Mechanism Of Dai Formula Yajieshaba Detoxifying Food Poison For Food Allergy Treatment

ObjectiveDetoxifying "food poison" of Dai medicine(TDM)is one of the important elements in"Ya Jie" theory.In recent years,detoxifying "food poison" and its herb formulae have become focuses in research of the TDM under the guidance of "Ya Jie" theory.In the previous studies,we have found that the "food poison" of TDM is closely related to food allergy.Yajieshaba(YJSB)is one of the prescriptions for detoxifying "food poison".Clinical observation and experimental studies have proved that it has a good detoxifying "food poisoning" effect.However,the recent research cannot fully reveal the effects and mechanism of YJSB for detoxifying "food poison".Therefore,on the basis of previous studies,this study intends to achieve the following objectives:1 The paper applies the statistical methods to analyze the empirical herbal formulae for detoxifying "food poison" of TDM to confirm the evidence of correlation between "food poison" and food allergy,food allergy is a typical disease that can reflect the connotation of"food poison",and YJSB is one of the representative Dai formulae detoxifying "food poison".Further,we verify intestines symptoms as indicators to investigate "food poison".So the paper can provide evidence for the clinical application and experimental research.2 According to the above theoretical research,the paper replicates a food allergic mouse model and investigates how YJSB mediating intestinal barrier function in terms of biological,physiological,and immune barrier in order to preliminarily discover the mechanisms of YJSB for treatment of food allergy,and to reveal its mechanism for detoxifying "food poisoning".Method1 Research on theoryThe paper applies the document analysis method to search,then normalize,filter,and summarize single and empirical formulae.Further,the paper applies the frequency statistic to analyze and summerize the distribution in terms of classification of the formulae,property,taste,four elements properties,classification,treatment scope and symptoms.Based on the frequency analysis,we also use clustering method to analyze the herb distribution pattern.2 Research on Experiments2.1 Replication the model of oval allergyForty female mice,six to eight old,were randomly assigned into 5 groups:control group,OVA food allergy(FA)group,YJSB treatment groups(low dose:0.39g/kg,middle dose:1.17g/kg,high dose:3.51g/kg).Mice for FA model were sensitized on days 0 by intraperitoneal injection of 10μg OVA and 1 mg aluminum in Hyper-Pure water and on days 14 by intraperitoneal injection of 10μg OVA.On days 30 and 34,mice were orally administrated OVA 80mg.Hyper-Pure water was used for control group for sensitization and challenge.2.2 Drug intervention experimentOne hour before each challenge,YJSB groups accepted treatment(0.39g/kg,1.17g/kg,3.51g/kg)for 5 days(day 30-34)through introgastric administration,while control group and model group were administrated in saline.2.3 Evaluation of food allergy syndromeFood allergy symptoms were evaluated by allergy symptoms and allergic diarrhea severity 0-60 minutes after two challenges.60 minutes after the second challenge,blood samples were collected to measure the level of serum OVA-specific IgE by ELISA kit.2.4 Detection for intestinal floraBy the end of the experiment,feces were collected and stored in liquid nitrogen.We used llumina high-throughput to sequence the 16S rRNA gene V3-V4 region in the fecal samples of each group to qualitatively analyze and explore the change of intestinal micro flora structure.2.5 Detection for the changes in intestinal mucosal permeabilityReal-time PCR was used to detect the mRNA expression of TJ protein Occludin,Claudin3 from Jejunum tissue after the second sensitization event.2.6 Immunodetection for the changes in intestinal mucosaELISA was used to detect he levels of IL-4、IFN-γ、IL-4/IFN-γ and sIgA.Real-time PCR was used to detect the mRNA expression of IL-17,TGF-β1 TGF-β1/IL-17 in jejunurn.Result 1 The regularity of compound herbal formulae for detoxifying "food poison"Among related formulae,we have collected 57 formulae,258 used requencies,and115 species for single and empirical formulae.Through frequency analysis,45.74%herbs belong to antidotes;48 herbs are used more than twice,accounting for 41.74%;the herbs with cool and cold properties account for 40.31%and 20.54%respectively;herbs with bitter taste is dominant,making up 45.35%;major herbs are with the property of wind element,taking up 29.20;major category of "food poison" are accidental toxicant and sitotoxism,accounting for 35.27%and 31.40%;the majority of treatment scope are for the digestive system,accounting for 54.64%.The main symptoms are diarrhea and vomiting,accounting for 18.06%respectively.By frequencies and variable-cluster analysis,we can classify the formulae into 7 genres,and among formulae in 7 categories,YJSB is proved to