Effects of p38 mitogen-activated protein kinase on osteogenic differentiation of human periodontal ligament stem cells in inflammatory microenvironment.

Abstract

To investigate the expression of mitogen-activated protein kinase (MAPK) in the chronic periodontitis tissue-derived in the periodontal ligament stem cells (PDLSCs) and explore its effect on the osteogenic differentiation of human PDLSCs in inflammatory microenvironment. PDLSCs were obtained from human healthy individuals (H-PDLSCs) and patients with periodontitis (P-PDLSCs). The tumor necrosis factor (TNF)-Α and interleukin (IL)-1Β secretion and mRNA expression levels of H-PDLSCs and P-PDLSCs were detected using enzyme-linked immunosorbent assay and real-time quantitative PCR. Immunofluorescence staining was used for determining the protein levels of p38 in PDLSCs. The levels of p38 and p-p38 following culture in osteogenic medium for 7 d of H-PDLSCs and P-PDLSCs were detected using Western blotting. After the PDLSCs were stimulated with SB-203580,the p38 MAPK specific inhibitor, for 30 min and then in osteogenic induction process for 7 days,the expression levels of the osteogenic gene Runx2 and alkaline phosphatase (ALP) were determined by real-time quantitative PCR, and bone formation ability of PDLSCs was tested by alizarin red (AR) staining. The secretions of TNF-Α and IL-1Β were significantly higher in P-PDLSCs compared with H-PDLSCs (68.80 ± 6.70 vs. 34.10 ± 3.07,P=0.001;57.10 ± 4.23 vs. 26.90 ± 2.58,P=0.000). The same trend was seen in the gene expression levels of both TNF-Α and IL-1Β in PDLSCs (PTNF-Α=0.011,P IL-1Β=0.009). p38 was more strongly induced in P-PDLSCs cells than in H-PDLSCs.The basal level of p38 in H-PDLSCs was lower than that in P-PDLSCs cells cultured in the basic medium. However,the level of p-p38 was increased in H-PDLSCs than in P-PDLSCs under osteogenic condition. Treatment of PDLSCs with SB-203580 and then cultures under osteogenic differentiation lead to significantly decreased expressions of Runx2 and ALP in both H-PDLSCs and P-PDLSCs (H-PDLSCs:P(Runx2)=0.044, PALP=0.036;P-PDLSCs:P(Runx2)=0.017, PALP=0.004). p38 MAPK is involved in the inflammatory response of PDLSCs in the chronic inflammatory microenvironment. The inhibition of p38 by SB-203580 also remarkably suppresses the osteogenic differentiation of PDLSCs in a chronic inflammatory microenvironment.