的主要致类过敏物质。3.吐温80的致类过敏机理初步研究吐温80可导致Beagle犬血浆总补体活性(CH50)降低和补体激活片段复合物(C5b-9)含量增加,注射药物后10 min较明显。BALA／C小鼠静脉注射2.0%吐温后,血浆组胺含量明显升高。结果提示,吐温80致类过敏机理可能与补体激活和组胺释放有一定关系。[结论]1.鱼腥草注射液导致的过敏反应可能是一种不需要抗体工gE介导的类过敏反应。2.鱼腥草注射液的超敏反应与鱼腥草二次蒸馏液关系不大；而其助溶剂0.5%吐温80给药后表现为明显的超敏反应症状,表明吐温80是鱼腥草注射液中的主要致敏物质。3.吐温80导致类过敏反应的发生是多条途径共同作用的结果：①通过激活补体系统,产生过敏毒素,诱发过敏反应的发生；②在不依赖工gE抗体的情况下,直接诱导肥大细胞脱颗粒,释放组胺等活性介质。更多还原

【Abstract】 The sensitization research of Yuxingcao InjectionYuxingcao Injection is the sterilized solution of redistilled ordate houttuynia in water, it contains over 50 ingredients, which are mainly volatile organic compounds, including methyl nonyl ketone, decanoyl acetaldehyde, laurel aldehyde, etc. The pharmacological research proves that Yuxingcao Injection can counteracts pathogenic microorganism, diminishes inflammation, antianaphylaxis, built up health, diuresis, relieves pain, relieves cough, counteracts active oxygen, stanches blood, it was widely used to treat respiratory tract infection, urinary infection, skin disease, otorhinolaryngologic infection and gynaecologic infection. However, as Yuxingcao Injection was used more and more, much adverse reaction was reported. In the investigation of them, it was found that these adverse reaction can affect several systems in human’s body, the clinical features are different. Serious adverse reaction include dyspnoea, acute pneumonedema, laryngeal edema, cardiopulmonary arrest and death, the direct reason of death is anaphylactic shock. What is the action features of sensitization of Yuxingcao Injection, what is the sensitizer and what is the mechanism of hypersensitivity? It is necessary to have a close study of the reason and to look for the prevention way of Yuxingcao Injection’s adverse reaction, then to guarantee the people’s safety.[Objectives]Clarification of the action features of the hypersensitivity of Yuxingcao Injection, decision of the sensitizer and preliminary investigation of the mechanism of hypersensitivity. [Methods]1. The study of action features of the Yuxingcao Injection’s anaphylactic reaction1.1 Guinea pigs’and Brown Norway rats’positive systemic anaphylaxis experimentGuinea pigs or Brown Norway rats were divided into five groups randomly:1) Negative control(C); 2) Positive control(P); 3) Yuxingcao Injection group(YT); 4) Tween-80 group(T); 5) Distilled Yuxingcao Injection group(Y). The experiment contains two stage:sensitization stage and initiation stage.Sensitization:All these groups used 1mL corresponding drug by intraperitoneal[i.p.] in day 1,3, 5. physiological saline(C); 0.6g/mL bovine serum albumin(BSA) (P); Yuxingcao Injection with 0.5% Tween-80 (YT); physiological saline with 0.5% Tween-80(T); 5) Distilled Yuxingcao Injection (The permeation pressure was controlled with NaCl)(Y).Initiation:All groups were initiated by injection of the same antigen. All these groups used 1mL corresponding drug by i.p. on the 14th and 21st day after the last sensibilization to initiation, physiological saline(C); 2.4% bovine serum albumin(BSA) (P); Yuxingcao Injection (YT); 1% Tween-80(T); 5) Distilled Yuxingcao Injection (Y).Index:The symptom of each animal’s anaphylactic reaction was observed everyday during the sensitization time. In the 60 minutes after the initiation, the intensity of anaphylactic reaction was judged according to the related observation standard of positive systemic anaphylaxis’s symptom and evaluation standard of systemic anaphylaxis.1.2 Brown Norway rats’passive systemic anaphylaxis experimentThe preparation of sensitized blood serum:Brown Norway rats were divided into five groups randomly:1) Normal control(C); 2) Adjuvant control(P); 3) Positive control(P); 4) Yuxingcao Injection group(YT); 5) Tween-80 group(T); 6) Distilled Yuxingcao Injection group(Y). Each group has 6 animals. Yuxingcao Injection,0.5% Tween-80 solution and Distilled Yuxingcao Injection was mixtured with Freund complete adjuvant (1:1) respectively and sensitized sample in each group was prepared.Brown Norway rats used 1mL corresponding drug by intraperitoneal[i.p.] in day 1,3,5. On the 21st day after the last injection, these animals were anaesthetized with PentobarbitalSodium (30 mg/kg, ip), blood was obtained from celiac artery and centrifugated in 3000r/m speed. Blood serum was separated and refrigerated at-80℃as a back up.Passive skin sensitization:SD rats were divided into corresponding 6 groups with Brown Norway rats’5 groups. Each group has 6 animals. The above sensitized blood serum was diluted to 1:1,1:2,1:4,1:8,1:16,1:32。