Avenanthramide-C Shows Potential to Alleviate Gingival Inflammation and Alveolar Bone Loss in Experimental Periodontitis

Abstract

Periodontal disease is a chronic inflammatory disease that leads to the gradual destruction of the supporting structures of the teeth including gums, periodontal ligaments, alveolar bone, and root cementum. Recently, interests in alleviating symptoms of periodontitis (PD) using natural compounds is increasing. Avenanthramide-C (Avn-C) is a polyphenol found only in oats. It is known to exhibit various biological properties. To date, the effect of Avn-C on PD pathogenesis has not been confirmed. Therefore, this study aimed to verify the protective effects of Avn-C on periodontal inflammation and subsequent alveolar bone erosion in vitro and in vivo. Upregulated expression of catabolic factors, such as matrix metalloproteinase 1 (MMP1), MMP3, interleukin (IL)-6, IL-8, and COX2 induced by lipopolysaccharide and proinflammatory cytokines, IL-1β, and tumor necrosis factor α (TNF-α), was dramatically decreased by Avn-C treatment in human gingival fibroblasts and periodontal ligament cells. Moreover, alveolar bone erosion in the ligature-induced PD mouse model was ameliorated by intra-gingival injection of Avn-C. Molecular mechanism studies revealed that the inhibitory effects of Avn-C on the upregulation of catabolic factors were mediated via ERK (extracellular signal-regulated kinase) and NF-κB pathway that was activated by IL-1β or p38 MAPK and JNK signaling that was activated by TNF-α, respectively. Based on this study, we recommend that Avn-C may be a new natural compound that can be applied to PD treatment.

Keywords: alveolar bone loss; avenanthramide-C; gingival fibroblasts; inflammation; periodontitis.

Fig. 1. Avn-C inhibits pathological condition-induced inflammatory factors expression in both human GF and PDL cells.

(A-C) Quantitative real time-PCR analysis of transcript levels treated with the indicated concentration of Avn-C in the presence of 1 µg/ml Escherichia coli LPS (A) or 2 ng/ml IL-1β (B) or 50 ng/ml TNF-α (C) for 24 h in human GF (n = 4). (D-F) mRNA levels in PDL cells treated with indicated concentration of Avn-C in the presence of 1 µg/ml E. coli LPS (D) or 2 ng/ml IL-1β (E) or 50 ng/ml TNF-α (F) for 24 h (n = 4). Data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.005. All experiments were repeated at least three times. Avn-C, Avenanthramide-C; GF, gingival fibroblasts; PDL, periodontal ligament; HGF, human gingival fibroblasts; LPS, lipopolysaccharide; Veh, vehicle.

Fig. 2. Avn-C suppresses the expression of the inflammatory factors and activities of MMPs during PD-like pathogenesis in vitro in human GF.

(A, C, and E) Protein levels in human GF treated with indicated concentration of Avn-C in the presence of 1 µg/ml Escherichia coli LPS (A) or 2 ng/ml IL-1β (C) or 50 ng/ml TNF-α (E) for 24 h (n = 3). (B, D, and F) The MMP1 and MMP3 activities in human GF treated with Avn-C in the presence of LPS (B) or IL-1β (D) or TNF-α (F) (n = 3). Data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.005. Avn-C, Avenanthramide-C; MMPs, matrix metalloproteinases; PD, periodontitis; GF, gingival fibroblasts; Veh, vehicle; LPS, lipopolysaccharide.

Fig. 3. Avn-C blocks IL-1β signaling by inhibiting the ERK and NF-κB pathways.

(A) Human GF were treated with 2 ng/ml IL-1β for the indicated time. Phosphorylation of ERK, p38 MAPK, JNK, and IκBα, was determined by Western blot. (B) Protein levels in human GF treated with Avn-C in the presence of the 2 ng/ml IL-1β. (C and D) Quantitative real time-PCR of the indicated genes in human GF treated with 2 ng/ml IL-1β in the absence or presence of the selective ERK inhibitor PD98059 (C) or the NF-κB inhibitor Bay11-7082 (D) for 24 h (n = 3). Data are presented as mean ± SD. ns, not significant. *P < 0.05, ***P < 0.005. Avn-C, Avenanthramide-C; GF, gingival fibroblasts; PD, periodontitis.

Fig. 4. Avn-C blocks TNF-α signaling by inhibiting the p38 MAPK, JNK, and NF-κB pathway.

(A) Human GF were treated with 50 ng/ml TNF-α for the indicated time. (B) Protein levels in human GF treated with Avn-C in the presence of the 50 ng/ml TNF-α. (C and D) Quantitative real time-PCR of the indicated genes in human GF treated with 50 ng/ml TNF-α in the absence or presence of the selective p38 kinase inhibitor, SB203580 (C) or the JNK inhibitor, SP600125 (D) for 24 h (n = 3). Data are presented as mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.005. Avn-C, Avenanthramide-C; GF, gingival fibroblasts.

Fig. 5. The therapeutic effects of Avn-C on alveolar bone loss in ligature-induced PD in mice.

Maxillary samples were divided into four groups (n = 8): Control (non-ligature), PD (ligature), PD + Veh (ligature + DMSO), and PD + Avn-C. (A) Representative µ-CT images of view of three-dimensional (3D), longitudinal, and transverse-sectional of the maxillae. BMD (B) and BV/TV (C) were measured from the bone between the first and second molars. (D) The distance of the cemento-enamel junction (CEJ) to the alveolar bone crest (ABC) was analyzed. (E) Representative images of H&E staining. Magnification, ×200. Scale bar = 200 µm. (F) Representative images of TRAP staining. Magnification, ×200. Scale bar = 200 µm. (G and H) Immunohistochemistry (IHC) staining for IL-6 (G) and IL-8 (H). Magnification, ×400. Scale bar = 50 µm. Data are presented as mean ± SD. *P < 0.05, ***P < 0.005. Avn-C, Avenanthramide-C; PD, periodontitis; Veh, vehicle; µ-CT, micro-computed tomography; BMD, bone mineral density; BV/TV, bone volume/tissue volume; CEJ-ABC, cementum-enamel junction-alveolar bone crest; PD, periodontitis; T, tooth; A, alveolar bone.