Research Of Mast Cells Degranulation And Complement Activation-related Pseudoallergy Induced By Traditional Chinese Medicine Injections

1 Purpose and significancePurpose:This study was to investigate the mast cells degranulation in anaphylactoid reactions induced by traditional Chinese medicine injections(TCMI)and its complement activation mechanisms.Through the preliminary experimental basis and combining with literature analysis,we used TCMI which caused anaphylactoid reactions easily on the mast cells(MC)of mice and animal model,validating anaphylactoid reactions induced by TCMI were correlated or no with activation of the complement system,namely complement activation-related pseudoallergy(CARPA).Significance:We hoped that through the in-depth study about this subject could provide the new experimental basis for the sensitization mechanism of anaphylactoid reactions in vivo,and provide the basis for preclinical evaluation of anaphylactoid reactions induced by TCMI.2 Content2.1 The experiment in vitro of anaphylactoid reactions induced by traditional Chinese medicine injectionsOn the basis of previous experiments,we extracted and purified peritoneal mast cells(MC)of mice,and MC were identified by toluidine blue and to study the growth characteristics at the same time.Taking MC as the cell model,six traditional Chinese medicine injections(TCMI)Xueshuantong,Xiangdan,Shengmai,astragalus,Shenmai,Shuanghuanglian were used in degranulation experiment,measuring IC50 of the drug and release percentage of histamine,beta hexosaminidase,tryptase of the sensitized target cells,and calculating MC degranulation rate to seek sensitive TCMI for measuring mechanism of anaphylactoid reactions.2.2 The research on complement activation mechanism of anaphylactoid reactions induced by traditional Chinese medicine injectionsOn the basis of determination which traditional Chinese medicine injections(TCMI)could cause anaphylactoid reactions in vitro,we observed whether there were behavioral changes associated with anaphylactoid reactions,and abdominal skin mucosa blue spots of mouse at 30 minutes after injecting drugs for the first time,and measuring the release percentage about histamine and beta hexosaminidase of mast cells(MC)in vivo induced by TCMI;then the activation of the complement system components was observed,including observing mouse serum hemolysis,measuring CH50 of serum and performing complement fixation test,and to determine whether there were complement activation in the pseudoallergy animal model and to compare with the hypersensitivity mouse model on the serum complement hemolytic reactions;finally,the changes of complement C3aR on the MC surface were measured by immunofluorescence assay and flow cytometry to confirm whether degranulation reactions were induced by the combination of complement C3aR on the MC surface and complement activation fragment.Based on the above experiments,the complement activation mechanism in anaphylactoid reactions induced by TCMI was studied.3 Results3.1 The experiment in vitro of anaphylactoid reactions induced by traditional Chinese medicine injections3.1.1 Isolating,staining peritoneal mast cells(MC)in mice and studying growth characteristics of MCMore than 90%of cells which were extracted and separated by Percoll were mast cells,and the activity of MC was more than 95%.The growth curve of MC was determined,and finding the cell growth was stable within 24 hours and the optimal drug treatment time was within 24 hours.3.1.2 The experiment of mast cells degranulation in vitro induced by traditional Chinese medicine injectionsThe IC50 of Xueshuantong,Xiangdan(XD),Shengmai,astragalus,Shenmai and Shuanghuanglian(SHL)were 1.559?l/ml,42.799?l/ml,5.371?l/ml,42.200?l/ml,36.573 ?l/ml and 8.431?l/ml on mast cells(MC).The release percentage of histamine,beta-hexosaminidase,tryptase and percentage of degranulation about MC induced in vitro by traditional Chinese medicine injections(TCMI)in IC50 concentration were statistically significant for SHL and XD injections,and sensitization of SHL injections was stronger than XD injections;the release percentage about histamine,beta-hexosaminidase and tryptase of SHL group were respectively 37.61%,32.71%and 17.71%;the percentage of MC degranulation began to increase at 0.5 hour,and gradually decreased after 2 hours.3.2 The research on the complement activation mechanism of anaphylactoid reactions induced by traditional Chinese medicine injections3.2.1 The observation on the behavior and blue spots of mouse model,and the determination of mast cells degranulationShuanghuanglian(SHL),Xiangdan(XD),C48/80 and normal saline(NS)group were divided into four groups for establishment of anaphylactoid reactions mouse model.After first injection within 0.5 hours,the activity of mice in SHL group reduced,but were not up to the evaluation criteria about abnormal behavior of anaphylactoid reactions;SHL group and XD group were more than three mice which appeared abdominal skin mucosa blue spots;the release percentage about histamine,beta-hexosaminidase about MC degranulation induced by traditional Chinese medicine injections in vivo showed that SHL group were respectively 35.90%,32.71%,XD group were respectively 25.97%and 24.09%;and the release percentage of bioactive medium in SHL group were higher than XD group.3.2.2 The determination results of complement activationThe result of complement hemolytic reactions:the OD of SHL and XD group were respectively 0.253 and 0.241,P value were less than 0.01 compared with the negative group;CH50 of SHL group was 78 U/mL;the experimental results of SHL and XD group were negative in the complement fixation test,but it was confirmed again that there were activated complement components in the serum of mice in SHL and XD group.3.2.3 The comparison of complement hemolytic reactions between the pseudoallergy and hypersensitivity miceThe observation of behavior and abdominal mucosa blue between pseudoallergy and hypersensitivity mice in SHL group were not different;the results of complement hemolytic reactions in pseudoallergy and hypersensitivity mice in SHL group showed:OD value were respective 0.196 and 0.182,and P value were less than 0.01 compared with the negative control group;but there were no significant difference between the two groups.3.2.4 The detection of complement C3a receptor on the surface of mast cellsThe results of measuring complement C3a receptors on the surface of mast cells(MC)by immunofluorescence showed that fluorescence were existed on the surface of MC in all experimental groups,and the fluorescence intensity of MC in the SHL and XD group were stronger than NS group.The results of measuring complement C3a receptors on the surface of MC by flow cytometry showed that the percentage about fluorescent expression of MC in SHL group was 27.60%,and 5.41 times that of negative group;XD group was 6.60%,and 1.29 times that of negative group.4 Conclusion(1)It was confirmed by measuring the active substance and the percentage of degranulation on the mast cells model which were confirmed by staining and activity assays that the Shuanghuanglian injection and Xiangdan injection could cause mast cells degranulation in vitro to induce anaphylactoid reactions.(2)It was found that traditional Chinese medicine injections could also cause mast cells to produce degranulation in vivo on mouse animal model.It was confirmed again that the Shuanghuanglian and Xiangdan injection could induce anaphylactoid reactions.(3)Measuring complement biological activity in mice model of pseudoallergy induced by traditional Chinese medicine injections,it was found that Shuanghuanglian injection and Xiangdan injection could cause the hemolytic activity of the complement system increased,and complement activation-related pseudoallergy(CARPA)was existing.Comparing the pseudoallergy and hypersensitivity,we hold that there were complement activation in serum of pseudoallergy and hypersensitivity mice induced by traditional Chinese medicine injections,but there were no significant difference between the two groups.(4)By further measuring the changes of complement C3aR on the surface of mast cells,the results indicated that the complement activation fragment could bind to complement C3aR on the mast cells during degranulation of target cells induced by traditional Chinese medicine injections,leading to the degranulation reaction of the mast cells,and further elucidating that anaphylactoid reactions induced by traditional Chinese medicine injections were closely related to CARPA.