| Traditional Chinese medicine injection has many virtues just as higher bioavialability,more efficiency etc.It often plays an important role in treating cardiovascular disease,cancer and other severely acute diseases.Duing to the complex compositions of TCM injection,the backward preparation technology and many other reasons lead to the clinical adverse reaction.The highest proportion of seriously adverse reactions of TCM injection is anaphylactic reaction.Adverse reaction,occur frequently,which is an important reason resulting in restricting in extensive clinical application.The key to solve this problem is improving preclinical safety evaluation,establishing standard animal model and looking for the biomarkers of allergenic reaction.In this study,Shuanghuanghlian injection and Qingkailing injection were used as allergen,Ovalbumin was used to positive control.We have observed the SD rats’,the ICR mouses’,the BN rats’ and Guinea pigs’ incidence rates,degree of allergenic reaction,tryptase,histamine,β-hexosaminidase,IgE release and passive cutaneous anaphylaxis test(PCA test),compared the applicability of allergenic reaction in four kinds of animals,selected suitable animal model whih was used to evaluating TCM injection’s allergenic reaction.On the basis of it,we established a set of Metabonomics studie methods of animal models of allergenic reaction.Our research was supported by national nature science foundation of China(81173540).1 The research on animal model for evaluating allergenic reaction induced by TCM injection1.1 The preliminary study on animal model for evaluating allergenic reaction induced by TCM injectionIn this section,we researehed the applicability of BN rat,SD rat,ICR mouse and guineaPig in evaluating allergenic reaction of TCM injeetion.Qingkailing injection,Shuanghuanglian injection as allergens,four kinds of animals were sensitized directly.We observed the symptoms,the degree of allergenic reaction,the level of IgE,HIS,TPS and β-Hex in plasma and PCA test in SD rats.The testing results:four kinds of animals sensitized by OVA,Qingkailing injection,Shuanghuanglian injection,but the allergic rates were obviously different among the animals.(1)The allergic rates of BN rats for every sample were 87.50%,62.50%,75.00%;The allergic rates of guineaPigs for every sample were 87.50%,62.50%,50.00%;The allergic rates of SD rats for every sample were 12.50%,25.00%,12.50%;The allergic rates of ICR mouses for every sample were 25.00%,0.00%,25.00%;The allergic rates of BN rats for every sample were higher than guinea Pigs,SD rats and ICR mouses.The allergic degree of BN rats is more serious than that of other three kinds of animals.(2)Increase rates of IgE,tryptase,β-hexosaminidase in guinea Pigs OVA group were higher than that in SD rats,BN rats and ICR mouses.Increase rates of IgE,tryptase,histamine,β-hexosaminidase in BN rats Qingkailing injection group and Shuanghuanglian injection group were higher than SD rats,guinea Pigs and ICR mouses.(3)PCA test by using sera from OVA group,Qinkailing injection group,Shuanghuanglian injection group of BN rats after irritations showed the strong positive result characterized as large amount of subcutaneous effusions of Evans blue in SD rats.Rates of positive response were 75.00%,37.50%,50.00%,that were higher than that of other three kinds of animals.According to analysis above mentioned,we can draw a conclusion that BN rat is more sensitive than the other three kinds of animals for evaluating allergenic reation induced by TCM injection,and BN rats maybe a suitable candidate as animal model for allergenic reaction induced by TCM injection.1.2 The Confirmatory study on animal model for evaluating allergenic reaction induced by TCM injectionIn this section,we foucuse on the applicability of BN rat,SD rat,ICR mouse and guineaPig in evaluating allergenic reaetion of TCM injeetion.Yinzhihuang injection,Yuxingcao injection,Chuanhuning injection,Mailuoning injection,Shengmai injection as allergens,four kinds of animals were sensitized directly.We observe the animals’ allergenic reaetion,the level of IgE,tryptase,histamine,β-hexosaminidase in plasma and passive cutaneous anaphylaxis test(PCA test)in SD rats.The testing results:four kinds of animals sensitized by OVA,Yinzhihuang injection,Yuxingcao injection,Mailuoning injection,Shengmai injection,Chuanhuning injection,but the allergic rates were obviously different among the animals.