18β-glycyrrhetinic acid inhibits periodontitis via glucocorticoid-independent nuclear factor-κB inactivation in interleukin-10-deficient mice

Abstract

Background and objective: 18β-Glycyrrhetinic acid (GA) is a natural anti-inflammatory compound derived from licorice root extract (Glycyrrhiza glabra). The effect of GA on experimental periodontitis and its mechanism of action were determined in the present study.

Material and methods: Periodontitis was induced by oral infection with Porphyromonas gingivalis W83 in interleukin-10-deficient mice. The effect of GA, which was delivered by subcutaneous injections in either prophylactic or therapeutic regimens, on alveolar bone loss and gingival gene expressions was determined on day 42 after initial infection. The effect of GA on lipopolysaccharide (LPS)-stimulated macrophages, T cell proliferation and osteoclastogenesis was also examined in vitro.

Results: 18β-Glycyrrhetinic acid administered either prophylactically or therapeutically resulted in a dramatic reduction of infection-induced bone loss in interleukin-10-deficient mice, which are highly disease susceptible. Although GA has been reported to exert its anti-inflammatory activity via downregulation of 11β-hydroxysteroid dehydrogenase-2 (HSD2), which converts active glucocorticoids to their inactive forms, GA did not reduce HSD2 gene expression in gingival tissue. Rather, in glucocorticoid-free conditions, GA potently inhibited LPS-stimulated proinflammatory cytokine production and RANKL-stimulated osteoclastogenesis, both of which are dependent on nuclear factor-κB. Furthermore, GA suppressed LPS- and RANKL-stimulated phosphorylation of nuclear factor-κB p105 in vitro.

Conclusion: These findings indicate that GA inhibits periodontitis by inactivation of nuclear factor-κB in an interleukin-10- and glucocorticoid-independent fashion.

Figure 1. GA inhibits the induction and progression of infection-induced alveolar bone loss in IL-10 deficient mice

A. Representative images of defleshed hemi-mandibles. Negative: non-infected, vehicle alone, n=9; Positive: P. gingivalis infected, vehicle alone, n=8; Prophylactic: P. gingivalis infected, GA 30mg/kg, day 0 to 34, n=9, Therapeutic: P. gingivalis infected, GA 30mg/kg, day 9 to 34, n=8. Area enclosed by black line: area of exposed cementum. B. Effect of GA on alveolar bone loss. Groups are as described above. Vertical bars: SD, * p<0.05 by ANOVA/Bonferroni test.

Figure 1. GA inhibits the induction and progression of infection-induced alveolar bone loss in IL-10 deficient mice

A. Representative images of defleshed hemi-mandibles. Negative: non-infected, vehicle alone, n=9; Positive: P. gingivalis infected, vehicle alone, n=8; Prophylactic: P. gingivalis infected, GA 30mg/kg, day 0 to 34, n=9, Therapeutic: P. gingivalis infected, GA 30mg/kg, day 9 to 34, n=8. Area enclosed by black line: area of exposed cementum. B. Effect of GA on alveolar bone loss. Groups are as described above. Vertical bars: SD, * p<0.05 by ANOVA/Bonferroni test.

Figure 2. GA does not inhibit gingival gene expression of HSD1 or HSD2

Gingival gene expression of HSDs was determined by qPCR on day 42. Change-fold values are based on positive control. Closed columns: positive control (infected, no treatment), Shaded columns: negative control (no infection, no treatment), Open columns: (infected, treated with prophylactic regimen). Vertical bars: SD. No significant differences were detected by ANOVA/Bonferroni test.

Figure 3. GA suppresses proinflammatory cytokine production and osteoclastogenesis in vitro

A. The effect of GA on cytokine production by LPS-stimulated IL-10^−/− macrophages. Negative: no LPS, no GA; Positive: LPS, no GA; GA: LPS, GA 10μM or 1μM, n=4/group. Vertical bars: SD, * p<0.05 by ANOVA/Bonferroni test. B. The effect of GA on T cell proliferation and IFNγ production. Negative: no ConA, no GA; Positive: Con A, no GA; GA: Con A, GA 10μM or 1μM, n=4/group, Vertical bars: SD, * p<0.05 by ANOVA/Bonferroni test. C. The effect of GA on RANKL-stimulated osteoclastogenesis by RAW264.7 cells. Negative: no RANKL, no GA, Positive: RANKL, no GA, GA: RANKL, GA 10μM or 1μM. n=4/group. Vertical bars: SD, * p<0.0005 by ANOVA/Bonferroni test.

Figure 4. GA inactivates the phosphorylation of NF–κB p105

A. Inactivation of NF–κB in LPS-stimulated IL-10^−/− macrophages. Lane 1: Negative (vehicle alone), Lane 2: Positive (LPS, vehicle), Lane 3: GA (LPS, 1μM GA). B. Inactivation of NF–κB in RANKL-stimulated RAW264.7 cells. Lane 1: Negative (vehicle alone), Lane 2: Positive (RANKL, vehicle), Lane 3: GA (RANKL, 1μM GA).