Studies On Hypersensibility Of Traditional Chinese Medicine Injections Such As Shuanghuanglian With RBL-2H3 Cells

With widely applications of traditional Chinese medicine injection(TCMI),its adverse reactions are rising,including skin,respiratory and cardiovascular system damages,and drug fever.Allergic reactions,frequently happened adverse,are usually considered to be severe,especially for anaphylactic shock.11 in 53 cases of reported allergic reactions, percent of 20.8%,caused by Chuanhuning injection are proved to be anaphylactoid reactions.Presently,there is no standard method for evaluating the anaphylactoid reaction, neither for discriminating between the allergic reaction and anaphylactoid reaction according to GLP.In fact,anaphylactoid reaction is different from allergy comparatively, which is caused by not only anaphylactogen,the compositions of drug itself can effect on mast cell and basophilic cell directly to release active mediums.Then series of allergic symptoms appear 30 minuets after the injection.The method of detecting the allergies caused by TCMI is animal experiment in the current Chinese pharmacopoeia;the objects are usually guinea pigs.Those TCMI cause allergy frequently have had undergone strict preclinical evaluation,which is not sensitive enough for emerges of false negative results and can not achieve the desired purpose.Thus, it's necessary to optimize the current method of detecting the allergies caused by TCMI and differentiate anaphylactoid reaction and allergy to find a new way to evaluate it accordingly.RBL-2H3 cells are basophilic leukemia cell line of Wistar rat,which can release granules containing histamine and Leukotriene to produce series of biological reaction when stimulated by the antigen or other stimulants.Therefore,this study observed RBL-2H3 cell's degranulation caused by several common TCMI combined with the clinical reports and animal experiments to explore how to use RBL-2H3 to the research of anaphylactoid reaction and hypersensitive response reaction caused by TCMI,such as Shuanghuanglian and Chuanhuning.At the same time,the influence of Tween-80 at different concentration on RBL-2H3 cells and rats was observed,to find the effect of Tween-80 in anaphylactoid reaction caused by TCMI,combining with the different anaphylactoid reactions caused by TCMI contained different residual Tween-80.1.Experimental Research1.1.Optimization of RBL-2H3 cells' culture conditionsThis study was based on RBL-2H3 cells.First of all,the cells culture condition was optimized and the growth curves were measured to choose the inoculum density of RBL-2H3 by CCK-8 and MTT.RBL-2H3 cells will be better when cultured in the incubator with 37℃,saturated humidity,5%CO₂,and RPMI 1640 containing 10%FBS as the culture medium. Subcultured a generation about every two days according the ratio of 1:3,1×105/mL is the better inoculum density for our experiments,and the experimental drugs are given after 24h when the cells were inoculated.It turned out to be the CCK-8 was more sensitive and convenient than MTT,but the latter is cheaper,it should better use MTT if the background (except formazam formed in experiment) has an influence on the results,we can chose one according to our experiment.1.2.The IC₅₀ of TCMIRBL-2H3 cells were treated with the TCMI at different concentration,and cultured in the incubator with 37℃,saturated humidity,5%CO₂ for 24hrs,the optical density was test by MTT,inhibition ratio(%) was caculated,and the IC₅₀ was caculated by statistical software.The IC₅₀ of TCMI is Mailuoning 11.1±3.0μL/mL,Xuesaitong 43.6±1.0μL/mL, Chaihu 59.9±1.5μL/mL,Shuanghuanglian 34.9±6.0μL/mL,Chuanhuning 57.4±1.2μL/mL,Qingkailin 18.3±4.0μL/mL,Xiangdan 1.0±0.2μL/mL(μL/mL is volume ratio of TCMI and culture fluid),administration in our experiment is according to this.1.3.The method of detecting hypersensitive response caused by TCMI in vitroThe RBL-2H3 cells were incubated with blood serum of antigenic specificity for being sensitized,and stimulated with the same antigen after 24 h,then the degranulated mast cells were counted with neutral red,P-hexosaminidase and histamine in supernatant were also tested,which were used to observe the effect of TCMIs on RBL-2H3 cells.Then compared with the clinical reports,to find the method of evaluating hypersensitive response caused by TCMI in vitro.It turned out to be that the TCMI can cause RBL-2H3 cells to degranulate,and release P-hexosaminidase and histamine,the degree of this is Shuanghuanglian>Chuanhuning> Chaihu,it's coincident with the clinical reoports.This experiment hinted that the RBL-2H3 also can be used in evaluating the hypersensitive response caused by TCMIs in vitro.1.4.Experiment of hypersensitive response caused by TCMI with animalGuinea pigs were sensitized with TCMI,id,and again with the same dose after 14 days,i.v.Hypersensitive response was observed and the blood serum was segregated in 30 minutes and tested by ELISA for quantitating the histamine,the result was compared with the cells' experiment.It turned out to be coincident the Chuanhuning injection can caused more RBL-2H3 cells to degranulate,and more animals' hypersensitivity and histamine releasing,but Chaihu injection was less than Chuanhuning injection,which further proved that the methods of detecting degranulation of sensitized