| ObjectiveHuman periodontal ligament cells are key cells of the periodontium playing important roles in promoting the regeneration and restoration of periodontal tissue. The purpose of our study was to explore whether the scutellarin had protective effects on human periodontal ligament cells, so as to provide theoretical foundation and explore a new way for periodontal disease therapy.Methods1 Human periodontal ligament cells were primarily cultured in vitro and examined by immunohistochemical method.2 Experimental sub-groups: The experiment was designed into normal control group, LPS group, scutellarin plus LPS group. Normal control group was only treated with 2% FBS DMEM; LPS group was treated with 100μg/ml LPS of E.Coli; scutellarin and LPS group was treated with different concentration (0.001μg/ml, 0.01μg/ml, 0.1μg/ml, 1μg/ml, 10μg/ml) scutellarin and 100μg/ml LPS of E.Coli respectively.3 MTT colorimetric assay was applied to investigate the effects of on the proliferative activity of the human periodontal ligament cells treated with LPS.4 Flow cytometry was applied to monitor the effects of scutellarin on the cell cycle of the human periodontal ligament cells.5 Enzyme dynamics method was applied to observe the alkaline phosphatase (ALP) activity of the human periodontal ligament cells.6 Enzyme-Linked immunosorbent assay (ELISA) was applied to detect the production of TNF-αin the culture supernatant of the human periodontal ligament cells.7 Western-blot method was applied to examine the expression of MMP-1 and MMP-2 in the human periodontal ligament cells.8 The statistical analysis was performed with the SPSS 11.5 using one-way analysis of variance (ANOVA) and LSD-t test. P < 0.05 and P < 0.01 were considered statistically significant.Result1 After primary HPDLCs were cultured successfully, the third generation of HPDLCs was examined by immunohistochemical staining with vimentin and keratin antibody. Expression of vimentin was found and expression of keratin was not found in the HPDLCs.2 The effects of scutellarin on the proliferative activity of HPDLCsThe proliferative activity of HPDLCs was remarkably decreased after treating with 100μg/ml LPS at the same time (P < 0.05, P < 0.01). Compared with the normal control group, there was remarkable statistical significance (P<0.05, P<0.01). The results show that LPS could inhibit the proliferative activity of HPDLCs. After adding different concentration (0.001μg/ml, 0.01μg/ml, 0.1μg/ml, 1μg/ml, 10μg/ml) of scutellarin, compared with the LPS group, the proliferative ability of HPDLCs was increased significantly when treated with 0.01μg/ml, 0.1μg/ml, 1μg/ml, 10μg/ml scutellarin for 24 hours,0.001μg/ml, 0.01μg/ml, 0.1μg/ml, 1μg/ml scutellarin for 48 hours and 0.1μg/ml, 1μg/ml scutellarin for 72 hours (P < 0.05, P < 0.01).3 The effects of scutellarin on the cell cycle of HPDLCsTreated with 100μg/ml LPS for 24 hours, the proportion of every phase (G0/G1, S, G2M) HPDLCs which compared with the normal control group, there was no statistical significance (P > 0.05). After adding 1μg/ml scutellarin, the G0/G1 phase cells proportion decreased, while G2M phase cells proportion and the cells proliferation index (PrI) increased. Compared with LPS group, there was remarkable statistical significance (P < 0.05).4 The effects of scutellarin on the ALP activity of HPDLCsCompared with the normal control group, the ALP activity of HPDLCs was remarkably inhibited when treated with 100μg/ml LPS for 72 hours (P < 0.01).The ALP activity of HPDLCs was promoted after adding 0.1μg/ml, 1μg/ml, 10μg/ml scutellarin (P < 0.05, P < 0.01).Moreover, the concentration of 1μg/ml scutellarin had the most remarkable promotion on ALP activity of HPDLCs treated with LPS. The low concentration (0.001μg/ml, 0.01μg/ml) of scutellarin had no statistical significance (P > 0.05).5 The effects of scutellarin on the production of TNF-αTNF-a secretion in the normal control HPDLCs was at low level. While the TNF-a production was remarkably increased by stimulation with 100μg/ml LPS for 24 hours. Compared with the normal control group, there was remarkable statistical significance (P < 0.01). Compared with LPS group, the TNF-a production was inhibited after adding different concentration (0.001μg/ml, 0.01μg/ml, 0.1μg/ml, 1μg/ml, 10μg/ml) of scutellarin (P < 0.01). Moreover, the concentration of 1μg/ml scutellarin had the most remarkable inhibition on the TNF-a production of HPDLCs. Compared with the low concentration (0.001μg/ml, 0.01μg/ml) of scutellarin, there was remarkable statistical significance (P < 0.01).6 The effects of scutellarin on the the expression of MMP-1 and MMP-26.1 The expression of MMP-1 proteinExaminated by Western-blot, the MMP-1 protein expression of HPDLCs was increased at some extent after treating with 100μg/ml LPS for 24 hours. However, compared with the normal control group, there was no statistical significance (P>0.05). After adding different concentration (0.001μg/ml, 0.01μg/ml, 0.1μg/ml, 1μg/ml, 10μg/ml) of scutellarin, the MMP-1 protein expression of HPDLCs had the decreasing tendency. While compared with the LPS group, there was no statistical significance (P > 0.05).6.2 The expression of MMP-2 proteinExaminated by Western-blot, the MMP-2 protein expression of the normal control group HPDLCs was at low level. Treated with 100μg/ml LPS for 24 hours, the MMP-2 protein expression of HPDLCs was increased. Compared with the normal control group, there was remarkable statistical significance (P<0.05). After adding different concentration (0.001μg/ml, 0.01μg/ml, 0.1μg/ml, 1μg/ml, 10μg/ml) of scutellarin, 10μg/ml scutellarin was able to decrease the MMP-2 expression of HPDLCs, compared with the LPS group, there was remarkable statistical significance (P < 0.05).Conclusion1 At the concentration of 100μg/ml, LPS can inhibit the proliferative activity and alkaline phosphatase activity of HPDLCs. Moreover, LPS can induce the TNF-αproduction as well as the MMP-2 protein expression.2 In the presence of different concentration of scutellarin, the proliferative activity and alkaline phosphatase activity of HPDLCs were greatly promoted. In addition, the TNF-αproduction, as well as the MMP-2 protein expression, remarkably decreased after adding different concentration of scutellarin.3 We conclude that scutellarin has protective effects on HPDLCs treated with LPS.
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