Abstract:

Objective To investigate the role of cimifugin in regulating macrophage polarization in periodontitis. Methods The effects of cimifugin at various concentrations (0, 20.4, 40.8, 81.6, 163.2, 326.5, 653.0, 1 306.0 μmol/L) on the viability of RAW264.7 cells were assessed using the CCK-8 and live/dead cell staining to determine the optimal concentration for subsequent experiments. Immunofluorescence staining was employed to observe the impact of cimifugin on M1 and M2 macrophage polarization phenotypes (CD86 and CD206). RT-qPCR was utilized to analyze changes in the expression levels of inducible nitric oxide synthase (iNOS), interleukin (IL)-1β, CD86, IL-6, tumor necrosis factor-α (TNF-α), CD206, and arginase-1 (Arg-1) in macrophages treated with cimifugin. A murine periodontal inflammation model was established, and the animals were divided into a periodontitis group (PD group), a periodontitis + cimifugin group (PD+Cimifugin group), and a sham surgery control group (Control group). Micro-CT was used for scanning and reconstruction, and CTAn software was employed to measure the cemento enamel junction-alveolar bone crest distance (CEJ-ABC), bone mineral density (BMD), relative bone volume/total volume (BV/TV), and trabecular separation (Tb.Sp). HE staining was performed to observe changes in periodontal tissues, while TRAP staining was used to quantify osteoclasts. Immunohistochemistry (IHC) was conducted to analyze the expression of IL-1β and IL-10. Results Cimifugin concentrations≤326.5 μmol/L did not significantly affect macrophage proliferation and viability (P>0.05). Cimifugin at 326.5 μmol/L inhibited the expression of M1 macrophage markers iNOS, IL-1β, CD86, IL-6, and TNF-α, while promoting the expression of M2 macrophage markers CD206 and Arg-1 (P<0.000 1). Compared to the PD group, the PD+Cimifugin group exhibited significant improvements in CEJ-ABC, BMD, BV/TV, and Tb.Sp (P<0.01), reduced inflammatory cell infiltration and osteoclast numbers in periodontal tissues (P<0.05), significantly elevated IL-10 expression (P<0.000 1), and markedly decreased IL-1β expression (P<0.01). Conclusion Cimifugin alleviates periodontal inflammation and reduces bone defects by inhibiting M1 macrophage polarization and promoting M2 macrophage polarization.

Key words: periodontitis, cimifugin, macrophage polarization