The Immunomodulatory Effect Of Chinese Herbal Formula Biminne And Its Components On Respiratory Allergy

BackgroundRespiratory allergy is chronic airway inflammation which is caused by antigen-induced immunological imbalance.In addition to IgE-induced mast cell degranulation,lymphocyte and eosinophils and other inflammatory cells and cytokines all contribute to chronic airway inflammation.The imbalance of T lymphocytes differentiation subsets, such as Th2 polarize and Treg(regulatory r cell) dysfunction,play an important role in the process of asthma and allergy.In treating respiratory disease,glucocorticoids andβ₂-adrenergic receptor stimulators and histamine antagonist may improve symptoms in the short term,but its side-effects can not be ignored.Therefore,it is necessary to explore safer and effective methods for treating respiratory allergy.Traditional Chinese medicines can be effective in treating allergic diseases based on symptom-specific treatment.A randomized,double blind, placebo-controlled,clinical trial was conducted to evaluate the effectiveness of the herbal formulation Biminne in the management of perennial allergic rhinitis.The results showed that Biminne significantly improved some of the symptoms,reduced serum IgE,and showed some potential long-term benefits in the treatment of allergic rhinitis. Our team previously observed that Biminne significantly prolonged the incubation period of asthma attack in allergic guinea pigs,and reduced the nose membrane permeability in allergic Brown Norway rats.The Biminne formulation is effective in treating allergic disease with its effect observed both in patients and respective animal models,However its mode of action is as yet unknown.The aim of this study is to explore the immunomodulating effect of the formulation Biminne and its components in the management of allergy,and use animal or cell models to identify its effective components.If this aim is achieved,we can use the effective components to form new formulations for more effective treatment.For this end,our team designed a series of experiments.This study is part of a larger study aimed at exploring the immunomodulating effect of the formulation Biminne,and looks at its components Baicalin and EF in treating respiratory allergic diseases,especially in regulating T helper cell differentiation。PARTâ… A Study of the immunomodulating effect of the traditional Chinese formulation Biminne in treating respiratory allergyObjective To test the effect of the formulation Biminne in treating mice model of airway allergy,and investigate its immunomodulating mechanism.Methods 96 BALB/C mice were randomly divided into 6 groups: normal group,model group,Biminne high-dose group,medium-dose group, low-dose group and Bidouyan group.The respiratory allergic model was induced by intraperitoneal and intranasal administration of ovalbumin (OVA) into mice.The occurrences of sneezing and nasal rubbing was counted; the proliferative capacity of splenocyte was tested with ³H-TdR and the levels of serum total IgE and OVA specific IgE were measured with ELISA.Results Biminne showed a positive effect in treating allergic rhinitis.The amount of sneezing and nasal rubbing increased markedly after allergy-induction modeling(P＜0.01),the stimulation index(SI) of splenocyte proliferation stimulated by PHA and OVA elevated(P＜0.05 and P＜0.01 respetively),and the levels of serum total IgE and OVA specific IgE increased significantly(both P＜0.01).However the elevation in serum total and OVA specific IgE were able to be reduced by Biminne(P＜0.01 and P＜0.05 respectively).Conclusion Respiratory allergy mice model could mimic clinic the symptoms and immuno-imbalance status of the disease,and could be used in the study on the effect and mechanism of formulations and components. Biminne has been shown in previous studies to be effective in the management of allergic rhinitis,and this study shows its immuno-modulating effect may well be related to inhibiting the proliferation of splenocyte and reducing the level of serum IgE which were increased by allergy.PARTâ…¡Effects of Baicalin and EF in the treatment of respiratory allergy and regulation of T helper cell differentiation in mice model of airway allergyObjective To test the effect of Baicalin and EF in the treatment of a mice model of airway allergy,and investigate its immunomodulating effect on T helper cell differentiation,including regulating Th1/Th2 balance and the number and function of T regulatory cells.Methods BALB/C mice were randomly divided to 6 groups:normal group, model group,Baicalin group,EF group,Baicalin plus EF group and Dexamisone group,with 12 mice in each.The respiratory allergy model was induced by the intraperitoneal and intranasal