(1)The allergic rates of BN rats for every sample were 87.50%,50.00%,50.00%;75.00%,62.50%,37.50%.Except the Chuanhuning injection group,the allergic rates of BN rats for every sample were higher than guinea pigs,SD rats and ICR mouses.The allergic degree of BN rats was more serious than that of other three kinds of animals.(2)Increase rates of IgE,tryptase,β-hexosaminidase in guinea Pigs OVA group were higher than that in SD rats,BN rats and ICR mouses.Increase rates of tryptase,histamine,β-hexosaminidase in BN rats Mailuoning injection group were higher than that in SD rats,guinea Pigs and ICR mouses.The release rate of IgE,histamine andβ-hexosaminidase in BNrats Shengmai injection group was higher than that in SD rats,guinea Pigs and ICR mouses.The release rate of IgE,histamine,tryptase and β-hexosaminidase in BNrats Yuxingcao injection group was higher than that in other three kinds of animals.(3)PCA test by using sera from OVA group,Yinzhihuang injiection group,Yuxingcao injection group,Mailuoning injection group,Chuanhuning injection group and Shengmai injection group of BN rats after irritations showed the strong positive result characterized as large amount.of subcutaneous effusions of Evans blue in SD rats.Except Chuanhuning injection group,rates of positive response were were higher than other three kinds of animals.According to analysis above mentioned,we can draw a conclusion that BN rat is more sensitive than guinea Pig for evaluating allergenic reaction induced by TCM injection,but maybe some certain varieties of TCM injections need specific animal models.2 The research of the way to improve allergenic reaction2.1 The research of adjuvants improve allergenic reaction of Yinzhihuang injection and Shengmai injectionIn this section,Yinzhihuang injection and Shengmai injection were mixtured with Freund complete adjuvant(1:1)respectively,and sensitized samples in each group.We observed incidence rates,degree of allergenic reaction,tryptase,histamine,β-hexosaminidase,IgE release and PC A test,compared the difference between adjuvant groups and normal injection groups.The testing results:(1)The allergic rates of adjuvant groups were higher than normal injection groups and the allergic degree is more serious than that of normal injection groups.(2)IgE,histamine,tryptase,β-hexosaminidase concentration of normal injection group with the corresponding normal saline group were no significant differences.Compared with normal saline group,the level of IgE,histamine and β-Hex in the plasma of Yinzhihuang injection adjuvant groups increased significantly,and the level of tryptase,β-hexosaminidase in the plasma of Shengmai injection adjuvant groups increased significantly.The release rate of IgE,histamine,tryptase and p-hexosaminidase in the plasma of adjuvant groups were higher than that in normal injection groups.(3)PCA test by using sera from adjuvant group after irritations showed the strong positive result characterized as large amount of subcutaneous effusions of Evans blue in SD rats and the rates of positive response were higher than that of normal injection groups.According to analysis above mentioned,we can draw a conclusion that Freund complete adjuvant can improve allergenic raection of TCM injection and improve the sensitivity of the animal model.2.2 The research of the artificial antigen improving allergenic reaction of Chlorogenic acidIn this section,The artificial antigen was synthesized by conjugating Chlorogenic acid(CA)and the BN rats serum in vitro.The BN rats were injected hypoderm ically with the mixture(mixture group)to rasearch the artificial antigen improveing allergenic raection of Chlorogenic acid.The testing results:(1)The allergic rates of mixture groups were higher than CA groups and the allergic degree was more serious than that of CA groups.(2)IgE,histamine,tryptase andβ-hexosaminidase concentration of CA group with the corresponding normal saline group were no significant differences.Compared with normal saline group,the level of IgE andβ-hexosaminidase in the plasma of mixture group increased significantly and the release rate of IgE,histamine,tryptase,β-hexosaminidase in the plasma of mixture groups was higher than that in CA groups.