Force-induced Hydrogen Sulfide Enhances M1 Macrophage Polarization to Promote OTM

Objectives: This study aimed to elucidate whether and how force-induced hydrogen sulfide (H₂S) regulated M1 macrophage polarization, and thus contributed to orthodontic tooth movement (OTM).
Methods: OTM models were established in male C57BL/6J mice by the application of approximately 30 g of force. To examine the influence of H₂S on the macrophage polarization and OTM under mechanical force stimuli, H₂S level was increased by injection of H₂S donor GYY4137 (1 mg/mouse) and decreased by injection of cystathionine b-synthase (CBS) inhibitor hydroxylamine (HA) (100 μg/mouse) every other day during the seven-day course of force application. The maxillae of the three groups of mice (Force, Force+GYY4137, Force+HA) were harvested. The expression of macrophages was detected using immunofluorescence staining. In vitro, the supernatant of force-treated PDLSCs with or without HA (FS+HA or FS) was applied to THP-1 macrophages to detect the influence of force-induced H₂S on macrophage polarization. Finally, we confirmed the mechanism of H₂S on M1 macrophage activation in THP-1 macrophages.
Results: Systemic injection of H₂S donor GYY4137 increased the OTM distance and the expression of force-induced CD68⁺iNOS⁺M1 macrophages, whereas injection of CBS inhibitor HA reduced the OTM distance and the expression of M1 macrophages. Meanwhile, the expression of CD68⁺CD163⁺M2 macrophages was hardly detected. In vitro, the H₂S production in PDLSCs was enhanced after force loading, which was reversed by application of HA. Moreover, the supernatant of force-treated PDLSCs with or without HA (FS+HA or FS) was applied to THP-1 macrophages. The expression of M1 marker TNF-α increased in the FS group and decreased in the FS+HA group, whereas M2 marker arginase-1 remained unchanged. Further experiments showed that force-induced H₂S contributed to M1 macrophage polarization via STAT-1 signaling pathway.
Conclusions: These data suggest that force-induced H₂S production in PDLSCs promotes M1 macrophage polarization, and therefore contributes to the OTM process.