Daphne jejudoensis Attenuates LPS-Induced Inflammation by Inhibiting TNF-α, IL-1β, IL-6, iNOS, and COX-2 Expression in Periodontal Ligament Cells

Abstract

Periodontitis is a common disease involving inflammation and tissue destruction in the periodontal region. Although uncontrolled long-term inflammation in the gingiva may lead to loss of the periodontal ligament, treatments or preventive solutions for periodontitis are scarce. The aim of this study is to find anti-inflammatory material from a natural source that can be used to treat or protect against periodontitis. Daphne species (Thymelaeaceae) are important and popular components of traditional Chinese medicine and are used as anti-inflammatory agents. Daphne jejudoensis is an endemic plant that grows on Jeju Island and was identified as a new species in 2013. In this study, for the first time, we investigated the anti-inflammatory effect of D. jejudoensis leaf extract (DJLE) on human periodontal ligament cells. The gene expression levels of pro-inflammatory cytokines (interleukin-1β and 6 and tumor necrosis factor-α) and inflammation-inducible enzymes (inducible nitric oxide synthase and cyclooxygenase-2) were reduced after DJLE treatment with/without lipopolysaccharide stimulation. The findings of this study indicate that D. jejudoensis possesses anti-inflammatory activities, suggesting that DJLE may be a potential preventive and therapeutic agent for periodontitis.

Keywords: Daphne jejudoensis; Thymelaeaceae; anti-inflammatory activity; lipopolysaccharide; periodontitis.

Figure 1

Effects of Daphne jejudoensis leaf extract (DJLE) on the viability of human periodontal ligament cells (hPDLCs). The cells grown in serum-free medium were treated with different concentrations (10, 50, and 100 μg/mL) of DJLE for 24 and 48 h using the MTT assay. Cell viability was expressed as the average of experiments relative to the untreated control (100%). * p < 0.05 vs. control.

Figure 2

Tumor necrosis factor (TNF)-α gene expression levels with different concentrations of lipopolysaccharide (LPS) (0.1, 1, 5, and 10 μg/mL) in human periodontal ligament cells. *** p < 0.001 vs. control.

Figure 3

Tumor necrosis factor (TNF)-α gene expression levels in human periodontal ligament cells with different concentrations of Daphne jejudoensis leaf extract (DJLE) (1, 10, 50, and 100 μg/mL). *** p < 0.001 vs. control.

Figure 4

Effect of Daphne jejudoensis leaf extract (DJLE) on tumor necrosis factor (TNF)-α gene expression under LPS-induced inflammation in human periodontal ligament cells. *** p < 0.001 vs. control.

Figure 5

Effect of Daphne jejudoensis leaf extract (DJLE) on the gene expression of the proinflammatory cytokines interleukin-1β (A) and interleukin-6 (B) in lipopolysaccharide (LPS)-stimulated human periodontal ligament cells. *** p < 0.001 vs. control.

Figure 6

Effect of Daphne jejudoensis leaf extract (DJLE) on the gene expression of inducible nitric oxide synthase (iNOS) (A) and cyclooxygenase (COX)-2 (B) in lipopolysaccharide (LPS)-stimulated human periodontal ligament cells. *** p < 0.001 vs. control.

Figure 7

Growth inhibition effect of Daphne jejudoensis leaf extract (DJLE) against Porphyromonas gingivalis: the broth turbidity comparison with positive control, chlorhexidine (A), and the optical density at 600 nm (B).

Figure 8

Base peak chromatogram of Daphne jejudoensis leaf extract (DJLE) using LC-QToF in negative ESI mode: daphnin (1), rutin (2), daphnetin (3), and cynaroside (4).

Figure 9

Structure of compounds identified from Daphne jejudoensis leaf extract (DJLE): daphnin (1), rutin (2), daphnetin (3), and cynaroside (4).

Figure 10

Picture of Daphne jejudoensis plant (A) and the front (B) and back (C) of its leaf.