Effects Of Paeonol On Differentiation And Maturation Of Mouse Bone Marrow Dendritic Cells And Secretion Of Cytokines

Objective To study the effect of Paeonol on the differentiation and maturation of bone marrow dendritic cells and the secretion of cytokines in mice,and to develop new ideas and theoretical basis for the prevention and treatment of atopic dermatitis.Methods Dendritic cells cultured in vitro using mouse bone marrow cells,cultured dendritic cells were divided into three groups,group A as control group,the control group with 2 ml RPMI1640 medium;B group 1 stimulation group,2ml stimulation group with 100ng/ml containing RPMI 1640 FSL-1 conditioned medium;group C high dose intervention paeonol low in paeonol group,low medium and high dose intervention group were 25 g/ml,50 g/ml,100 g/ml paeonol incubated 2 hours after adding 100ng/ml FSL-1 were incubated for 24 hours.Flow cytometry was used to detect DC surface molecules(CD40,CD86 and MHC-II)the level of detection of DC,ELISA amount of IL-10 in culture supernatant,IL-12 and TNF-a.Results 1.Dendritic cells cultured bone marrow cells of mice after separated by RM,GM-CSF and RM induced by IL-4 after cultured for 48 hours,under the microscope can be observed in most cells adherent growth,cell morphology and size,after 72 hours of incubation,cell suspension increased than before,and some cells were clustered into groups the phenomenon of.After fourteenth days of culture were observed when the cell volume significantly,cell proliferation was significantly increased,and the formation of cell clones,high magnification were observed on the cell surface without typical dendritic protrusions,with immature morphology of DC.2.The expression level of dendritic cell surface molecule FSL-1 after treatment with FSL-1 compared to DC,CD40,CD86 and cell surface expression of MHC-Ⅱ increased significantly(p<0.05)(Fig 2 A,Table 1).In order to investigate the effect of Paeonol on the role,we are adding paeonol low,medium and high dose in cells;and incubated with 2H,and then added to the FSL-1 culture expression of surface molecules of DC by FACS after 24h.Compared with FSL-1 group,Paeonot in the high dose group of DC,CD86 and MHC-Ⅱsurface CD40 expression was significantly decreased(Fig 2 B,Table 1),and the difference was statistically significant(p<0.05),but in the low dose group,CD40 expression also decreased,but the difference was not statistically significant(p>0.05).The above results suggest that FSL-1 can induce the formation of mature dendritic cells through TLR2 signaling pathway,while adding paeonol can reverse the process to.inhibit the maturation of dendritic cells,and this inhibition effect is concentration dependent,i.e.the concentration of paeonol is higher,the stronger the inhibition ability.3.Cytokine levels in the supernatant ofgroups:FSL-1 treatment group and FSL-1 training group compared with the untreated,proinflammatory cytokines IL-12 and TNF-levels were significantly higher(p<0.05)(Fig 3A,Table 2),the anti-inflammatory cytokine IL-10 decreased significantly(p<0.05)(Fig 3 A,Table 2).At the same time,in the low,medium and high dose of Paeonol in treatment group,compared with the FSL-1 group,IL-12 was significantly decreased(p<0.05)(Fig 3B,Table 2),and the levels of TNF-α in paeonol group and high dose of decreased significantly(p<0.05)(Fig 3 B,Table 2),and the concentration of TNF-in the low dose group of Paeonol in alpha also decreased,but the difference was not statistically significant(p>0.05).For the change of the anti-inflammatory cytokine IL-10,we found that compared with the FSL-1 group,the concentration of low,medium and high dose of paeonol group IL-10 increased significantly(p<0.05)(Fig 3 B,Table 2),and with the increasing concentration of paeonol,the stronger the ability to secrete IL-10.The above results showed that paeonol can affect the secretion of DC cytokines to change the differentiation of lymphocyte subsets,it can inhibit DC secretion of proinflammatory cytokines such as IL-12,while promoting the anti-inflammatory cytokine production of IL-10 from the original ThO cell differentiation to Thl cells.Conclusion 1.Paeonol can inhibit the lipoprotein of pathogenic microorganism to activate DC through the TLR2 path,inhibit DC maturation;2.Paeonol can inhibit TLR2 signaling pathway of atopic dermatitis and chronic inflammation of the development;3.Paeonol can inhibit IL-12 and TNF-alpha proinflammatory cells due to the release of IL-10,promote the release of anti-inflammatory cytokines,prevent naive Th cells to differentiate into Th1,improve the proportion of Th1/Th2 offset state,chronic inflammation further control of AD,so as to achieve the purpose of treatment of atopic dermatitis.