Osmunda japonica Extract Suppresses Pro-Inflammatory Cytokines by Downregulating NF-κB Activation in Periodontal Ligament Fibroblasts Infected with Oral Pathogenic Bacteria

Abstract

Periodontal diseases are caused by bacterial infection and may progress to chronic dental disease; severe inflammation may result in bone loss. Therefore, it is necessary to prevent bacterial infection or control inflammation. Periodontal ligament fibroblasts (PDLFs) are responsible for the maintenance of tissue integrity and immune and inflammatory events in periodontal diseases. The formation of bacterial complexes by Fusobacterium nucleatum and Porphyromonas gingivalis is crucial in the pathogenesis of periodontal disease. F. nucleatum is a facultative anaerobic species, considered to be a key mediator of dental plaque maturation and aggregation of other oral bacteria. P. gingivalis is an obligate anaerobic species that induces gingival inflammation by secreting virulence factors. In this study, we investigated whether Osmunda japonica extract exerted anti-inflammatory effects in primary PDLFs stimulated by oral pathogens. PDLFs were stimulated with F. nucleatum or P. gingivalis. We showed that pro-inflammatory cytokine (IL-6 and IL-8) expression was induced by LPS or bacterial infection but decreased by treatment with O. japonica extract following bacterial infection. We found that the activation of NF-κB, a transcription factor for pro-inflammatory cytokines, was modulated by O. japonica extract. Thus, O. japonica extract has immunomodulatory activity that can be harnessed to control inflammation.

Keywords: Fusobacterium nucleatum; Osmunda japonica; Porphyromonas gingivalis; periodontal ligament fibroblast; pro-inflammatory cytokines.

Figure 1

Osmunda japonica extract (OJE) inhibits pro-inflammatory cytokine expression in response to E. coli LPS (ecLPS). Human PDLFs were stimulated with ecLPS in the presence or absence of the plant extracts. (A,B) PDLFs were pre-treated with 100 µg/mL of various plant extracts and then incubated with ecLPS (1 µg/mL) for 12 h. After the incubation, real-time polymerase chain reaction (PCR) was performed to detect messenger RNA (mRNA) expression of IL-6 (A) and IL-8 (B). (C,D) Dose-dependent effects of the OJE on IL-6 (C) and IL-8 (D) mRNA expression after 12 h of ecLPS (1 µg/mL) stimulation. The values are the mean ± standard deviation of triplicate assays.

Figure 2

Dose-dependent inhibitory effects of OJE on pro-inflammatory cytokine expression in PDLFs infected with oral bacteria. (A–C), IL-6 mRNA; (D–F), IL-8 mRNA. Human PDLFs were pre-treated with 100 µg/mL OJE prior to bacterial infection (M.O.I = 100). The PDLFs were infected with F. nucleatum (A,D), P. gingivalis (B,E), or a mixture of F. nucleatum and P. gingivalis (C,F). The values are the mean ± standard deviation of triplicate assays. F. nucleatum: Fn; P. gingivalis: Pg; F. nucleatum and P. gingivalis: Fn_Pg.

Figure 3

The inhibitory effect of the OJE on pro-inflammatory cytokine expression in PDLFs infected with oral bacteria for the indicated times. (A–C), IL-6; (D–F), IL-8. Human PDLFs were pre-treated with 100 µg/mL OJE prior to bacterial infection (M.O.I = 100). The PDLFs were infected with F. nucleatum (A,D), P. gingivalis (B,E), or a mixture of F. nucleatum and P. gingivalis (C,F). The values are the mean ± standard deviation of triplicate assays. F. nucleatum: Fn; P. gingivalis: Pg; F. nucleatum and P. gingivalis: Fn_Pg.

Figure 4

The effect of OJE on NF-κB phosphorylation and host cell invasion. (A,B) NF-κB, phosphorylated NF-κB (p-NF-κB), and beta actin (b-actin) were blotted to detect and normalize the NF-κB activation in response to various stimuli in the absence or presence of OJE. Western blotting (A) was performed to detect the activation of NF-κB after ecLPS (1 µg/mL) stimulation or bacterial infection (MOI = 100) and then analyzed by using ImageJ software (B). (C) The bacterial invasion assay was performed with F. nucleatum single or mixed strains (MOI = 200). O. japonica; Oj; No treatment; NT.

Figure 5

Inhibitory effect of the O. japonica crude extract and its fractions on pro-inflammatory cytokine expression. (A) Cytotoxicity of the O. japonica crude extract and its fractions. The fractions were separated as described in Materials and Methods. (B,C) IL-6 (B) and IL-8 (C) mRNA was detected after ecLPS stimulation. (D,E) PDLFs were infected with single strains of bacteria or their mixture, and the secretion of IL-6 (D) and IL-8 (E) was measured. The values are the mean ± standard deviation of triplicate assays. Ethyl acetate: EtOAc; Butanol: BuOH; No treatment: NT.

Scheme 1

Systematic purification using solvent partitioning from O. japonica.