Xanthine Derivative KMUP-3 Alleviates Periodontal Bone Resorption by Inhibiting Osteoclastogenesis and Macrophage Pyroptosis

Abstract

Aim: This study investigated the function effects of KMUP-3, a self-developed synthetic xanthine-based derivative, in suppressing Porphyromonas gingivalis (Pg-LPS)-aggravated osteoclastogenesis and pyroptosis as a potential treatment for periodontitis.

Methods: In vitro, the effects of Pg-LPS and KMUP-3 on osteoclast formation and macrophage pyroptosis were investigated using the receptor activator of nuclear factor-κB ligand (RANKL)-primed RAW264.7 macrophages. In vivo, the therapeutic effects of KMUP-3 were evaluated in a model of experimental periodontitis induced by gingival ligature placement.

Results: We reveal that KMUP-3 suppressed osteoclastogenesis, inducible nitric oxide synthase activation, and reduced nitric oxide production enhanced by Pg-LPS in RANKL-primed RAW264.7 cells while also decreasing TLR4/NF-κB p65 pathway activation and decreased pro-inflammatory cytokine production; moreover, Pg-LPS promoted NLRP3 activation and exacerbated pyroptosis induction effects that were abolished by KMUP-3. Finally, KMUP-3 ameliorated alveolar bone loss and IL-1β levels in the gingival crevicular fluid in the rat ligature periodontitis model.

Conclusions: Our study demonstrated that KMUP-3 attenuates Pg-LPS-enhanced osteoclastogenesis and macrophage pyroptosis. Notably, KMUP-3 alleviates alveolar bone loss in experimental periodontitis rats and thus suggests its certain role in safeguarding against periodontal bone resorption.

Keywords: alveolar bone loss; nitric oxide; osteoclastogenesis; pyroptosis; xanthine derivative KMUP‐3.

FIGURE 1

Xanthine derivative KMUP‐3 suppresses osteoclast differentiation augmented by Pg‐LPS in RANKL‐primed macrophages. (A) Chemical structure of KMUP‐3. (B) Cell viability was measured by MTT assay (n = 6). (C) Outline of the experimental protocol of the in vitro study. (D) Representative images of TRAP staining were observed under the optical microscope. (E) Quantification of TRAP‐positive multinucleated cell (TRAP⁺ MNC) number from the images in (D) performed using Carl Zeiss AxioVision software (n = 6). (F) Quantitation of TRAP activity assay (n = 6). Yellow arrows indicate TRAP⁺ MNCs. All scale bars represent 100 μm. Data are expressed as mean ± SEM. Nonsignificant (n.s.) p > 0.05 and ^$ p < 0.05 compared with the control group. ^### p < 0.001 compared with the RANKL‐only group. **p < 0.01 and ***p < 0.001 compared with the RANKL + Pg‐LPS group.

FIGURE 2

KMUP‐3 reduces Pg‐LPS‐enhanced osteoclast multinucleation by inhibiting iNOS‐derived nitric oxide. (A) Representative images of F‐Actin rings in osteoclasts by immunofluorescence staining. (B) Quantification of the area of F‐Actin ring per field (n = 6). (C) Measurement of the nucleus number per multinucleated cell (MNC) using Carl Zeiss AxioVision software (n = 6). (D) Griess assay was used to identify nitrite products in the culture medium (n = 6). (E) Soluble enzyme, Inos, in the culture medium was evaluated by ELISA assay (n = 6). (F) Protein expression of iNOS was detected by western blotting and (G) quantitatively analyzed (n = 5). All scale bars represent 100 μm. Data are expressed as mean ± SEM. Nonsignificant (n.s.) p > 0.05 compared with the control group. ^### p < 0.001 compared with the RANKL‐only group. ***p < 0.001 compared with the RANKL + Pg‐LPS group.

FIGURE 3

KMUP‐3 inhibits inflammatory cytokines secretion via suppressing TLR4/NF‐κB p65 pathway aggravated by Pg‐LPS. (A) Protein expression of TLR4 and TRAF6 were detected by western blotting and (B) quantitatively analyzed (n = 5). (C) Protein expression of phospho‐IκBα/IκBα and phospho‐p65/p65 were detected by western blotting and (D) quantitatively analyzed (n = 5). (E) TNF‐α, (F) IL‐6, (G) MCP‐1, and (H) IL‐1β in the culture medium were evaluated by ELISA assay (n = 6). Data are expressed as mean ± SEM. ^$ p < 0.05, ^$$ p < 0.01, and ^$$$ p < 0.001, compared with the control group. ^## p < 0.01 and ^### p < 0.001 compared with the RANKL‐only group. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the RANKL + Pg‐LPS group.

FIGURE 4

KMUP‐3 treatment mitigates macrophage pyroptosis via suppressing NLRP3 inflammasome activation. (A) Protein expression of NLRP3 (B), ASC (C), and Caspase‐1 (D) were detected by western blotting and quantitatively analyzed (n = 5). (E and F) Protein expression of IL‐1β was detected by western blotting and quantitatively analyzed (n = 5). Data are expressed as mean ± SEM. ^$ p < 0.05 and ^$$$ p < 0.001, compared with the control group. ^### p < 0.001 compared with the RANKL‐only group. *p < 0.05, **p < 0.01 and ***p < 0.001 compared with the RANKL + Pg‐LPS group.

FIGURE 5

KMUP‐3 alleviates periodontal bone loss and reduces gingival IL‐1β in ligature‐induced periodontitis rats. (A) Representative 3D micro‐CT images of rat palatal maxilla. (B and C) Distance (as indicated by red lines) between the cementoenamel junction (CEJ) to the alveolar bone crest (ABC) at the second maxillary molar (M2) of mesiolingual (ML) and M2 of distolingual (DL) was measured (n = 6). (D) Representative micro‐CT images of the second molar ligature placement in the maxilla. The red circles indicate the area quantitatively analyzed. (E) Bone volume/tissue volume (BV/TV), (F) trabecular thickness (Tb.Th), and (G) trabecular separation (Tb.Sp) were measured (n = 6). (H) Representative images of TRAP staining in the rat maxillary alveolar bone sections were observed under the optical microscope. (I) Statistical analysis of TRAP‐positive (TRAP⁺) osteoclast numbers distributed in the alveolar bones (n = 6). (J) IL‐1β in the gingival crevicular fluid (GCF) was evaluated by ELISA assay (n = 6). The red squares indicate the area quantitatively analyzed. Yellow arrows indicate TRAP⁺ osteoclast. All scale bars represent 50 μm. Dox means doxycycline. Data are expressed as mean ± SEM. Nonsignificant (n.s.) p > 0.05 and ^### p < 0.001 compared with the sham group. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the ligature group. Nonsignificant (N.S.) p > 0.05 compared with the ligature + KMUP‐3 group.

FIGURE 6

Proposed mechanisms of KMUP‐3 treatment alleviate periodontal bone loss. KMUP‐3 treatment ameliorates Pg‐LPS‐enhanced osteoclastogenesis, NO production, pro‐inflammatory cytokine secretion, and pyroptosis in RANLK‐primed macrophages. These effects lead to the inhibition of periodontitis‐induced alveolar bone loss. The image was created using BioRender.com (Nov. 2024).