Research On Animal Models Of Anaphylactoid Reactions In TCM Injections

Background:TCM injections adverse reactions primarily are anaphylactoid reaction,clinical symptoms similar to allergic reactions but unrelated with antigen-specific immune response.When initial contact with these drugs anaphylactoid reaction can happen.And it has correlation with dose and injection speed.The distinction between allergic reactions and anaphylactoid reaction are very important clinically.Such as allergic reactions,when patients exposure to allergens react again they may be fatal.While anaphylactoid reactions is different,when patients re-exposure to the allergen reaction is not necessarily increased,even no reaction possibly.Anaphylactoid reaction mechanism is not clear.It may lead to activation of the inflammatory response.Anaphylactic shock ismost harmfulland skin flushing,urticaria-like rash is most commonin TCM injections anaphylactoid reactions.According to the technical guidance of the natural medicine natural medicine,traditional Chinese medicine of immunotoxicity studies,allergen study is one of a rule in traditional Chinese medicine injection safety study.But so far,allergic reactions have not yet developed technical guidelines and relevant experimental methods were few.The interim evaluation method of TCM injections allergy reactions is mainly for allergic reactions not anaphylactoid reactions.And it relation to the immune status of the patient,Tween-80 levels of TCM injections and many other factors.The current method of allergy evaluation is based on normal physiological state of the animal,clinical user of traditional Chinese medicine injections are pathological state,the difference between the two is easily lead to false results of the evaluation.Objectives:This paper relies on "study on key technologiesofgeneral character safety of traditional Chinese medicinal injections",Guang dong major projects(project number:2010A090200076).We focuses on allergic reactions of traditional Chinese medicine injection,which is one of a problem in general character safety.We screen potentially sensitizing substances of traditional Chinese medicine injections and establish relevant large animal model,in order to improve evaluation system.1.Applicate mouse popliteal lymph node assay to evaluate potentially sensitizing substances of Xueshuantong injection.2.Exploring allergies model of Wuzhishan miniature pig under fever state.3.Exploring allergies model of Beagle dogs under immunocompromised state.4.Impact on blood biochemical indicators in immunocompromised Beagle dogs when anaphylactoid reactions occurs.Methods:In this paper,we use mouse popliteal lymph node assay to evaluate potentially sensitizing substances of Xueshuantong injection and to explore allergies model of Wuzhishan miniature pig under fever state.We study on allergies model of Beagle dogs under immunocompromised state and to search allergies biochemical indicators of Beagle dogs when anaphylactoid reactions occurs.1.Rats were randomly divided into saline group,20%DMSO vehicle group,diclofenac group,Xueshuantong injection group,PNS group,ginsenosides Rg1 group,ginsenoside Rb1 group,ginsenoside Rd Groups,ginsenoside Re group,monomer 1(unknown)groups,each group were 10 rats.Each separate samples were dissolved in 20%DMSO vehicle,the dose of diclofenac is 0.75mg/rat,the dose of each separate samples is 1mg/rat,the administered volume is 50μl/rat.The dose of Xueshuantong injection is a single clinical dose.The first day of the experiment,rat’s toe were disinfected,drawing 50μl of each test substance injected into rats right toe,left toe was no treatment,then observe six days.The 7th day of of the experiment,rats were sacrificed,removing PLN,placed in an ice bath for 1%BAS-PBS,and remove excess tissue and fat,drain water and weighed.Then a single cell suspension was prepared according to the following formula,in order to calculate the weight index and cell index.WI = administration side of the lymph node weight/weight is not administered side of the lymph nodes;CI=administration side of the lymph node cells/lymph node cells are not administered side.2.The Wuzhishan miniature pigs were randomly divided into six groups,n = 3,they were saline group(G1),Tween-80 group(G2),Zhongjiefeng injection group(G3),fever model saline group(G4),fever model Tween-80 group(G5),fever model Zhongjiefeng injection group(G6).Experimental dose setting:2.4-dinitrophenol dose of 0.14g/kg,intraperitoneal injection;Zhongjiefeng Injection:0.48ml/kg,intravenously;Tween-80:2.5 ml/kg,intravenously.After quarantine observation 2 days.The 3th day each animal draw blood,blood samples were centrifuged at 3000rpm for 10min,light vampire clear,sealed freezer at-80 ℃ preservation.Fever model group were injected with 2.4-dinitrophenol test solution,after successful modeling,intravenous administration.Rectal temperature were recorded before and after modeling.Non-manufacturing group directly administered intravenously.Drawing blood before administration,after modeling,after administration of 0.5h,1h.Observed animal behavioral changes after administration immediately and recorded it.Last we determinate histamine,complement C3 content.3.Beagle