PI3K Regulates IgE Switching By Controlling Pax5

Background and Objectives:Allergic diseases mainly lead to disorders of their own physiological functions,accompanied by an increased class of immunoglobulin E?IgE?in the serum,such as allergic rhinitis,asthma,etc.These disorders have occurred in the past 10 years.Rapid growth has reached the proportion of near epidemics^[1].Phosphatidylinositol 3-kinase?PI3K?plays an important regulatory role in the conversion of B lymphocytes.Paired box protein 5?Pax5?is an important regulator in the early stages of B cell maturation.It can express B-cell lineage specific activator protein?BSAP?in the early stage..Its abnormal expression may also lead to the formation of the body's lymphoma.The presence of this molecule has an important regulatory effect on the expression of antibody transcripts.However,the specific mechanism of the regulation of IgE switching by PI3K has not yet been explored.Therefore,this study aims to explore the relationship between PI3K signaling pathway and Pax5 and other transcription factors in IgE switching.Methods:1)In the study,we choose manly the two cell lines of RPMI8266 and U266,called human Multiple myeloma cells.Firstly,I choose two kinds of PI3K inhibitors in order to pick out the more effective one.After 48h with stimulations of IL-4?50ng/m L?and anti-CD40?1ug/ml?in the presence of LY294002 or PIK-293,we extracted the RNA of cells,then measured the relative expression of AID and?GLT.Meanwhile,we measured the IgE⁺cells'percent by flow cytometry.2)To determine whether PI3K can switch the IgE class though p-AKT,we treated cells with PI3K inhibitor?15?M LY294002?,p-AKT inhibitor?20?M PHT-427?and the co-inhibition of the above two substances accompanied by stimulations of IL-4?50ng/mL?and anti-CD40?1?g/ml?.After 48h,we extracted the RNA of cells,then measured the relative expression of AID and?GLT.3)We suppressed the PI3K and Pax5 expression from the level of nucleic acid by the siRNA.After 6h with the conduction of si-PIK3-CD,si-Pax5 and the co-transfection of above,the cells were stimulated with IL-4?50ng/mL?and anti-CD40?1?g/ml?.After 48h,we extracted the RNA of cells,then measured the relative expression of AID and?GLT.4)In order to further confirm the role of Pax5protein in the PI3K pathway,we established sensitization model in the C57B/L mice.All mice were bought one week earlier and the animals were stable.After one week,each mouse was given intraperitoneal injection of OVA and AL?OH?3 mixed PBS solution 100?L.After one week,treatment group mice were injected intraperitoneally at a concentration of 4 mg/kgLY294002.After one week or two weeks,we picked up the mice spleen and eyeball blood.Spleen equally divided into 4%paraformaldehyde preservation for immunohistochemistry,the other half preserved in-80?Results:1)We chose a stronger PI3K-inhibitor?LY294002?by analyzing AID and?GLT through flow cytometry and QPCR assays.2)We found that IgE production has a concentration-dependence of LY294002 by ELISA and flow cytometry.3)We performed a single PI3K-inhibitor,a single AKT-inhibitor or mixed inhibitors treatment.We concluded that PI3K could regulate IgE switching,but not by p-AKT?P<0.05?.And We found that the expressions of Pax5 increased in LY294002-treated cell lines.4)We performed a PIK3CD/Pax5 siRNA experiment.The mRNA expression levels of AID and?GLT were decreased when cells were transfected only with Pax5si RNA?P<0.05?.However,when cells were co-transfected with PIK3CD and Pax5si RNA,the levels of AID and?GLT and the secretion of IgE correspondingly increased but were significantly lower than those found for PIK3CD siRNA transfection alone?Figure 4,P<0.05?.5)The acute sensitization model of mice was made with LY294002,and the content of serum IgE in the experimental group was higher than that of thecontrol group?n=10,F=107.463,P<0.01?.The expression of Pax5 in the experimental group was higher than that in the control group;Id2 expression was decreased?P<0.05?.Conclusion:Inhibition of PI3K activity enhances the class switching of IgE and regulates IgE production by controlling the expression of Pax5 molecules.