SD rats were firstly denuded by vertebral column, (3×4cm2),0.1mL 1:1,1:2,1:4,1:8,1:16,1:32 blood serum was injected by intracutaneous respectively in the upper left, upper right, middle left, middle right, down left, down right site of denuded zone to cause passive sensitization.The initiation and observation of blue spot:48 hours after SD rats’s passive sensitization, each group were initiated by injection of the same volume of antigen as sensitizer (with out adjustvor) and the same amount of Evans dye(the total volume is 1 mL). After 30 minutes, animals were killed by anesthesia, the skin on back was obtained and the inner spot was measured. The spot larger than 5mm is judged as positive. The diameter of irregular spot is a half of total of long diameter and short diameter. The positive rate of skin’s anaphylactic reaction is worked out. The blue spot is obtained and sheared, soaked in cool acetone, the content of Evans blue was measured by spectrophotometer in 610nm.1.3 Beagle dogs’sensitization action Healthy male Beagle dogs were divided into two groups randomly:control group (C) and Yuxingcao Injection group (YT),5 animals in each group. Group C was injected physiological saline and group YT was injected Yuxingcao Injection, the injection volume is 3.3mL/Kg. Another injection was performed after two weeks. Immediately and 30 minutes after the first injection, animals’sensitization action was observed and evaluated according to the reaction degree.30 minutes after the injection, Systolic blood pressure(SBP)and Diastolic blood pressure(DBP) was measured, the breath frequency was observed by eyes.2. The study of sensitizer in Yuxingcao Injection2.1 Beagle dogs’anaphylactoid reactionHealthy male Beagle dogs were divided into 4 groups randomly:group C, group T, group Y, group YT.4 animals in each group. The blood pressure, heart rate and breath frequency were measured before the use of drug. Group C was injected physiological saline, Group T was injected 0.5% Tween-80, group YT was injected Yuxingcao Injection and group Y was injected distilled Yuxingcao Injection, the injection volume is 3.3mL/Kg and the injection speed was controlled in order that it would be finished in 2 minutes. The symptom of animals’sensitization reaction and the appearance time was noted immediately after the injection. The measure of blood pressure, heart rate and breath frequency were performed 10minutes and 30 minutes after the injection. The above:experiment was performed again after the first injection.2.2 Small mice’s anaphylactoid reactionICR mice were divided into 10 groups randomly,4 animals in each group, group C(physiological saline), group Y (Yuxingcao Injection), group T1 (0.5% Tween-80), group T2 (2.5% Tween-80), group T3 (5% Tween-80), group T4 (10% Tween-80), group YT1 (Yuxingcao Injection with 0.5% Tween-80),group YT2(Yuxingcao Injection with 2.5% Tween-80),group YT3(Yuxingcao Injection with 5% Tween-80),group YT4(Yuxingcao Injection with 10% Tween-80), all physiological saline used in these groups were made with 0.4% Evans blue. The injection volume is 20mL/Kg. The change of action and the blue appearance of ear was observed 1h after the injection. The ears were obtained, soaked in 3mL cool acetone, the content of Evans blue was measured by spectrophotometer in 610nm.3. The preliminary mechanism study of Twain 80’s anaphylactoid3.1 The role of Tween-80 in Beagle dogs’make-up systemIn 2.1 experiment, blood was obtained 10 and 30 minutes after the use of drug and the blood serum was separated. The blood serum’s total activity (CH50) was measured by half hemolysis test method and the end activation complex molecule (C5b-9) was measured by ELISA. 3.2 The role of Tween-80 in SD rats’histamine content in blood serum and complement activitySD rats were divided into 5 groups randomly, group C,0.5% group T,2.0% group T, YT group, positive-control group. Each group was injected corresponding drug by iv. The volume is 10 mL/kg, it is finished in 1 minutes. Group C is injected the same volume of physiological saline. The blood was obtained and the serum was separated 10,20,30 minutes after the injection. The role of Tween-80 in total activity (CH50), activation complex molecule (C5b-9) the histamine and trypsinase content was measured by ELISA.3.3 The influence of Tween-80 on the IgE content in BN rats’and Guinea pigs’serumBN rats and Guinea pigs were divided into 4 groups randomly. group C, adjuvant group,0.2% Tween-80 group, adjuvant with Tween-80 group. All animals were injected 1mL 0.2% Tween-80 by ip to sensitized on day 1,3,5.7 and 14 days after the last sensitization, The Tween-80 group was injected 1mL 0.8% physiological saline emulsion and control group was injected the same volume of physiological saline. 