The Sensitization Research Of Yuxingcao Injection

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;...