Expression of interleukin-6 stimulated by mechanical pressure in human periodontal ligament fibroblasts and the effect of p38 mitogen activated protein kinase inhibitor / 中国组织工程研究

ABSTRACT

BACKGROUND: Human periodontal ligament fibroblasts (HPLF) is the crucial cells in maintaining the configuration and function of periodontium. Adverse stress may cause HPLF to synthesize more inflammatory agents, which may cause the damage of periodontium.OBJECTIVE: To investigate the role of p38 MAPK of HPLF in the expres sion of inflammatory cytokine of interleukin-6 (IL-6) subjected to mechanical pressure, and explore the mechanism of the occlusal trauma to periodontium.DESIGN: A randomized and controlled trial.SETTING: Pathological Laboratory of the Fourth Military Medical Univer sity of Chinese PLA.MATERIALS The HPLF were obtained from the middle part of 1/3 pe riodontium of 12 to 16-year-old youth whose 20 healthy permanent premo lar teeth should be extracted for orthodontic need. Main reagents and ap paratus IL-6 enzyme-linked immunoabsorbent assay (ELISA) kit (Staff Room of Immunology of the Fourth Military Medical University of Chinese PLA); ELISA apparatus (Huadong Electronic Tube Factory); p38 MAPK specific inhibitor of SB203580 (produced by Biochemical Company, ob tained as a present from Professor Jiang, Staff Room of Pathophysiology, Southern Medical University).METHODS: The cells were primarily cultured till the 4-5 passages, and randomly divided into four groups ①pressure-loading control group the cell s were not subjected to pressure-loading and without pretreatment; ② pressure-loading group the cells were subjected to continuous pressure-load ing (200 kPa) but without pretreatment; ③ pretreatment control group the supernatant were added with 10 g/L dimathyl sulfoxide (DMSO, SB203580 solvent) at 1 hour before pressure-loading, the method and time of pressure loading were the same as those in the pressure-loading group; ④ pretreated group the cells were pretreated with 1 μmol/L SB203580 (a specific in hibitor of the p38 MAPK) at 1 hour before pressure-loading, the method and time of pressure-loading were the same as those in the pressure-loading group. The cytosol and the supernatant in each group were sampled at 16 and 24 hours after pressure-loading respectively. The IL-6 expressions at different time points were detected with ELISA.MAIN OUTCOME MEASURES: The amount of IL-6 expression in HPLF induced by pressure with or without pretreatment by SB203580, a specific inhibiter of p38 MAPK.RESULTS: The expressions of IL-6 after continuous pressure-loading for 16 and 24 hours in the pressure-loading group were (143.1±0.42) and (49.46±1.01) ng/L, which were obviously higher than those in the pres sure-loading control group [(18.36±0.43), (18.78±0.50) ng/L, P ＜ 0.05]. The expressions of IL-6 after continuous pressure-loading for 16 and 24 hours in the pretreatment group were (56.39±0.72) and (21.52±1.39) ng/L, which were obviously higher than those in the pressure-loading control group [(137.96±0.54), (48.47±0.79) ng/L, P ＜ 0.05].CONCLUSION: p38 MAPK of HPLF acts as important cooperative mechanism to regulate IL-6 synthesis induced by mechanical pressure.