RBL-2H3 cells can be used in evaluating the allergies caused by TCMI.1.5.Research on anaphylactoid reactions caused by TCMI with RBL-2H3 cellsRBL-2H3 cells were cultured and treated with TCMI like Xuesaitong in vitro,the degranulated mast cells were counted with 0.5%neutral red,β-hexosaminidase and histamine in supernatant were also tested,which were used to observe the effect of TCMI on RBL-2H3 cells,in order to find the method of evaluating anaphylactoid reaction caused by TCMI in vitro.The results from three methods turned out to be coincident,which caused more adverse effects in clinic,results in conspicuous degranulation of RBL-2H3 cells,the degree of RBL-2H3 cells' degranulation caused by TCMI is Xuesaitong>Qingkailing>Chuanhuning> Shuanghuanglian>Xiangdan>Mailuoning>Chaihu.It's also coherent with the clinical reoports,that is,the TCMI which caused more adverse effects in clinic,resulted in conspicuous degranulation of RBL-2H3 cells.This result hinted that the RBL-2H3 cells may be used in evaluating the anaphylactoid reaction caused by TCMIs.1.6.Experiment of anaphylactoid reaction caused by TCMI with animalWistar rats and guinea pigs were treated with TCMIs according to weight by i.v., anaphylactoid reaction was observed,and blood serum was segregated in 0.5h and tested by ELISA for quantitating the histamine,the result was compared with the cells' experiment.It turned out to be coincident in the animals and cells' experiment on the whole,the degree of RBL-2H3 cell degranulating caused by TCMI is coincident with the symptoms and blood serum histamine experimentalized with animals,which further proved that the methods of detecting degranulation of RBL-2H3 can be used in evaluating the anaphylactoid reaction caused by TCMI.The inter group disparity of anaphylactoid reaction is more obvious in the experiment with guinea pigs than with wistar rats,but the histamine in the blood serum is nearly identical.So,guinea pigs may be more sensitive on the experiment of evaluating the anaphylactoid reaction caused by TCMI if only symptoms are observed.It turned out to be the Shuanghuanglian and Chuanhuning injection had an obvious effect on sensitized RBL-2H3 cells degranulating and animal's hypersensitiveness,but which was not obvious in anaphylactoid experiment.It indicated that the hypersensitive response caused by Shuanghuanglian and Chuanhuning injection involved in IgE,which may be initiated by the antigen or hapten in the TCMI,not simply the mast cells degranulating caused by the drug itself.1.7.Effect of Tween-80 in anaphylactoid reaction caused by TCMITo find out the effect of Tween-80 in anaphylactoid reaction caused by TCMI in vitro, by investigating its influence on RBL-2H3 cells at different concentration,combining with the different anaphylactoid reactions caused by TCMI contained different residual Tween-80.Tween-80 at 0.2～40mg/mL can cause RBL-2H3 cells to degranulate,to releaseβ-hexosaminidase and histamine,they are positive correlation.Also cause animals' anaphylactoid reaction when the concentration was more than 112 mg/kg.Further experiments found that the TCMI contained excess Tween-80 was exposed to cause RBL-2H3 cells to degranulate.This result indicates that excess Tween-80 may be one of the reasons for anaphylactoid reaction caused by TCMI.2.Conclusion2.1.Hypersensitive response and anaphylactoid reaction can be caused by TCMIIn this study,those which caused more hypersensitive response and anaphylactoid reaction in clinic,resulted in conspicuous degranulation of RBL-2H3 cells.2.2.The sensitized mechanism of different TCMI is distinctIt indicated that the hypersensitive response caused by Shuanghuanglian and Chuanhuning injection involved in IgE,which may be initiated by the antigen or hapten in the TCMI,not simply the mast cells degranulating caused by the drug itself.The adverse effect caused by TCMI such as Xuesaitong injection partly was anaphylactoid reaction, which should be paid attention to when hypersensitive response is evaluated caused by the TCMIs.2.3.RBL-2H3 cells can be used in evaluating the anaphylaxis of TCMIThe sensitized RBL-2H3 cells can be used in evaluating hypersensitive response caused by TCMI,by testing their degranulation;RBL-2H3 cells also can be be used in evaluating anaphylactoid reaction caused by TCMI,by testing their degranulation. 2.4.Tween-80 may be one of the reasons for anaphylactoid reaction caused by TCMITween-80 at 0.2～40mg/mL can cause RBL-2H3 cells to degranulate,to releaseβ-hexosaminidase and histamine,they are positive correlation.Also cause animals' anaphylactoid reaction when the concentration was more than 112 mg/kg.This result indicates that excess Tween-80 may be one of the reasons for anaphylactoid reaction caused by TCMI.3.Significance of the ResearchThis paper studied effect of several TCMI on RBL-2H3 cells' degranulation,combined with the animal experiments and statistical results of clinical reports,to explore how to use RBL-2H3 to distinguish and evaluate the hypersensitive response and anaphylactoid reactions.We also found that the excess Tween-80 may be one of the reasons for anaphylactoid reaction caused by TCMI.All of this paper provided the new referred methods of evaluating and anaphylactoid reactions caused by TCMI,which will be good for clinical safety of medication.