| Qingkailing injection is a traditional Chinese medicine which is widely used in clinical practice for the treatment of upper respiratory inflammation,pneumonia,high fever and viral encephalitis.However,with the increase of clinical application of Qingkailing injection,the frequency of its adverse reactions is gradually increasing,and most of the adverse reactions induced by Qingkailing injection were anaphylaxis.Although the study of Qingkailing injection-induced anaphylaxis has reported,the pathogenesis of it is still unclear.In this study,we first screened sensitization method of Qingkailing injection and the time point of IgE antibody produced based on passive cutaneous anaphylaxis experiment.Then screened the responders from Qingkailing injection-sensitized rats and established accurate allergy model of rats.Meanwhile,a dynamic large-scale nontargeted metabonomics approach based on ultra-performance liquid chromatography coupled with a quadrupole time-of-flight mass spectrometry(UPLC Q-TOF/MS)was developed to investigate the dynamic changes of serum metabolic profiles of rats from the early,middle and late stage of anaphylaxis.The multivariate analysis was used to find the perturbed endogenous metabolites associated with anaphylaxis,and according to the pathways analysis of these perturbed endogenous metabolites deciphering the overall pathogenesis of Qingkailing injection-induced anaphylaxis.Finally,a two-stage partial least squares(PLS)modeling was calculated based on the data of predose urine metabolites to predict the allergy index and to select the metabolites that substantially contributed to such prediction.At the same time,biology analysis was applied to establish the relationship between predose urine metabolites and Qingkailing injection-induced anaphylaxis.The main research contents and results are as follows:1.Screening the preparation method of antiserum and prepare antiserumUsing the passive cutaneous anaphylaxis experiment method explored the sensitization method of Qingkailing injection and the time of IgE antibody produced.Screening the preparation method of antiserum according to the diameter of blue spot and the absorbance of dye leakage extracted in the supernatant of saline control group,Qingkailing injection control group,Al(OH)3 gel control group,FCA control group,Qingkailing injection+Al(OH)3 gel group,Qingkailing injection+FCA group.The results showed that the diameter of blue spot in Qingkailing injection+ Al(OH)3 gel group was more than 5 mm.Besides,the absorbance of dye leakage extracted in the supernatant of Qingkailing injection+Al(OH)3 gel group were higher than all control groups.The result indicating that rats which were sensitized by QKLI+Al(OH)3 gel is the most effective sensitization method,and the level of IgE were highest at 25th day after the first sensitization.So the QKLI+Al(OH)3 gel group was selected to prepare antiserum and blood samples were collected at 25th day.Furthermore,in this study,we found the diameter of blue spot in rat inner skin was 2 mm larger than the outside.The findings were extremely important for the allergy model and the study of the pathogenesis of Qingkailing injection-induced anaphylaxis.2.The study of SD rats model of Qingkailing injection-induced anaphylaxisIn this study,we screened the responders from Qingkailing-sensitized rats according to the diameter of blue spot based on the passive cutaneous anaphylaxis experimental.The experiment result showed that only 28 rats were responders among the 65 rats after Qingkailing injection administration,which indicated that the success rate of animal modeling is not 100 percent.The responders in this study were used to establish allergy model.Meanwhile,additional rats of control groups were established.After modeling the clinical allergy index in rat serum were detected.The result showed that the levels of histamine and β-hexosaminidase in allergy group were evidently higher than that of control group.These results indicated that all rats of allergy groups experienced anaphylaxis whereas rats of control groups did not.Besides,the significantly increased levels of histamine andβ-hexosaminidase in rats of allergy groups confirmed that the allergy model we established was accurate.3.Study of the pathogenesis of Qingkailing injection-induced anaphylaxis in serum with metabolomics strategyIn this study,a dynamic large-scale nontargeted metabonomics approach based on ultra-performance liquid chromatography