| Houttuynia cordata is a Chinese traditional medicine with long history with various beneficial biological activities,such as antibiosis,elevating immunity, antiinflammatory,antivirus and diuresis.The injections of houttuynia cordata are used widely clinicaly,but its adverse reaction is outstanding,the majority of which is allergic reaction,inclouding serious allergic reaction,such as allergic shock.The adverse reaction threaten the health of patients seriously and interfere the clinical application of the injections of houttuynia cordata.The injections of houttuynia cordata contain houttuynin cordata injection and neohouttuynin injection,houttuynin cordata injection contains 48 components,the main effective compont is houttuynin(HOU);the only component in neohouttuynin injection is neohouttuynin(NEO).some researchers presume that there are some direct relationship between HOU,NEO and the allergic reaction of the injections of houttuynia cordata.But there is not any effective evidence to this kind of realationship. So we investigate sensitization of HOU and NEO.Haptan with low molecule weight will initiate allergic reaction only after coupling with carrier protein.Coupleing haptan with low molecule can be used to prevent allergic reaction.so,we try to block the hydroxide radical with which haptan couple with carrier protein with low moleculer and detect the antibosis activity of production after blocking.We established guinea pig model of allergic reaction caused by houttuynin and neohouttuynin and explained the sensitization mechanism.We also try to provid some method to prevent the allergic reaction.Materials and Methods1.Coupling houttuynin and neohouttuynin with serum albumin We used BSA and HSA as carrier protein and identification the holoallergen using UV scaning.2.Establishment of guinea pigs model of allergic reaction We divided 40 guinea pigs(200～250g) into 4 groups:HOU,NEO,Positive and Negative group.We ip HOU-BSA,NEO-BSA with FCA for HOU and NEO group at the frist day and ip HOU-BSA,NEO-BSA at the 3rd and 5th day to sensitize guinea pigs,and iv HOU-HSA and NEO-HSA to provoke them.We ip BSA with FCA at 1st and BSA at 3rd day to sensitize,and iv HSA to provoke negative group;We ip OVA at 1st,3rd and 5th day to sensitize and iv OVA to provoke positive group.We observe the allergic symptom after provoking and we make and the pathological section of lung and liver tissue of the dead guinea pigs.3.Specfic IgE and IgG of guinea pigs sensitized We divided 12 guinea pigs into HOU and NEO groups,and get the serum at the 11,15,19,23,27,31,35th day. We use PCA to detect the antibodies in sensitized guinea pigs and identify the type of antibodies;We also detect specific IgG using ELISA.4.Degranulation of peritoneal mast cell(PMC) and contraction of isolated ileum We observe degranulation of PMC after incubation with the serum of sensitized guinea pigs and provoking.We also calculate the rate of dagranulation.We detect the contraction of isolated ileum after provoking.5.Reforming the form of houttuynin We try to eliminate the sensitization of houttuynin through refoming the form of houttuynin.We block the hydroxide radical of houttuynin using ED,and detect the antibosis activity of houttuynin-ED.6.The antibosis activity of the injection of houttuynia cordata The concentration of hottuynin in houttuynia cordata injection is very low,which is 6.3%of neohouttuynin injection.We try to measure the rationality of clinical dosage of houttuynia cordata injection. 6.1 Antibosis activity in vitro bacteria:staphylococcus aureus;culture medium: mediumâ…¡;we tile the medium on the plate,perforate with pinhole plotter(3mm). Incubate at 37℃for 18h after spotting and determine the diameter of inhibition zone.6.2 Antibosis activity in vivo We iv staphylococcus aureus to mouse and take the quantity of bacterium as MLD;We treat MLD for mouse and ip houttuynin, neohouttuynin injection and houttuynia cordata injection to treat.Observe the survival rate for 7 days.7.statistics and analysis The analyses were Performed by using the SPSS(10.0) statistical progrma.The measurement data were analysed with t-test.The analysises of correlation were Performed with Spearman test.A value of P＜0.05 was considered statistically significant.ResultsMethods and results1.The preparation of coupling of hapten and proteion UV scanning spectrum demonstrate that absorption of the antigens in 280nm was reinforced significantly,which demonstrsting coupling of hapten and proteion is succeed.2.Establishment of guinea pigs model of allergic shock Allergic shock symptom,including dyspnea,tumbleing