be safe,applicable,and accessible for clinic.2 Syndrome of food allergy2.1 General syndromeAfter first challenge,YJSB treatment groups all decreased scores,however without significant difference(P>0.05).After second challenge,YJSB treatment groups significantly decreased scores compared with the control group,with significant difference(P<0.01).2.2 Severity of diarrheaAfter first challenge,YJSB treatment groups all decreased diarrhea score,and only YJSB at medium dosage has significant difference(P<0.05).After second challenge,YJSB treatment groups all decreased diarrhea score significantly,with significant difference(P<0.01).2.3 Serums OVA-specific IgE levelsThe levels of serums OVA-specific IgE are decreased markedly in YJSB groups,with significant difference(P<0.05).3 Intestinal flora3.1 The Alpha bacterial flora of fecesCompared with the control group,the indicators of ACE,Chaol in YJSB groups are with no difference.But YJSB high dose shows the trend to increase both.Meanwhile,Compared with the control group,indicator Shannon in YJSB groups reveals the trend of increase,and Simpson indicator is of decrease trend.However,only YJSB with medium dose is with significant difference(P<0.05).3.2 Intestinal micro biotaAt Phylum level,the ratio of Firmicutes was decreased in YJSB groups,especially it in high dose group was statistically significant(P<0.05),whereas that of Bacteroidetes was increased;also there were significant difference especially in YJSB high dose group(P<0.05).At genus level,the relative proportions of Lactobacillus was lower,however without significant difference(P>0.05);contrarily,Barnesiella was higher in YJSB groups,specially there was significant difference in group of high(P<0.05),compared with the OVA group.3.3 Microbial community structureCompared with the OVA group,the microbial community structure of YJSB samples were distributed independently,and can be separated easily.4 Intestinal permeabilityCompared with the model group,the Occludin mRNA expression levels of Ocln were significantly increased in YJSB group,with statistic significance(P<0.05);however,the levels of Claudin 3 mRNA were not markedly changed,without statistic significance(P>0.05、).5 Intestinal mucosal immunityCompared with the model group,YJSB groups at low and medium doses increased IL-4,without statistic significance(P>0.05);but YJSB at high doses decreased IL-4,with statistic significance(P<0.05);compared with the model group,YJSB increased IFN-y markedly,with statistic significance(P<0.01);also YJSB groups could down-regulate the ratio of IL-4/IFN-y,but only at high doses there was significant difference(P<0.05).Compared with the model group,the differences of IL-17 and TGF-β1 mRNA expression,TGF-β1/IL-17 ratio and sIgA level in YJSB groups were not statistically significant(P>0.05).Conclusion1 The antidote formulae are the majority in detoxifying "food poison" formulae;the herbs with cool properties and bitter taste are the majority in formulae for treating "food poison".In addition,formulae with two or more ingredients,combining cold and hot properties,and balancing "four elements" are also common regularities.2 The "food poison" of Dai medicine is partly associated with food allergy;also food allergy is a typical disease that can reflect the connotation of "food poison".In this case,YJSB is a representative formula of detoxifying "food poison".Intestines symptoms are an important indicator of "food poison",while diarrhea is one of main symptom.3 The Dai medicine formula YJSB could attenuate symptoms of food allergy in an OVA-allergic murine model by reducing the overall symptom scores,diarrhea scores and serum OVA-IgE levels.4 The Dai formula YJSB could modulate the balance of gut microbiota in mice by increasing the Shannon index and the diversity of colonies,while contrarily reducing the Simpson index.5 At Phylum level,the Dai formula YJSB could reduce the Firmicutes bacteria proportion,increase the proportion of Bacteroides,and restore the imbalance of intestinal flora of mice,especially at high dose.6 At genus level,the Dai formula YJSB could reduce the proportions of Lactobacillus,increase the proportion of Barnesiella,and restore the imbalance of intestinal flora of mice,especially at high dose.7 The Dai formula YJSB could recover the microbial community structure.8 The Dai formula YJSB treated-mice showed increased mRAN expression of TJ protein Ocln and decreased intestinal permeability,but it has no significant effects on Cldn3 mRNA expression.9 The Dai formula YJSB could treat the allergic diarrhea by reversing the Th1/Th2 imbalance,the immune unlbalance caused by Th2 advantage,by increasing the levels of IFN-y production,and decreasing the levels of IL-4.10 The Dai formula YJSB has no significant effects on IL-17 mRNA,TGF-β1 mRNA expression,ratio of TGF-β1/IL-17 and sIgA.11 The Dai formula YJSB might be a promising therapy for food allergy,especially at high dose.