coupled with a quadrupole time-of-flight mass spectrometry(UPLC Q-TOF/MS)was used to investigate the changes of serum metabolic profile of anaphylaxis rats.We studied the dynamic process of Qingkailing injection-induced anaphylaxis from the early,middle and late stage,expecting to find perturbed endogenous metabolites associated with anaphylaxis and decipher the overall pathogenesis of Qingkailing injection-induced anaphylaxis.The result of PCA indicated that the changes of serum metabolic profiles of rats experiencing anaphylaxis were associated with the anaphylactic reaction progressed.The OPLS-DA model was used to analyze the allergy and control rats matched by time point and find out the significantly altered metabolites at each time point which made rats metabolic profiles changed based on P<0.05and VIP>2.Fifty-eight potential biomarkers were screened and identified at 10 min post-challenge,including 33 metabolites in polar part and 25 metabolites in weak polar part.Fifty-nine potential biomarkers were screened and identified at 30 min post-challenge,including 33 metabolites in polar part and 26 metabolites in weak polar part.Forty-one potential biomarkers are screened and identified at 120 min post-challenge,including 23 metabolites in polar part and 18 metabolites in weak polar part.Among which 24 same metabolites were found in the three time points post-challenge,including AA,12-HETE,15-HETE and some phosphatidylcholines.What is more,the same fluctuating trend was also found comparing with corresponding control group.All potential biomarkers of three time points were analyzed by MetPA,respectively.The result indicated that that disturbed pathway of AA metabolism,glycerolphospholipid metabolism,fatty acid metabolism,sphingomyelin metabolism and tryptophan metabolism were involved in Qingkailing injection-induced anaphylaxis to some extent,among which the disturbed AA metabolism pathway was closely associated with the pathogenesis of Qingkailing injection-induced anaphylaxis.4.Study of Qingkailing injection-induced anaphylaxis bansed on pharmacometabolomicsIn the study,UPLC-Q-TOF/MS analysis was performed on the predose urine samples both in positive mode and negative mode.A two-stage PLS analysis was employed to build a statistical mode that can predict Qingkailing injection-induced anaphylaxis effectively using the measured metabolic data.The initial PLS analysis was performed on all 4,326 and 4,578 peak intensities(X block,prediction variables)related to the allergy marker(Y block,response variable)in positive and negative mode,respectively.On the basis of this analysis,we selected the metabolites(X variables)that made a large contribution to predicting the clinical allergy index(Y variable)based on VIP>2.According to the initial PLS analysis,179 and 188 variables of high VIP values(VIP>2)were highly relevant to histamine in positive and negative mode,respectively.156 and 138 variables of high VIP values(VIP>2)were highly relevant to β-hexosaminidase in positive and negative mode,respectively.181 and 192 variables of high VIP values(VIP>2)were highly relevant to AA in positive and negative mode,respectively.We then performed a second PLS analysis to predict individual clinical allergy index,using the selected metabolites.On the basis of these selected variables,we built the second PLS model to be used for predicting the clinical allergy index based on VIP>1.A set of 23 and 18 key metabolic features in positive and negative mode were selected,respectively,which characterizing Qingkailing injection-induced anaphylaxis,represented by the histamine as those with VIP>1 in the second PLS model.A set of 18 and 4 key metabolic features in positive and negative mode were selected,respectively,which characterizing Qingkailing injection-induced anaphylaxis,represented by the β-hexosaminidase as those with VIP>1 in the second PLS model.A set of 26 and 17 key metabolic features in positive and negative mode were selected,respectively,which characterizing Qingkailing injection-induced anaphylaxis,represented by theβ-hexosaminidase as those with VIP>1 in the second PLS model.To screening the metabolites which can be used for predicting the Qingkailing injection-induced anaphylaxis,metabolic pathways was used in our study.The result indicated that 1-methylhistidine and phosphorylcholine were associated with anaphylaxism,and can be used for predicting the Qingkailing injection-induced anaphylaxis. |
PDF Full Text Request