The Effects Of MAPKs Signaling Pathway On LPS Regulated Odonto/Osteogenic Differentiation Of HAPCs

AimBacterial endotoxin lipopolysaccharide (LPS) is a powerful virulence factor which is associated with the pathogenesis of apical periodontitis. LPS can be detected in the necrotic pulp. It permeates through apical foramen into the periapical tissue, resulting in periapical lesions. In immature permanent teeth with pulp necrosis or apical periodontitis, LPS can stimulate human dental apical papilla cells (HAPCs) from apical foramen, and the cell biological characteristics will be impaired by LPS inevitably.The purpose of this study was to explore the effects of LPS on the proliferation and odonto/osteogenic differentiation of HAPCs, and the role of MAPKs signaling pathways in LPS regulated the odonto/osteogenic differentiation of HAPCs.Methods and resultsPart I Culture and isolation of HAPCs and induced mineralizationHAPCs were cultured by enzymatic dissociation method. The cell had active proliferative capacity and showed "S" shape growth curve. HAPCs were cultured in odonto/osteogenic induction medium for 21 days. The alizarin red staining showed that calcium deposition were increased after induction compared with the control group.Part II The effects of LPS on the proliferation and odonto/osteogenic differentiation of HAPCs Cells were cultured with LPS in different concentrations, proliferation and alkaline phosphatase activity of HAPCs were determined. The results showed that low concentration of LPS promoted proliferation and ALP activity of HAPCs. Further, after cells were treated with low concentration of LPS, alizarin red staining was used to detect the formation of mineralized nodules, and the expression of odonto/osteogenic genes such as dentin sialophosphoprotein(DSPP), runt-related transcription factor 2(Runx2), bone sialoprotein (BSP), osteocalcin (OCN) were assessed by real-time PCR. The results showed that at 7d after induction, compared with the control group, the calcium deposition had no difference. But the expression of DSPP, Runx2, BSP were increased after stimulated by LPS and the difference was statistically significant. The expression of OCN had no difference compared with the control group. After 14 days of induction, the calcium deposition was increased in the group treated with LPS(p<0.05). The expression of DSPP, Runx2, BSP,OCN were all increased compared with the control group, however only the former three factors indicated significant difference. When the cells were induced for 21 days, only the expression of Runx2 was increased in the group treated with LPS and the difference was statistically significant. The calcium deposition and the expression of DSPP, BSP, OCN all had no difference compared with the control group. In summary, LPS promoted odonto/osteogenic differentiation of HAPCs.Part III The effects of MAPKs signaling pathway on LPS regulated odonto/osteogenic differentiation of HAPCsThe phosphorylation of ERK, p38 and JNK was examined by Western blotting in HAPCs treated by LPS. The results showed that LPS could activate the phosphorylation of ERK and p38MAPK. The ERK inhibitor U0126 and the p38 inhibitor SB203580 were used to inhibit the activity of ERK and p38MAPK signaling pathway during the induction of LPS on HAPCs. Then the ALP activity and the expression of DSPP, Runx2, BSP were detected.The results demonstrated that not only the ALP activity, but also the expression of DSPP, Runx2, BSP mRNA/protein were all decreased after inhibition of ERK and p38MAPK signaling pathway. In conclusion, inhibition of ERK and p38MAPK signaling pathway could reduce odonto/osteogenic differentiation of HAPCs induced by LPS.ConclusionLow concentration of LPS could promote the proliferation and odonto/osteogenic differentiation of HAPCs. The ERK and p38 MAPK signaling pathways were involved in the LPS regulated the odonto/osteogenic differentiation of HAPCs.