[Mechanical stimulation disrupts mitochondrial homeostasis and compromises biological functions of inflamed human periodontal ligament cells].

Objective: To investigate the effect and mechanism of mechanical stimulation on the proliferation, migration, and osteogenic differentiation of periodontal ligament cells (PDLC) under inflammatory microenvironment by regulating mitochondrial homeostasis. Methods: An inflammatory cell model was established by treating PDLC with tumor necrosis factor-α (TNF-α, 10 ng/ml) combined with interleukin-1β (IL-1β, 5 ng/ml) for 12 h. Normal and inflamed cells were exposed to pressures of 0, 60, 120 and 180 kPa using a multi-functional pressure loading system. The expression of the osteogenic transcription factor Osterix (OSX) was detected by Western blotting and real-time fluorescence quantitative PCR to identify the most inhibitory pressure condition. PDLC were divided into control, pressure, inflammation, and inflammation+pressure groups. The mitochondrial fission inhibitor Mdivi-1 was used for intervention. The following parameters were measured: apoptosis rate by flow cytometry; indicators of mitochondrial homeostasis by fluorescence staining; and the expression of runt-related transcription factor 2 (RUNX2), OSX, collagen type I (COL-Ⅰ), interleukin-6 (IL-6), dynamin-related protein 1 (DRP1), mitofusin 1/2(MFN1/2), PTEN-induced putative kinase 1 (PINK1) and Parkin by Western blotting. Results: Under inflammatory conditions, the 180 kPa pressure group showed the most significant reduction in the relative protein and mRNA expression levels of OSX (t=8.34, P<0.05; t=4.91, P<0.05), whereas no significant differences were observed under other pressure conditions.Therefore, 180 kPa was used for the subsequent experiments. Compared with the control group, the pressure group showed no significant changes in mitochondrial homeostasis parameters. Compared with the inflammation group, the inflammation+pressure group showed disruption of mitochondrial homeostasis, and significantly elevated expression of DRP1, PINK1, and Parkin (t=3.21,P<0.05,t=2.84,P<0.05;t=3.70, P<0.05), and significantly decreased expression of MFN1 and MFN2(t=3.50,P<0.05;t=3.68,P<0.05) After Mdivi-1 treatment, the migration rate (at 3, 6, and 12 h) and proliferation rate of PDLC were significantly increased (migration: t=6.52, P<0.05;t=5.97, t=10.07, P<0.05; proliferation: t=4.73, P<0.05), and the apoptosis rate was significantly decreased (t=4.53, P<0.05). Moreover, the protein expression levels of RUNX2, OSX, and COLI were significantly increased (t=6.11, P<0.05;t=4.97, t=3.22, P<0.05), while the protein expression level of IL-6 was significantly decreased (t=3.98, P<0.05). Conclusions: Mechanical stimulation can inhibit the migration, proliferation and osteogenic differentiation of inflammatory periodontal ligament cells by disrupting mitochondrial homeostasis.