Hydrogen-saturated Sterile Water Influences Anti-DNP-IgE Mediated Mast Cells Activation

Objective: To study the influence of type Ⅰ allergic disease is a kind of allergic diseases mediated by IgE,mainly including bronchial asthma,rhinitis,diseases such as conjunctivitis.Main show is abundant on Th2 cytokines,acidophilic granulocyte and mast cells infiltration,and inflammatory reaction.In recent years the incidence of typeⅠallergic disease significantly increased,increasing the same degree of care.TypeⅠallergic disease is a kind of need from the multiple perspectives multi-level comprehensive treatment of disease,and its treatment is still not perfect at present stage,it has become a global health problem.Recently a number of studies have found that mast cells in the pathogenesis of type Ⅰ allergic disease throughout,in the process of the core role.Mast cells are the primary effect of anaphylaxis,the activation directly affect secondary effector cells,eosinophils and raise and activation of neutrophils,are the important regulatory immune cells,is mediated speed important pivot new hairstyle allergy and inflammation,can trigger and expand the inflammatory reaction.At the same time,mast cells also involved in many diseases and inflammation process,particularly with some allergic diseases such as urticaria,psoriasis,and closely related to the occurrence of systemic lupus erythematosus(sle).But for the mast cell activation mechanism is not yet clear,if it can be proved mast cells activation mechanism,type ofⅠallergy disease prevention and control is of great significance.Hydrogen as a kind of new gas signal molecule,with intervention in a variety of inflammatory reaction,such as acute pancreatitis,acute pneumonia).Hydrogen could also has an intervention effect of immune regulation,The research group has proved the hydrogen-saturated sterile water has therapeutic effect on asthma in mice.So to mast cells of mice P815 as the research object,mast cell activation model is set up,from the Angle of the mast cell research hydrogen-saturated sterile water treatment typeⅠ allergic disease became our research direction.By observing the hydrogen-saturated sterile water effect on the mast cell activation,from the perspective of mast cells to explore the possibility of hydrogen-saturated sterile water for the treatment of allergic disease for hydrogen and proved the molecular mechanism of regulation of mast cells activation,used to treat type Ⅰ allergy disease to provide new ideas.Methods: Mast cells(P815 mouse mast cell lines,buy from the United States)in 1640 in the culture medium for culture,which contains 10% newborn calf serum,design of experiment group: normal control group(Con group),the Anti-DNP-IgE model group(Anti-DNP-IgE group),hydrogen-saturated sterile water treatment group(H2+Anti-DNP-IgE group),pure hydrogen-saturated sterile water group(H2 group).Anti-DNP-IgE was determined by the determined by MTT experiment the optimum concentration and intervention in time.Of groups of toluidine blue stain from the perspective of morphology revealed hydrogen-saturated sterile water whether influences Anti-DNP-IgE induction of mast cell activation,preliminary observations of hydrogen-saturated sterile water of Anti-DNP-IgE induction of mast cells activation;By ELISA kits of mast cells activation key indicators including histamine(His),β-hexosaminidase(β-Hex)and tryptase(TPS)in each level from the perspective of immune adjustment validation hydrogen-saturated sterile water for Anti-DNP-IgE induction of mast cells activation might affect;Further research from the perspective of morphology applied immunofluorescence staining techniques hydrogen-saturated sterile water intervention mechanism of mast cells activation water three key tyrosine protein kinase(Lyn,Syk and Fyn)and the receptor(FceRIβ)combining with the situation.Paper further from the Angle of molecular hydrogen regulation of the molecular mechanism of mast cell activation,Western Blot method to observe whether hydrogen saturation water sterilization intervention mast cells activation mechanism of initial stage of the three key tyrosine protein kinase(Lyn,Syk and Fyn)and downstream channel PI3K-AKT molecular expression of signal transduction pathways.Results:1 Anti-DNP-IgE optimal intervention time and concentration.Determined by MTT experiment to determine the optimal intervention time Anti-DNP-IgE activate mast cells of the optimum concentration and intervention time 10 h 1000 ng/ml,respectively.2 Mast cells of toluidine blue stain morphology observation.Normal controls with pure hydrogen saturation sterilized water group has no obvious change,Anti-DNP-IgE cytoplasm endocrine vesicle model group was obviously increased,and the hydrogen saturation sterilization in cytoplasm vacuoles within the water treatment group decreased significantly.From the point of view of morphological preliminary evidence that the hydrogen saturation sterilized water hydrogen is influential to mast cells activation.3 Hydrogen-saturated sterile water suppresses the Anti-DNP-IgE induction of mast cells activation.This topic selection of mast cells activation three key indicators(histamine,β-hexosaminidase and trypsin),extracting supernatant on each cell,testing each cell by ELISA experiments of histamine,β-hexosaminidase and tryptase levels of ELISA detection: Anti-DNP-IgE histamine in model group,β-hexosaminidase and tryptase levels compared with normal control group increased significantly,while hydrogen saturation sterilized water treatment compared with Anti-DNP-IgE model group significantly reduced.That hydrogen saturation sterilizing water for mast cells activation index has obvious inhibitory effect.4 Morphological perspective observe hydrogen-saturated sterile water influences mast cells activation of three key tyrosine protein kinase.Immunofluorescence result shows: the Anti-DNP-IgE stimulation,found Syk,Lyn and Fyn three proteins may occur in the redistribution of cells,all can shift to membrane;And after hydrogen-saturated sterile water intervention,suppresses the transfer.Further instructions from the morphology of hydrogen-saturated sterile water may inhibit three tyrosine kinase pathway to inhibit mast cell activation.5 Hydrogen-saturated sterile water suppresses the mast cells activate the initial stage of the three key tyrosine protein kinase activationWestern blot results show that the Anti-DNP-IgE stimulation,P-SYK,P-LYN,P-FYN expression significantly higher;And the hydrogen-saturated sterile water above index expression decreased significantly after intervention.That hydrogen-saturated sterile water may inhibit the expression of three kinds of tyrosine protein kinase,so as to achieve the role of mast cells activation inhibition.6 Hydrogen-saturated sterile water inhibit mast cell activation pathways downstream of PI3K-AKT molecular signal transduction pathways activated.Western blot results show that compared with the Anti-DNP-IgE model group,hydrogen-saturated sterile water intervention group of P-PI3 K and P-AKT expression decreased significantly.Description hydrogen-saturated sterile water inhibition of Anti-DNP-IgE mast cell activation,stimulation may be inhibit mast cell activation classic downstream pathway PI3K-AKT activation.Conclusions:1 Hydrogen-saturated sterile water can inhibit Anti-DNP-IgE induction mast cell activation.2 Hydrogen-saturated sterile water inhibit mast cell activation the initial stage of the three key tyrosine excitation protease(Lyn,Syk and Fyn)membrane transfer and the process of phosphorylation.3 Hydrogen-saturated sterile water inhibit mast cell activation pathways downstream of PI3K-AKT molecular signal transduction pathways activated.To sum up,the hydrogen –saturated sterile water can be through the regulation of mast cells activation initial stage three tyrosine excitation protease(Lyn,Syk and Fyn)and downstream pathway of PI3K-AKT pathways,thus inhibiting mast cell activation.