| Objective: To evaluate the allergic reaction caused by Sanqi Panax Notoginseng for Injection(abbreviation: Xuesaitong),to clarify the nature of its reaction.Screen the main components of the allergic symptoms and explore the mechanisms by which they induce allergic reactions.In order to reveal the essence of allergic reaction caused by Xuesaitong,it provides experimental reference for further scientific research and allergy evaluation of traditional Chinese medicine injection.Method: 1.Allergic reaction test: Evaluation of allergic reactions to Xuesaitong by the guinea pig ASA and the mouse allergy test method based on blood biomarker detection.The NS was used as the negative control,OVA as the positive control,and set the corresponding adjuvant group.After 3 times of sensitization and re-excitation,the symptoms of each group were observed and recorded,and the symptoms were evaluated according to the corresponding systemic sensitization evaluation criteria.On this basis,the mice collected blood samples to detect blood biomarker content and prepared antiserum for rat heterogeneous PCA test.The two methods were used to comprehensively evaluate the allergic reaction of Xuesaitong.2.Anaphylactoid reaction detection: In vitro,use P815 and RBL-2H3 cells to detect the levels of degranulation medium released into the supernatant after incubation with Xuesaitong,to preliminary evaluation the anaphylactoid reaction.Use MSAR test method to evaluate the symptoms and detect the changes in blood biomarker levels.Combining overall experiments with cell experiments to comprehensively evaluate the allergenicity of Xuesaitong.3.Screening of anaphylactoid components: Using P815,RBL-2H3 cell and ICR mice,use this model to detect the anaphylactoid reactions of the three components Rb1,Rg1,and R1,which are the most abundant in Xuesaitong,in order to screen the substances in which allergic reactions were induced.4.Pathway study: using P815 and RBL-2H3 cell models to study the activation of direct stimulation of MC/BAS pathway by Xuesaitong and allergens Rb1 and Rg1.Decting the changes of characteristic factors C5a(complement pathway),C1q(classical pathway)and FD(alternative pathway)in the plasma to analyze the relationship between each index and the complement pathway,in order to speculate the mechanism of allergic reactions.Results:1.Allergic reaction test:(1)Systemic allergy test in guinea pigs: The allergic reaction to the Xuesaitong was negative.(2)The establish of mouse anaphylaxis methods which based on detecting biomarkers: After multiple sensitization with Xuesaitong,it was no increase in serum Ig E content.After challenge,only some mice showed weak positive reaction.Only the level of b-hexosaminidase was increased after the stimulation of blood biomarkers,and other indicators of histamine,tryptase and Ig E did not change significantly.The serum of the mouse blood-suppressed antibody was sensitized to the skin of the rat,and no blue spot appeared after the blood stasis was injected by intravenous injection.There was no significant increase in Ig E in the mouse antibody serum samples.2.Anaphylactoid reaction test:(1)In vitro cell test: Xuesaitong 1-8 mg/m L is incubated with cells.There was no significant change in the level of degranulation medium in the supernatant of P815 cells.The concentrations of histamine,b-hexosaminidase and trypsin in the supernatant of RBL-2H3 cells increased in a concentration-dependent manner,among which the increases of histamine,trypsin,and b-hexosaminidase at 4-8 mg/m L were statistically significant.(2)The mouse model of systemic anaphylactoid reaction test: Single intravenous injection of xuesaitong showed different degree of symptom response in each dose group.At 60-120 mg/kg,the intensity of symptomatic response was weakly positive/suspicious;at 240-480 mg/kg,the intensity of symptomatic response was positive and strongly positive.Blood biomarker test results showed no significant changes in the indicator level at 60-120 mg/kg;histamine at 240 mg/kg,and histamine,b-hexosaminidase and tryptase at 480 mg/kg elevation was statistically significant.3.Screening for anaphylactoid ingredients: 3.1 in vitro cell test(1)P815 cell test: a.The main component composition of Xuesaitong 1-8 mg/m L was co-cultured with the cells,the histamine in the supernatant showed an irregular decrease,and there was no significant change in trypsin,?-hexosaminidase was elevated only at 8mg/m L.b.R1 at 0.15-1.2 mg/m L was co-cultured with cells,and there were no significant changes in histamine,?