Slit guidance ligand 2 promotes the inflammatory response of periodontitis through activation of the NF-κB signaling pathway.

BACKGROUND AND OBJECTIVES: Periodontitis is a chronic multifactorial inflammatory disease associated with dental plaque biofilms. Slit guidance ligand 2 (SLIT2) has been shown to guide neuronal migration, regulate the inflammatory response and cancer progression. However, the role of SLIT2 in periodontitis is poorly understood. In this study, we investigated the expression of SLIT2 in the gingiva of periodontitis and its role in periodontitis progression. METHODS: Gingiva and gingival crevicular fluid (GCF) were collected from healthy people and periodontitis patients. Immunohistochemistry and enzyme-linked immunosorbent assay (ELISA) were used to analyze SLIT2 secretion level. Healthy human gingival fibroblasts (hGFs) were isolated and the expression of SLIT2 in lipopolysaccharide (LPS)-treated hGFs was detected. The effect of SLIT2 on inflammation was analyzed using western blot and immunofluorescence. SLIT2 knockdown (KD) and overexpression assays in hGFs were performed to investigate the role of SLIT2 in the LPS-induced inflammatory response. RESULTS: Gingival tissues and GCF of periodontitis patients displayed higher expression of SLIT2. Similarly, SLIT2 was upregulated in hGFs in an inflammatory environment (LPS treatment). In addition, SLIT2 treatment increased the expression of the inflammatory mediators interleukin-6 (IL-6) and IL-8 in hGFs. Mechanistically, SLIT2 stimulated the activation of nuclear factor-κB (NF-κB) signaling, as well as LPS. Lastly, SLIT2 KD impaired LPS-induced IL-6 production in hGFs, while SLIT2 overexpression amplified the inflammatory response. CONCLUSION: SLIT2 may be involved in the aggravation of periodontitis by activating NF-κB signaling in hGFs.

Substance P participates in periodontitis by upregulating HIF-1α and RANKL/OPG ratio.

BACKGROUND: Both substance P and hypoxia-inducible factor 1 alpha (HIF-1α) are involved in inflammation and angiogenesis. However, the relationship between substance P and HIF-1α in rat periodontitis is still unknown. METHODS: Ligation-induced rat periodontitis was established to observe the distribution and expression of substance P and HIF-1α by immunohistochemistry. Rat gingival fibroblasts were cultured and stimulated with Porphyromonas gingivalis lipopolysaccharide (LPS). Recombinant substance P was applied to elaborate the relationship between substance P and HIF-1α in gingival fibroblasts in vitro. Primary mouse bone marrow-derived macrophages (BMMs) were isolated and cultured to observe the effect of substance P on receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis by TRAP staining. Western blotting was used to investigate the expression of HIF-1α, osteoprotegerin (OPG) and RANKL. RESULTS: Rat experimental periodontitis was successfully established 6 weeks after ligation. Gingival inflammatory infiltration and alveolar bone loss were observed. Positive expression of substance P was found in the infiltrating cells. Higher HIF-1α levels were observed in periodontitis compared to that of normal tissues. Substance P upregulated the level of HIF-1α in gingival fibroblasts with or without 1 µg/ml LPS in vitro (*P < 0.05). Substance P upregulated the expression of HIF-1α in RANKL-stimulated BMMs in vitro. Substance P also increased the RANKL/OPG ratio in gingival fibroblasts (*P < 0.05). Both 10 nM and 50 nM substance P promoted RANKL-induced osteoclast differentiation (*P < 0.05). CONCLUSION: Substance P participates in periodontitis by upregulating HIF-1α and the RANKL/OPG ratio.

Periodontal ligament-associated protein-1 promotes osteoclastogenesis in mice by modulating TGF-ß1/Smad1 pathway.