dogs were randomly divided into two groups:normal control group,model immunocompromised groups,each group were four,half male and half female.Immunocompromised model group were injected by 12.0mg·kg-1 cyclophosphamide intraperitoneally 3 times(2 days/time).Record daily general clinical symptoms during the evaluation of the modeling results.After the modeling,the dose of Zhongjiefeng injection were 0.50ml·kg-1,intravenously.At the same time,drawing blood before modeling,before administration,after administration 5min,15min,30min and 60min.The blood samples were centrifugated at 3000rpm 10min,stored at-80 ℃ freezer.Last determinated histamine,IgE,IgG and IgM levels.4.The experimental groups and procedures consistent with the experimental one.After blood collection is complete,the serum sample is placed in a water bath at 37℃ standing 30min-50min,upon serum precipitation,centrifuge 3000 r/min lOmin.A portion of the serum is used to detect blood chemistry indicators,aspartate aminotransferase(AST),alanine aminotransferase(ALT),alkaline phosphatase(ALP),lactate dehydrogenase catalase(LDH),blood urea nitrogen(UREA),creatinine(CRE),total bilirubin(TBIL),creatine kinase(CK),total protein(TP),albumin(ALB),total cholesterol(CHOL),triglyceride(TG),glucose(GLU)measured by automatic biochemical analyzer;using electrolyte analyzer to measure Na,K and Cl ions.Results:1.Vehicle group showed no significant difference compared with the saline group,WI<2,CI<5.PNS,ginsenosides Rgl,ginsenoside Rbl were WI>2,CI>5,with a significant difference.The experiment results consistent with P815 cells results.2.Fever rectal temperature changes of each model group Wuzhishan miniature pig:the temperature of fever model Tween-80 group,fever model saline group,fever model Zhongjiefeng injection group after modeling is higher than before the experiment,with a significant difference.Histamine change:histamine content of Tween-80 group,Zhongjiefeng injection group and fever model Zhongjiefeng injection group after administrating 0.5h is higher than before the experiment,with a significant difference;histamine content of fever Tween-80 group of 1h lower than before.Complement C3 change:after administration of 0.5h,complement C3 content of Tween-80 group is higher than before the experiment,with a significant increase.Behavior Change:show positive anaphylactoid reaction:Tween-80 group,fever model Tween-80 group,Zhongjiefeng injection group;shows uspicious anaphylactoid reactions:fever model Zhongjiefeng injection group;show negative anaphylactoid reaction:saline group,fever model saline group.3.After intravenous injection,the behavior of immunosuppressed Beagle dogs changed,and the rating of overall behavior abnormality of immunosuppressed group is higher than normal control group;the histamine of both groups was gradually rising,and began to decline after 30min,gradually tended to be normal after 60min.During 5min-30min,the histamine of immunosuppressed Beagle dogs increased,and there was a significant difference between these two groups(P<0.01);while the overall variation regularity of IgE,IgG,IgM is not obvious4.Serum alanine aminotransferase(ALT):after modeling immunocompromised group lower than before,with a statistically significant(P<0.05);after drug 30min,immunocompromised group higher than normal control group,with a statistically significant(P<0.01).Aspartate aminotransferase(AST):after drug administration after 60min,normal control group higher than 30min,with a statistical significance(P<0.05).Conclusions:1.Comparing the results of P815 with direct popliteal lymph nodes of rat,we know both experimental results have some relevance.We can comprehensive evaluate Xueshuantong injection and sensitization potential of their separate components combined this two experiments.This method is a reliable,effective,and low false-negative method to screen sensitization potential of other traditional Chinese medicine injections.Based on previous work,ginsenoside Rbl and Rgl may be sensitization potential.2.In this experiment,compared anaphylactoid reactions of fever state Wuzhishan miniature pig with normal physiologica states,Sarcandrae group and Tween-80 group showed positive anaphylactoid reactions.While in the fever state conditions anaphylactoid reactions were expressed as suspicious.Showing that under fever state,the strength of anaphylactoid reactions declined.3.After administration of 5-30min,strength of anaphylactoid reactions of Beagle dogs is stronger than the normal control group.Suggesting that the state of body can affect anaphylactoid reactions strength.After experiment,we suggest that,different types of traditional Chinese medicine injections should establish happropriate pathological states allergy models of Beagle dogs.4.ALT and AST of immunocompromised groups were decreased after administration 30min.After administration 60min,they gradually increased.After administration 30min,normal control group were decreased.After administration 60min they basically unchanged.Combined with literature,suggesting that ALT and AST may have some relevance to anaphylactoid reactions,but the correlation with allergies need large sample experiment for further verification.