[Mechanism of low-concentration IL-6 in promoting osteogenic differentiation of human stem cells from apical papilla via the JAK2-STAT3 signaling pathway under inflammatory condition].

Abstract

Objective: To investigate the effects and underlying mechanisms of low-concentration interleukin-6 (IL-6) on the proliferation, migration and osteogenic differentiation of human stem cells from apical papilla (hSCAPs). Methods: hSCAPs were isolated from the apical papilla of third molars extracted in the Department of Oral Maxillofacial Surgery, the First Affiliated Hospital of Zhengzhou University, and identified by flow cytometry and immunofluorescence. Cell counting kit-8 (CCK-8) and Transwell migration assay were used to detect the effects of IL-6 in different concentrations (10, 20, 50 μg/L) on the proliferative and migration ability of hSCAPs. Alizarin red staining, cetyl pyridine chloride quantification, alkaline phosphatase (ALP) staining and ALP activity assay were performed to evaluate the osteogenic differentiation capacity of hSCAPs under inflammatory conditions induced by different concentrations of IL-6, so as to determine the optimal IL-6 concentration to simulate the inflammatory microenvironment of hSCAPs osteogenic differentiation. Real-time fluorescence quantitative PCR (RT-qPCR) and Western blotting were used to detect the osteogenic differentiation ability of hSCAPs treated with low-concentration (10 μg/L) IL-6. Western blotting, Transwell migration assay, ALP staining, ALP activity assay and RT-qPCR were applied to explore the mechanism by which low-concentration IL-6 promotes the migration and osteogenic differentiation of hSCAPs via the Janus kinase 2-signal transducer and activator of transcription 3 (JAK2-STAT3) signaling axis. Results: The isolated and cultured hSCAPs were positive for surface markers CD24 and vimentin, while negative for CD45 and cytokeratin. Different concentrations of IL-6 exerted no significant effect on the proliferation of hSCAPs (P0.05). The number of migrated cells (11 463.33±622.70), cetyl pyridine chloride quantification (2.087±0.121) and ALP activity (94.11±4.06) U/g of hSCAPs in 10 μg/L IL-6 treatment were all significantly increased than those in 0 μg/L IL-6 control group, respectively [8 246.00±674.02, 1.330±0.096, and (53.87±3.70) U/g] (all P0.05). Moreover, the cell migration number and ALP activity in the 10 μg/L IL-6 group were more remarkably increased than in 20 μg/L IL-6 group [9 503.33±275.03, (74.44±3.55) U/g] (both P0.01). After osteogenic mineralization induction of hSCAPs under low-concentration IL-6 intervention, the mRNA expression levels of osteogenic-related factors Osterix (OSX) and Runt-related transcription factor 2 (Runx2) at day 3 (2.033±0.308, 1.433±0.215) and day 7 (3.497±0.153, 3.360±0.061) were significantly upregulated compared to the control group (all P0.05), while the mRNA level of osteocalcin (OCN) was significantly elevated at day 7 (2.950±0.315) (P0.05). The protein expression levels of OCN were significantly increased at day 3 (2.077±0.447) and day 7 (5.023±0.537) than those in the control group (both P0.05), while the protein levels of OSX and Runx2 elevated significantly at day 7 (3.283±0.601, 3.740±0.613) (both P0.05). Compared with the control group, low-concentration IL-6 obviously upregulated the protein expression ratios of p-JAK2/JAK2 and p-STAT3/STAT3, cell migration capacity, ALP activity, as well as the mRNA and protein expression levels of OCN, OSX and Runx2 of hSCAPs (all P0.05). Nevertheless, the JAK2-STAT3 signaling pathway inhibitor AG490 could significantly reverse these promotive effects of IL-6 on cell migration and osteogenic differentiation (all P0.05). Conclusions: IL-6 at concentrations of 10, 20 and 50 μg/L has no obvious effect on the proliferation of hSCAPs. Low-concentration IL-6 promotes the migration and osteogenic differentiation of hSCAPs under inflammatory conditions through activating the JAK2-STAT3 signaling pathway.