Regulation function of NF-кB signaling pathway mediated by IL-34 on odontogenic/osteogenic directional differentiation of stem cells from apical papilla
Objective:To investigate the regulatory role of nuclear factor кB(NF-кB)mediated by interleukin-34(IL-34)on the odontogenic/osteogenic differentiation of stem cells from the apical papilla(SCAPs).Methods:Enzymatic digestion method was used to subculture SCAPs from mouse apical papilla tissue.Flow cytometry was used to assess the expression of CD44,CD90,CD45,and col-ony-stimulating factor 1 receptor(CSF-1R)on the surface of SCAPs.The experiment was carried out in four groups:control group,IL-34 group(100ng/ml IL-34),CSF-1R group(100ng/mL IL-34+25ng/ml anti-CSF-1R),and NF-кB group(100ng/ml IL-34+1μmol/L BMS-345541).Western blot analysis was conducted at 30,60,and 120 minutes to examine the expression levels of NF-кB key proteins(p-IкBα,IкBα,p-P65,and P65)in the cytoplasm of SCAPs.Mineralization induction was performed on the four groups of SCAPs for 3,7,and 14 days,and Alizarin Red staining and semi-quantitative analysis were used to compare the mineralization nod-ule formation capacity of the cells.RT-qPCR was used to measure the mRNA levels of the osteogenic markers Runt-related transcription factor-2(RUNX2),dentin sialophosphoprotein(DSPP),and osteocalcin(OCN)in SCAPs of each group.Results:Flow cytometry analysis showed that in SCAPs expressed CD44(90.4%)and CD90(93.5%)were positive and were negative for hematopoietic stem cell marker CD45(3.1%).SCAPs expressed CSF-1R was positive(23.2%).Compared with the control group,SCAPs in the IL-34 group demonstrated significant upregulation of p-IкBα and p-P65 expression(P＜0.05),increased mineralization nodule formation on the 3rd,7th,and 14th day,and a significant elevation in relative mRNA expression levels of RUNX2,DSPP,and OCN(P＜0.05).Compared with the IL-34 group,the CSF-1R group and NF-кB group showed decreased expression of p-IкBα and p-P65 in the cyto-plasm of SCAPs(P＜0.05),reduced mineralization nodule formation on the 3rd,7th,and 14th day,and a significant decrease in rel-ative mRNA expression levels of RUNX2,DSPP,and OCN(P＜0.05).Conclusion:IL-34 can regulate the NF-кB signaling pathway in SCAPs via the IL-34/CSF-1R axis,and modulate the differentiation capacity of SCAPs towards tooth/bone via the IL-34/CSF-1R/NF-кB signaling pathway.
Stem cells from apical papillaInterleukin-34Directional differentiationNF-кB signaling pathway
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