administration of ovalbumin (OVA) into mice.The occurrences of sneezing and nasal rubbing were counted.The lung and spleen tissues were fixed in buffered formalin for histopathology examination.Lung eosinophils per HP were counted under microscope.Serum cytokines IFN-γ,IL-4,IL-5,IL-10,TGF-βwere tested using ELISA.Spleen IFN-γ⁺CD4⁺ T,IL-4⁺CD4⁺T,CD4⁺CD25⁺T,and CD4⁺foxp3⁺T cells were determined using flow cytometry.Spleen CD4⁺CD25⁺T cells were isolated by magnetic separation.Under the stimulation of OVA,CD4⁺CD25⁺T cells of each group and APC co-cultured with and CD4⁺CD25^-T cells from model group,the level of IFN-γ,IL-4,IL-5 in culture supernatants were tested by ELISA,ceils proliferation was determined by ³H-TdR,to reflect the depressing function of CD4⁺CD25⁺T cells。The OVA-induced-CD4⁺CD25⁺T cells from each group were mixed with the antigen provider ceils(APC),and then co-cultured with the CD4⁺CD25^-T cells from the model group.The level of IFN-γ,IL-4,IL-5,TGF-βin the culture supernatants were tested by ELISA. Ceils proliferation was determined by ³H-TdR,to show the suppression function of CD4⁺CD25⁺T cells。Results 1,Behaviors,lung tissue histopathology,T cell differentiation of the allergic model miceCompared with normal control group,The level of sneezing and nasal rubbing increased markedly after allergy-induction modeling(P＜0.01). Examination of lung sections under light microscopy revealed an infiltration of eosinophils and lymphocytes into the airway of OVA-challenged mice in the immunized group.The percentage of Th2 cells and ratio of Th2/Th1 in the model group up-regulated significantly(P＜0.01),while the percentage of CD4⁺CD25⁺T and CD4⁺foxP3⁺T cells downregulated(P＜0.05).Meanwhile the level of IL-4andIL-5 increased significantly(P＜0.01);but the level of serum IFN-γ,IL-10 and TGF-βdecreased significantly(P＜0.01).The function of CD4⁺CD25⁺T cells decreased in depressing CD4⁺CD25^-T ceils(P＜0.01).2,The effect of Baicalin and EF treatment on behaviors and lung tissue histopathology in respiratory allergic miceCompared with the model control group,the number of sneezes in five minutes in the Baicalin group decreased significantly(P＜0.05).Lung tissue histopathology revealed that the infiltration of eosinophils was also reduced.In the EF group sneezing and nasal rubbing numbers decreased significantly(P＜0.05 and P＜0.01 respectively).Lung tissue histopathology revealed that the infiltration of eosinophils and lymphocytes were also reduced.In the Baicalin plus EF group,sneezing and nasal rubbing levels decreased significantly(P＜0.01).Lung tissue histopathology revealed that the infiltration of eosinophils and lymphocytes were reduced. 3,The regulating effect of Baicalin and EF on helper T cell differentiationBaicalin decreased the level of Th2 cytokine IL-5 significantly (P＜0.01),and increased the level of regulatory T cytokine TGF-β(P＜0.01)。In the Baicalin group,spleen CD4⁺CD25⁺T cells showed suppression of the CD4⁺CD25^-T cell proliferation,but did not reach statistical significance compared to the model group.EF increased the level of Th1 cytokine IFN- significantly(P＜0.05), decreased the level of Th2 cytokine IL-4 and IL-5(P＜0.05),up-regulated the percentage of spleen Th1(IFN-γ⁺CD4⁺) cells(P＜0.01),down-regulated the percentage of Th2(IL-4⁺CD4⁺) cells(P＜0.05),reduced the ratio of Th2/Th1(P＜0.05).Compared to the model group,the level of regulatory T cytokine IL-10 and TGF-βincreased significantly(P＜0.01),the percentage of nTreg(CD4⁺CD25⁺T,CD4⁺foxP3⁺T) increased significantly(P＜0.05),and the depression function of CD4⁺CD25⁺T versus CD4⁺CD25^-T increased(P＜0.01 and P＜0.05 respectively)。In the Baicalin plus EF group,the level of Thl cytokine IFN-γincreased significantly(P＜0.05),the level of Th2 cytokine IL-4 and IL-5 decreased(P＜0.05),the percentage of spleen Th1(IFN-γ2+CD4⁺) cell was up-regulated(P＜0.05),the percentage of Th2(IL-4⁺CD4⁺) cells was down-regulated(P＜0.05),and the ratio of Th2/ Th1 reduced(P＜0.01). Compared to the model group,the level of regulatory T cytokine IL-10 and TGF-βincreased significantly(P＜0.01),the percentage of nTreg (CD4⁺CD25⁺T,CD4⁺foxP3⁺T) increased significantly(P＜0.05),and the depression function of CD4⁺CD25⁺T versus CD4⁺CD25^-T increased(P＜0.01).Conclusion Both Baicalin and EF could reduce the allergic respiratory inflammation reaction,but their key functions are different. Baicalin was able to reduce serum Th2 cytokine IL-5 level,increase T regulatory cytokine TGF-β,and reduce the infiltration of eosinophils in the lung tissue。