Effects of different concentrations of γ-secretase inhibitor on osteogenic differentiation of human periodontal ligament stem cells

Abstract

Objective To evaluate the effect of gamma secretase inhibitor (DAPT) on osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) in different concentrations. Methods hPDLSCs cultured in vitro were randomly divided into, (1) two control groups: hPDLSCs group (without DAPT) and 0.173 25 μL/L dimethyl sulfoxide (DMSO) group, and (2) three experimental groups: 25, 50 and 75 μL/L DAPT groups. The effects of different concentrations of DAPT on the proliferation of hPDLSCs were evaluated by cell counting kit (CCK-8) in control group and three experimental groups (25, 50 and 75 μmol/L DAPT) . Notch letters of different concentrations of DAPT on hPDLSCs were detected by alizarin red staining, real-time fluorescence quantitative polymerase chain reaction (PCR) and western blot. SPSS 21.0 was used for statistical description and analysis, when α= 0.05 was set as the statistical significance level. Results CCK-8 results showed that DMSO concentration of 0.17325 ul/L had no significant effect on the proliferation of periodontal ligament stem cells, and DAPT at different concentrations inhibited the proliferation of hPDLSCs, and DAPT concentration of 50 μmol/L had the strongest inhibitory effect on cells (P<0.001) ; Alizarin red staining results showed that the mineralized nodules in SC and DMSO groups were large and dark. There was no significant difference between the two groups. In contrast, the mineralized nodules formed in the three experimental groups were less. Specifically, the mineralized nodules formed in the group of 50 μmol/L DAPT were less and smaller than that in the groups of 25 and 75 μmol/L. The results of real-time PCR showed that the expressions of CAP, OCN and Runt-related transcription factor 2 (Runx2) were 0.574 ± 0.182, 0.269 ± 0.100 and 0.470 ± 0.080, respectively, in the group of 50 μmol/L DAPT, less than that in other groups (P<0.05) . The expression levels of Hes-1 and Notch signaling pathway related molecules Notch1 and Hes-1 were 0.467 ± 0.118 (P<0.001) and 0.318 ± 0.015 (P<0.024) , respectively, the lowest among the groups. The expression of Notch protein, Hes-1 and Runx2 in the control group was higher than that of the three experimental groups, and the expression of Runx2 protein in the DAPT group at 50 μmol/L was 0.116 ± 0.006, which was significantly lower than that in the other groups (P<0.001) . Conclusions DMSO concentration of 0.173 25 μL/L had no significant effect on the proliferation and bone differentiation of periodontal ligament stem cells. DAPT may inhibit the proliferation and bone differentiation of hPDLSCs by blocking Notch signaling pathway. Such an inhibitory effect was found to be strongest in the group of 50 μmol/L DAPT. Key words: Periodontal ligament; Stem cells; Osteoblasts; Notch signal pathway