-hexosaminidase and trypsin in the supernatant.c.Rb1 at 0.4-3.2 mg/m L was co-cultured with the cells,the histamine in the supernatant showed no significant increase,and the concentration of ?-hexosaminidase and trypsin increased in a concentration-dependent manner.d.Rg1 at 0.45-3.6 mg/m L was co-cultured with cells,the histamine in the supernatant showed a decreasing trend,while the trypsin level increased in a concentration dependent manner,3.6 mg/m L increase was statistically significant.(2)RBL-2H3 cell test: a.The main component composition of Xuesaitong 1-8 mg/m L was co-cultured with the cells,the histamine at 2-8 mg/m L and the trypsin at 8 mg/m L increased significantly.b.R1 at 0.15-1.2 mg/m L was co-cultured with cells,the histamine in the supernatant showed a downward trend,and the ?-hexosaminidase and trypsin were no significant changes.c.Rb1 at 0.4-3.2 mg/m L was co-cultured with the cells,histamine at 0.4,1.6,3.2 mg/m L,?-hexosaminidase at 0.4,0.8,3.2 mg/m L,and trypsin at 0.8,1.6 mg/m L increase were statistically different.d.Rg1 at 0.45-3.6 mg/m L was co-cultured with cells,the supernatant in histamine,?-hexosaminidase,trypsin were not significantly increased.3.2The mouse model of systemic anaphylactoid reaction test:(1)Main component composition of Xuesaitong: Single intravenous injection,the symptoms were evaluated as weak positive/suspicious at 60 and 120 mg/kg,and the level of blood biomarker was not significantly changed;The symptom intensity was assessed as strongly positive at 240 and 480 mg/kg,and the blood histamine and ?-hexosaminidase were also significantly increased,but the pancreatic protein was only significantly increased at 480 mg/kg.(2)R1: after a single intravenous injection,the a anaphylactoid symptoms of mice were only positive at 72 mg/kg,but the level of blood biomarker was not significantly increased.(3)Rb1: single intravenous injection was evaluated as positive at 96 and192 mg/kg,and the blood histamine and ?-hexosaminidase were also significantly increased,but the change of trypsin was not significant.(4)Rg1: single intravenous injection,at the dose of 54,108,216 mg/kg,the symptom intensity was assessed as positive or strongly positive,the blood histamine also increased significantly,?-hexosaminidase and trypsin changes were not significant.4.Anaphylactoid reaction mechanism(1)Direct stimulation of MC/BAS degranulation pathway activation: a.Xuesaitong at 1-8 mg/m L was co-cultured with RBL-2H3 cells,the degranulation component in the supernatant increased,histamine at 4-8 mg/m L,?-hexosaminidase and tryptase at 8 mg/m L were significantly increased.b.Rb1 was co-cultured with RBL-2H3 cells at 0.4-3.2 mg/m L,and the histamine at 0.4,1.6,3.2 mg/m L,?-hexosaminidase at 0.4,0.8,3.2 mg/m L,and trypsin at 0.8,1.6 mg/m L in supernatant was significantly increased.c.Rg1 was co-cultured with RBL-2H3 cells at a 0.45-3.6 mg/m L,and the degranulated components in the supernatant did not change significantly.(2)Activation of complement-related pathways a.Intravenous injection of xuesaitong at 240 mg/kg,the blood levels of C3,C5 a and C1 q were decreased,in which the levels of C3 and C1 q were decreased significantly,while the levels of FD were not changed significantly.b.Rb1 was intravenously injected at 192 mg/kg,the levels of C3,C5 a and C1 q in the blood of mice were significantly decreased,while the levels of FD were not significantly changed.c.Rg1 was intravenously injected at 216 mg/kg dose,the blood levels of C3,C5 a,C1q and FD in mice all decreased to a certain extent,but the blood levels of C5 a in the 216 mg/kg dose group only decreased significantly.Conclusion: 1.The allergic of Xuesaitong was negative and the anaphylactoid was positive,and the initial dose of anaphylactoid positive was 240 mg/kg,which was 4 times of the clinical equivalent dose.2.The main components of Xuesaitong,Rb1 and Rg1,were positive,and the positive initial doses were 96 mg/kg and 54 mg/kg,respectively,which were 4 times and 2 times of the clinical equivalent doses.The anaphylactoid reaction of R1 was negative.It is suggested that Rb1 and Rg1 in the main components of Xuesaitong may be the main components that induce anaphylaxis.3.Anaphylactoid reactions induced by Xuesaitong and ginsenoside Rb1 are mainly through direct effects and classical approaches.The anaphylaxis induced by Rg1 should be further studied. |
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