BACKGROUND: Periodontal ligament-associated protein-1 (PLAP-1), an important target molecule of osteoarthritis research, may affect alveolar bone resorption. The aim of our study was to comprehensively and systematically detect the effect of PLAP-1 on alveolar bone resorption and the underlying mechanism in PLAP-1 knockout mouse models. METHODS: We used a PLAP-1 knockout (C57BL/6N-Plap-1-/- ) mouse model to investigate the effect of PLAP-1 on osteoclast differentiation and the underlying mechanism by adding Porphyromonas gingivalis lipopolysaccharide to stimulate bone marrow-derived macrophages. The effect of PLAP-1 on alveolar bone resorption and the underlying mechanism were studied using a ligature periodontitis model, with microcomputed tomography imaging, immunochemistry, and immunofluorescence. RESULTS: The in vitro analysis results demonstrated that PLAP-1 knockout significantly inhibited osteoclast differentiation under both normal and inflammatory conditions. Bioinformatic analysis, immunofluorescence, and co-immunoprecipitation showed colocalization and interaction between PLAP-1 and transforming growth factor beta 1 (TGF-ß1). The phosphorylation of Smad1 was reduced in the PLAP-1 knockout cells compared with that in the cells from wild-type mice. The in vivo analysis results demonstrated that PLAP-1 knockout decreased bone resorption and the levels of osteoclast differentiation markers in experimental periodontitis compared with those in wild-type mice. Immunofluorescence staining confirmed colocalization of PLAP-1 and TGF-ß1 in the experimental periodontitis model. The phosphorylation level of Smad1 was significantly reduced in PLAP-1 knockout mice compared with that in wild-type mice. CONCLUSIONS: This study revealed that the knockout of PLAP-1 inhibits osteoclast differentiation and decreases alveolar bone resorption through the TGF-ß1/Smad1 signaling pathway, which could serve as an innovative target for the prevention and treatment of periodontitis.

Downregulation of odontogenic ameloblast-associated protein in the progression of periodontal disease affects cell adhesion, proliferation, and migration.

OBJECTIVE: This work aimed to examine changes in odontogenic ameloblast-associated protein (ODAM) expression during the progression of periodontal disease and to investigate the effect of ODAM deficiency in vitro by RNA sequencing. DESIGN: Gingival tissue samples were collected from three groups, including healthy control, gingivitis and periodontitis patients, and ODAM expression was assessed by immunohistochemistry and quantitative reverse transcription PCR (qRT-PCR). Then, an Odam-knockdown cell line was established by lentiviral infection of small guide RNAs (sgRNAs) into an immortalized ameloblast-lineage cell line. RNA sequencing was carried out in Odam-knockdown and empty lentiviral vector-transfected cells. Differentially expressed genes were compared between these two cell groups to analyze functional enrichment, and a protein-protein interaction network was built. RESULTS: ODAM expression levels in gingival tissue samples were significantly lower in patients with periodontitis than in healthy controls as determined by immunohistochemistry and qRT-PCR. Transcriptomic analysis of Odam-knockdown cells identified 2801 differentially expressed genes, which were enriched in cell-substrate adhesion, proliferation, and migration pathways. The expression of a core of differentially expressed genes was confirmed by qRT-PCR in Odam-knockdown cells and by immunohistochemistry in clinical samples. Knocking down Odam significantly reduced cell adhesion but increased cell proliferation and migration capacity in vitro. CONCLUSIONS: ODAM is important in cell adhesion, proliferation, and migration, and its downregulation may contribute to periodontitis progression in humans.

Multiple idiopathic cervical root resorption involving all permanent teeth.

Multiple idiopathic external cervical root resorption is a rare condition with numerous predisposing factors that have not yet been clearly elucidated. In addition, its diagnosis and treatment pose challenges for clinicians, and thus, the extraction of the involved teeth is commonly performed. Here, we report a 29-year-old pregnant woman with no contributory medical or family/social history who experienced cervical root resorption that progressed aggressively and involved all permanent teeth. This case is unique owing to the involvement of all teeth. Reports of multiple idiopathic external cervical root resorption are rare in the literature, and the pathogenesis of the condition is poorly understood. This report aims to add an additional case to the existing literature, analyse the underlying mechanisms and provide clinicians with some guidance in diagnosing cervical root resorption.