(3)PCA test by using sera from mixture group after irritations showed the strong positive result characterized as large amount of subcutaneous effusions of Evans blue in SD rats and the rates of positive response are higher than that of CA groups.According to analysis above mentioned,we can draw a conclusion that CA can be used as a kind of hapten.The artificial antigen was synthesized by conjugating CA and the BN rats serum in vitro to stimulate the immunologic response and induce allergic reaction in BN rats and that is a new way to screening allergenic raection caused by hapten.3 The preliminary study on animal model for evaluating anaphylactoid reaction induced by TCM injectionIn this section,Yuxingcao injection was used as allergen,Tween 80 was used to positive control,we had observed the SD rats’,the ICR mouses’,the BN rats’ and Guinea pigs’ incidence rates,degree of anaphylactoid reaction,tryptase,histamine,β-hexosaminidase,IgE,Terminal compleoment complex(C5b-9),Complement Fragment 4b(C4b)release,compared the applicability of anaphylactoid reaction in four kinds of animals,selected suitable animal model which was used to evaluating TCM injeetion’s anaphylactoid reaction.The testing results:(1)The allergic degree of guinea Pigs is more serious than that of BN rats,SD rats,ICR mouses.After injection,6 guinea Pigs from Tween 80 group showed scratching noses,cough and other symptoms,and 5 guinea Pigs from Yuxingcao injection group showed alysmus,sneezing and other symptoms.(2)After injection the test sample,IgE,histamine,tryptase,β-hexosaminidase,C5b-9,C4b concentration of BN rats,SD rats and ICR mouses with the corresponding normal saline group were no significant differences.Cmpared with normal saline group,the release rate of IgE,histamine,P-Hex,C4b in the plasma of guinea pigs Tween 80 groups were higher than that in other three kinds of animals.Increasing rates of IgE,histamine,β-hexosaminidase,C5b-9 in guinea pigs Yuxingcao injection group are higher than SD rats,BN rats and ICR mouses.According to analysis above mentioned,we can draw a conclusion that guinea Pigs was relatively more sensitive than BN rats,SD rats and ICR mouses for evaluating anaphylactoid reaction induced by TCM injection.The multiple criterions of evalution with different animal species and increased observation targets for anaphylactoid reaction induced by TCM injections are more comprehensive and acceptable.4 Metabonomics study of allergy animal model4.1 Metabonomics study of allergy SD rats modelBy means of ovalbumin induced allergy animal model,we analyzed metabolic finger print data of SD rats’ albumin in the control and model group in order to obtain endogenous biomarkers.Through research by metabonomics,we found 11 endogenous biomarkers.Metabolomics can provide new ideas for establishing animal model for allergenic reaction induced by TCM injection.4.2 Metabonomics study of allergy BN rats modelBy means of ovalbumin induced allergy animal model,we analyzed metabolic finger print data of BN rats’albumin in the control and model group in order to obtain endogenous biomarkers.The results showed that 6 endogenous biomarkers were different between two groups.Therefore,metabonomies is very valuable as to be a new choice as studying the mechanism of allergenic reaction induced by TCM injection.5 Study of preparation of antibody kitThis experiment was conducted to research the methods of measurement of sensitization material in Huangqi injection to ensure clinical medication safety.The protein mixture(protein content is about 5.60%)was prepared from astragalus membranaceus as antigen to immune rabbits to produce specific antibodies.Through the use of specific antibodies,the ELISA was used to determine the content of sensitization material of Huangqi injection.The result shows:with residual protein,its scope of linear was 0.056-1.68μg·mL-1(r2=0.97).The LOD and LOP was 0.0055 and 0.0076 μg·mL-1 respectively.The method with simple pretreatment,good precision and promising specificity can be used for improving the manufacture process and for routine quality control of drug products. |
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