EF could regulate Th1/Th2 balance,increase serum T regulatory cytokine IL-10 and TGF-β,and up-regulate the percentage and suppressing function of nTreg.EF was shown to reduce the infiltration of eosinophils and lymphocytes in the lung tissue.The Baicalin plus EF group showed the total anti-inflammation effects of the two components,and improved normal immunity status of helper T cell differentiation. PARTâ…¢The immunomodulating effect and mechanism of Baicalin and EF on the differentiation of T helper cell line D10G4.1Objective To establish an allergic model of T helper cell,and study the immunomodulating effect and mechanism of baicalin and EF on the differentiation of T helper cell line D10G4.1.Methods Different concentrations of IL-2 were added to the culture supernants of T cell lineage D10.G4.1.The cell viability was tested by XTT to ensure the IL-2 addition concentration in the culture supernatants. Cytokines in the culture supernatants were examined using ELISA,cell membrane markers and cytokines in the cell were examined by flow cytometry, and foxP3mRNA expression was examined by semi-quantitation RT-PCR,to determine the characteristics of D10.G4.1.Antigen conalbumin(CA) and antigen presenting cell(APC) was added to the culture supernants of D10.G4.1,to establish an allergic T helper cell model.Cell proliferation was tested by ³H-TdR,with the level of IFN-γ,IL-4,IL-5,TGF-βin the supernatants examined using ELISA。In the presence of CA and APC,D10.G4.1 cultures were divided into: model group,Baicalin high dose group,medium dose group and low dose group, EF high dose group,medium dose group and low dose group,Baicalin plus EF high dose group,medium dose group and low dose group,and dexamethasone group.Cell proliferation was tested by ³H-TdR,with the level of IFN-γ,IL-4,IL-5 and TGF-βin the supernatants were examined by ELISA, and foxP3 mRNA expression was examined by semi-quantitation RT-PCR.Resultsâ… characteristics of D10.G4.11.Cell marker CD4 and CD25 express on the D10.G4.1 cell membrane. Without antigen stimulation,a certain level of IFN-γ,IL-4,IL-5 and TGF-βcould be tested out in the supernants,and foxP3mRNA expression was positive.2.At the present of CA and APC,D10.G4.1 culture supernatants IL-4 and IL-5 level increased,TGF-βlevel decreased,IFN-γlevel didn't change,showed Th2 polarization.But D10.G4.1 have no regulatory T cell activity and no depressing function to CD4⁺CD25^-T cell.â…¡Effect of Baicalin and EF on D10.G4.1 proliferation and differentiation1.Baicalin depressed D10.G4.1 proliferation significantly at the concentration of 50ug/ml.Baicalin decreased supernatant IL-4 levels, markedly decreased IL-5 levels,increased TGF-βlevels,but didn't change IFN-γlevels.2.EF depressed D10.G4.1 proliferation significantly at the concentration of 0.5-50ug/ml.EF decreased supernatants IL-4,IL-5 and IFN-γlevels,while increasing TGF-βlevels.3.Baicalin plus EF decreased supernatants IL-4 IL-5and IFN-γlevels, increased TGF-βlevels.Conclusion:D10.G4.1 is a T helper cell line that contains the potential for Th2 polarization.It is useful to employ this cell line to study the differentiation of allergic T helper cells and the drugs to modulate allergic T helper cell differentiation.Baicalin could depress the Th2 differentiation polarization of D10.G4.1,in particular the depression IL-5 levels.EF could depress the Th2 differentiation polarization of D10.G4.1,and improve some regulatory characteristics.Baicalin combined with EF showed the total effects of the two components, transforming the allergic Th2 polarization of D10.G4.1 and regulating the helper T cell differentiation to normal immunity status.Conclusion1,Our study confirmed the clinical treatment effect of Chinese herbal formulation Biminne in respiratory allergy model,and immuno-modulation act as an important effective mechanism,reducing serum total IgE and OVA-sIgE and suppressing allergic lymphocyte proliferation may be one of Biminne' s mode of action.This work formed foundation for further study of the effect and mechanism of other components.2,On the animal model level,baicalin and EF have the similar effect as Biminne on treating allergy,manifest that baiclin and EF are important effective components of Biminne.Both baiclin and EF could release respiratory allergic inflammation,regulate the allergic helper T cells and responding eytokines widely.But baiclin and EF focus on different aspects,the two together may make the T helper cell differentiation tending to normal.3,Baiclin and EF could suppress Th2 polarization characteristics of T helper cell line D10.G4.1,increase regulatory cytokines secretion, modulate the balance of T helper cell differentiation.The cell lineage experiment conformed further that Baiclin and EF could transform the allergic Th2 polarization.4,Use Baiclin and EF together,may suppress unnormal products,and promote normal immuno-modulation,embody the FuZhengQuXie regular of traditional Chinese medicine.