Study On Amlexanox Inhibited Maturation Of Mouse Bone Marrow-derived Dendritic Cells

Objective: Dendritic cell(DC)are currently known as the most powerful antigen presenting cell(APC),and play an important role in immune-mediated related diseases.DC was divided into mature dendritic cell(mDC)and immature dendritic cell(imDC)according to their degree of maturation.MDC could activate immune response;imDC were lowly expressed necessary factors which induce immunity response and induce immune tolerance.Therefore,inhibition of DC maturation is necessary for the induction of immune tolerance and treatment of autoimmune diseases.The important metabolic alteration of imDC maturation is the enhancement of glycolytic metabolism,mainly by TANK-binding kinase 1(TBK1)/ nuclear factor-?B kinase subunit-?(IKK?)–AKT pathway at early stages.Amlexanox(ALX)is a novel and specific inhibitor of IKK? and TBK1 that inhibits glycolysis metabolism,inhibits imDC maturation and induces immune tolerance.This study explores the effect of amlexanox on inhibiting DC maturation,in order to provide a new research ideas for the treatment of autoimmune diseases.Methods:1.Culture bone marrow-derived dendritic cells in vitroAfter the mice were sacrificed,the femur bone marrow was taken out and placed in complete medium containing 20ng/ml granulocyte-macrophage colony-stimulating factor(GM-CSF).Changed medium on the 3th and 6th days,respectively.On the 8th day,the cells were randomly divided into 4 groups,each group contained 4 petri dishes which were from 4 mice respectively.The control group was added GM-CSF 20ng/ml;the LPS group was added GM-CSF 20ng/ml,lipopolysaccharide(LPS)1?g/ml;low dose ALX group added GM-CSF 20ng/ml,LPS 1?g/ml,ALX 2?M;high dose ALX group added GM-CSF 20ng/ml,LPS 1?g/ml,ALX 10 ?M.On the 10 th day,each group of cells was collected for further experiments.2.The morphology of the cells was observed by fluorescence inverted microscope and scanning electron microscope.The ultrastructure of cells was observed by transmission electron microscope.3.Flow cytometry detected the expression of CD11c?MHC??CD80 and CD86 in each group on the 10 th day cultivation.4.Enzyme Linked Immunosorbent Assay(ELISA)detected the level of IL-6?IL-12 and IL-23 in the supernatant of each group on the 10 th day after cultivation.5.SPSS 21.0 was used for statistical analysis.Results:1.The cell surface protrusions in the control group were few and small;the low dose ALX group and high dose ALX group cells were large and irregular,has many surface protrusions,which were thick;the LPS group cells were large and irregular,and the surface protrusions were dense,thick and long.2.The golgi apparatus and mitochondria in the low dose ALX group and high dose ALX group cells were decreased compared with the LPS group on the 10 th day of culture.3.Compared with LPS group,MHCII and CD80 in the low dose ALX group were statistically decreased(P<0.05);MHCII,CD80,CD86 in the high dose ALX group were statistically decreased;(P<0.05).4.Compared with LPS group,the level of IL-12 and IL-23 in the low dose ALX group and high dose ALX group were significantly decreased(P<0.05).Conclusions:1.This experiment used GM-CSF successfully culture BMDC in vitro.2.Amlexanox may inhibitis the maturation of BMDC.