1h after each initiation, the blood was obtained from the eye and the serum was separated. The content of IgE was measured by ELISA test.[Results]1. The features of Yuxingcao Injection’s sensitization reaction1.1 Yuxingcao Injection doesn’t cause an anaphylactic reaction mediated by immunityGuinea and BN rats systemic hypersensitivity test results show that in group T, YT group and Y group, whether sensitization in day 1,3,5 by ip or secondary initiation, no typical sensitization symptom appears. But the result of positive control BSA is very different, the positive rate is 100%. The degree of sensitization is strong. The results shows that Yuxingcao Injection, Tween-80 and distilled Yuxingcao Injection does’t cause an anaphylactic reaction mediated by immunity.The rat passive skin allergy test results show that only the result of positive control (BSA) is positive, the positive rate is 100%. Other group including group T, YT group and Y group failed to induce passive skin allergy, all the result is negative. The results also shows that Yuxingcao Injection, Tween-80 and distilled Yuxingcao Injection does’t cause an anaphylactic reaction mediated by immunity.The two above experiment shows that the sensitization reaction of Yuxingcao Injection may not be an anaphylactic reaction mediated by immunity.1.2 Possible naphylactoid reaction caused by Yuxingcao InjectionAfter the first use of drug, all Beagle dogs in YT group quickly have (after 2-5min)the symptom of red lips, red ears, forelimb, scratching, etc, similar to the symptoms of an allergic reaction and positive rate is 100%, the reaction degree is strong.At the same time, the blood pressure decreased obviously and the breath turns fast. The same and no more symptom appears after the second use of drug. All the animals in group C has no unmoral reaction. Because obvious symptom appears just after the first use of Yuxingcao Injection, it can be reduced that the hypersensitivity of Yuxingcao Injection may be not anaphylactic reaction mediated by immunity, but a naphylactoid reaction.2. Tween-80 is the main sensitizer in Yuxingcao InjectionAccording to the results of Beagle dogs’anaphylactoid reaction experiment, there is no any hypersensitivity symptom appear, whether the redistilled Houttuyniae solution was used by intravenous injection once or twice. It means the hypersensitivity of Yuxingcao Injection has no much relation with redistilled Houttuyniae solution. However, Obvious symptom of hypersensitivity occur on all Beagle dogs after the first and the second injection in the Yuxingcao Injection (0.5% Tween-80 containing) group and 0.5% Tween-80 group, including the mucocutaneous symptom, digestive tract symptom, the respiratory system’s and circulatory system’s symptom, neural symptom’s symptom, etc. as the clinically symptoms of Yuxingcao Injection’s anaphylactic reaction. The results shows that Yuxingcao Injection’s anaphylactic reaction is related to Tween-80, Tween-80 is the main sensitizer in Yuxingcao Injection.After use of 0.5～10.0% Tween-80 with 0.4% Evans blue as indicator and Yuxingcao Injection with 0.5～10.0% Tween-80, mice’s auricle vasopermeability increases, auricle becomes blue in different degree and is present to be dose depentdant. But Yuxingcao Injection can’t cause the increase of auricle vasopermeability.This result also shows that Tween-80 is the main sensitizer in Yuxingcao Injection.3. The preliminary mechanism study of Twain 80’s anaphylactoidTween-80 can cause Beagle dogs’activity decrease of plasma total complement activity (CH50) and content increase of complement activation clips complex (C5b-9). This change of index appears 10 min after the intravenous injection. BALA/C mice’s plasma histamine content increased significantly after intravenous 2.0% Twain. The results shows that the mechanism of Twain 80’s anaphylactoid may be related to complement’s activation and histamine’s release.[Conclusions]1. The hypersensitivity of Yuxingcao Injection may be an anaphylactoid reaction, which not needed to be mediated by IgE antibody.2. The hypersensitivity of Yuxingcao Injection.has no much relation with redistilled Houttuyniae solution, but obvious symptom of hypersensitivity occur on all Beagle dogs after the injection in 0.5% Tween-80 group, which show that Tween-80 is the main sensitizer in Yuxingcao Injection.3. The occurrence of anaphylactoid reaction caused by Tween-80 is the outcome of combined action of different ways:①Activation of make-up system produces the anaphylatoxin, which induced the anaphylactic reaction;②Tween-80, not relying on IgE antibody, induces the mast cell degranulation directly to release the active medium such as histamine;更多还原