Effect Of IL-33 On Osteoclasts In Chionic Apical Periodontitis

Objective To investigate the effect of interleukin-33(IL-33)on osteoclast formation and bone resorption in chronic apical periodontitis,to study the vital regulating function and mechanism of IL-33 on osteoclast formation,and further prove the role of IL-33 in the mechanism of bone resorption in chronic apical periodontitis.Methods 1.To determine the effect of IL-33 on the proliferation and differentiation of mouse preosteoclast RAW264.7 cells.RAW264.7 cells were treated with 100ng/ml IL-33,and the cells were harvested 1.5h,3h,6h,1d and 2d after induction.The proliferation of RAW264.7 cells treated with IL-33 was detected by flow cytometry.RAW264.7 cells were treated with 100ng/ml IL-33,and the cells were harvested 0d,4d and 7d after induction.Identification of cell differentiation and osteoclast formation of RAW264.7 cell line treated with IL-33 by TRAP staining.2.To study the effect of IL-33 on the genes and proteins of osteoclast related factors.The experiment was divided into four groups:group A: blank control group(only medium without any drug),group B: 100ng/ml IL-33 interferes with RAW264.7 cells for 1.5 h,group C: 100ng/ml IL-33 interferes with RAW264.7 cells for 3h,group D: 100ng/ml IL-33 interferes with RAW264.7 cells for 6 h and harvested cells at 4 time points.The mRNA and protein expression of RANKL,NFATc1 and TRAP were examined by real-time quantitative PCR and Western-blot,and the effect of IL-33 on the mRNA and protein expression of osteoclast-related factors RANKL,NFATc1 and TRAP was analyzed.Results Flow cytometry showed that the proliferation of RAW264.7 cells treated with100ng/ml IL-33 was inhibited,and with the increase of the time of 100ng/ml IL-33 treatment,the distribution proportion of S phase decreased gradually,and the cell proliferation slowed down gradually.The results of TRAP staining showed thatRAW264.7 cells were differentiated by 100ng/ml IL-33.Compared with the control group,there were no TRAP positive multinucleated osteoclasts(? 3 nuclei)on the 4th and 7th day.The results of RT-qPCR showed that the gene expression of TRAP in 1.5h group was higher than that in control group,while that in 3h group and 6h group was lower than that in control group.(p<0.05)The gene expression of NFATc1 in 1.5h group was higher than that in control group,while that in 3h group and 6h group was lower than that in control group.(p<0.05)The gene expression of RANKL in 1.5h group was higher than that in control group,while that in 3h group and 6h group was lower than that in control group.(p<0.05)The results of Western-blot showed that the protein expression of TRAP in 1.5h group and 6h group was higher than that in the control group,while the protein expression in the 3h group was lower than that in the control group.(p<0.05)The protein expression of NFATc1 in 1.5h group was higher than that in control group,while that in 3h group and 6h group was lower than that in control group.(p<0.05)The protein expression of RANKL in 1.5h group was higher than that in control group,while the protein expression in 3h group and 6h group was lower than that in control group.(p<0.05)Conclusion IL-33 inhibits osteoclast formation and inhibits the mRNA and protein expression of osteoclast-related factors RANKL,NFATc1 and TRAP.IL-33 may inhibit the differentiation and proliferation of osteoclasts by directly inhibiting the formation of osteoclasts or indirectly inhibiting the expression of osteoclast-related factors RANKL,NFATc1,TRAPmRNA and protein,and then inhibit bone resorption,which plays a protective role in bone resorption in chronic apical periodontitis.