aconuresis,aconuresis,even to death appeared quickly in some sensitized guinea pigs after they were challenged.The incidences of Allergic shock of houttuyfonin and neohouttuyfonin group is 20%and 50%,respectively,and the incidences of death were 20%and 40%.In negative control group,any anaphylaxis didn't appearance;in positive group,the incidences of allergic shock and death is 100%.H & E staining told that the tissue of lung from guinea pigs of both sensitized groups were congested and the alveolar wall were destroyed severely.But the tissue of lung from negative control group was normal and the alveolar wall was normal,without inflammatory cell infiltrating.In addition,the liver from guinea pigs of both sensitized groups became dropsical more conspicuously compared with the negative control group and was accompanied with inflammatory cell infiltrating and congestion.The Pathological changes suggested the antigen induced a typical immediate hypersensitivit response.3.Specific IgE and IgG antibodies in guinea pigsWe determined the titers of antibody of guinea pigs from both sensitized group with PCA.It indicated the antibody could be determined on the 15th day and got maximum on the 23th day and then degraded gradually.PCA reaction induced by the injection of sensitized serum from both sensitized group heated at 56℃for 1 hour was positive after attacked after 24 hours and negtive after 72 hours.PCA reaction induced by serum not heated was positive after attacked after 72 hours.All these suggested both sIgE and sIgG existed in the sensitized serum from both sensitized groups.But there was no correlation between IgG and IgE antibodies determined by PCA(P＞0.05).ELISA was used to examine specific IgG antibodies of among both sensitized groups and 30 healthy guinea pigs.Specific IgG of houttuyfonin and neohouttuyfonin group is 0.36±0.18 and 0.51±0.19,respectively,both of which is higher than the negative group.we had shown a correlation between specific IgG level by ELISA and the PCA titers.4.PMC degranulation and contraction of isolated ileumA lot of uncomplete cell envelope was observed after PMC incubated with plasm from the both sensitized guinea pigs was challenged by houtttuynin-HSA and nonhoutuynin-HSA and the degranulation rate of PMC was(63.6±7.5)%, (70.8±13.6)%,respectively.It was higher than that incubated with plasm from negative control group(29.2±6.61%,P＜0.01).There was no statistical significance in the degranulated rate between both sensitized group and positive group(70.4±12.5%, P＞0.05).Otherwise,isolated ileum from sensitized guinea pigs anastoled conspicuously after it was challenged by houtttuynin-HSA and nonhoutuynin-HSA.However,the isolated ileum from negative control group did not anastoled.5.Attempt to change the chemcial constitution of houttuyfonin Houttuyfonin couple with serum protein by the hydroxy group.We try to block the group by ED with molecular weight.We employed microorganism standardize its antibosis activity. substing in standard curve,we found the corresponding concentration of houttuyfonin-ED is 0.13mg/ml.Comparing with the concentration of houttuyfonin added is 0.6mg/ml,the antibacterial activity of houttuyfonin degrade significantly after coupleing with ED.6.Pharmacodynamics of houttuynin cordata injection.6.1 Antibiosis activity in vitro Antibacterial study in vitro showed inhibition zone of houttuyfonin with concentration of 0.013mg/ml(corresponding to the concentration in houttuynin injection) and houttuynin injection is 0mm,which demonstrating that the concentration of houttuyfonin in houttuynin injection is very low,and the antibosis activity in vitro of the latter is poor.6.2 Antibiosis activity in vivio ED25 of houttuynin,neohouttuynin injection and houttuynia cordata injection is 4.58,4.29å'Œ0.56 mg·kg-1,respectively.The result demonstrate that to achieve identical antibosia action,the concentration in houttuynia cordata injection is 12.2%of houttuyfonin,19.2%of neohouttuyfonin.So we think there are other antibiotic component in houttuynia cordata injection and its clinical dosage reasonable to some extent.Conclusions1.Allergic reaction of houttuynin cordata injection is associated with allergenicity of houttuyfonin and neohouttuyfonin.2.The development of allergic response induced by houttuyfonin and neohouttuyfonin in guinea pigs is associated with specific IgE,IgG and the medium released by mast cell.3.To degrade the allergic reaction by blocking the hydroxide radical is useless.4.There are some other effective components in houttuynia cordata injection besides houttuynin. |
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