Recombinant Irisin Protects Against Alveolar Bone Destruction During Orthodontic Tooth Movement.

Patients with periodontitis have higher risk of alveolar bone loss when seek for orthodontic therapy. Inflammation and osteogenesis are key factors in alveolar bone destruction in periodontitis and orthodontic tooth movement (OTM). Here we evaluated the effects of irisin on alveolar bone destruction in rats with periodontitis and OTM. We isolated and cultured human periodontal ligament stem cells (PDLSCs). Irisin was administrated to PDLSCs. Cell proliferations, osteogenic differentiation, expression of RUNX2 and ALP, and the expression of OPG and RANKL were measured. We induced periodontitis and OTM in rats and treated rats with irisin. The alveolar bone loss, inflammatory cytokine levels, and expression of OPG and RANKL in gingival tissues were measured. Irisin promoted the cell proliferation and osteogenic differentiation of PDLSCs. Irisin elevated the expression of RUNX2, ALP, and OPG while decreased the expression of RANKL in PDLSCs. Irisin ameliorated the alveolar bone loss, suppressed cytokine levels, and increased OPG/RANKL expression ratio in rat with periodontitis and orthodontic tooth movement. Irisin prevented alveolar bone destruction during OTM in rats with periodontitis.

Effects of vitamin A in promoting proliferation and osteogenic differentiation of human periodontal ligament cells.

Human periodontal ligament cells (hPDLCs) are known as ideal seed cells for the regeneration of periodontal tissues. Several factors (i.e., vitamin D3, luteolin, and 6-bromoindirubin-3'-oxime) have been shown to promote osteogenic differentiation of hPDLCs. In this study, we aim to investigate the effect of vitamin A on cell proliferation, migration, and osteogenic differentiation of hPDLCs. hPDLCs were cultured in osteogenic induction medium supplemented with different concentrations of vitamin A. Cell proliferation and migration assays were conducted after 24, 48, and 72 h of incubation, whereas osteogenic differentiation and osteogenesis-related gene expression were assessed after 21 d only. Our results demonstrated that 1-µM vitamin A stimulation exerted the most potent promotion effect on cell proliferation, migration, and osteogenic differentiation of hPDLCs. It also induced significant upregulation of osteogenic differentiation-related genes and mitochondrial complexes II and IV in hPDLCs. Vitamin A may serve as a promising potential candidate for periodontal tissue regeneration.

HDAC5-Mediated Acetylation of p100 Suppresses Its Processing.

INTRODUCTION: Periodontitis is a condition involving chronic inflammation in the gums, periodontal ligaments, cementum, and alveolar bone. Nuclear factor-κB (NF-κB) activation is the prominent mediator of inflammation and osteoclast differentiation. The role of histone deacetylase 5 (HDAC5) in periodontitis development and NF-κB regulation is not fully understood. METHODS: We used primary mouse bone marrow-derived osteoclast cultures in vitro and a mouse model of chronic periodontists (CPD) treated with the HDAC4/5 inhibitor LMK-235. Real-time polymerase chain reaction, micro computed tomography, flow cytometry, western blot, and immunoprecipitation were used to study proinflammatory cytokines, NF-κB activation, HDAC5 activity, and the interaction of HDAC5 with NF-κB p100. RESULTS: LMK-235, a selective inhibitor of HDAC4 and HDAC5, reduced osteoclast marker gene expression (Cstk, Acp5, and Calcr) and tartrate-resistant acid phosphatase activity in primary osteoclast cultures. LMK-235 reduced the increase in cementoenamel junction-alveolar bone crest distance, inflammatory cell infiltration of gingival tissues, and expression levels of interleukin (IL)-1ß, tumor necrosis factor alpha, IL-6, and IL-23a, indicating an ameliorative effect on CPD. Immunoprecipitation experiments have further confirmed p100-HDAC5 interaction, acetylation levels of p100, and NF-κB activation. CONCLUSIONS: These results indicate that HDAC5 binds and deacetylates p100, leading to its activation, increased proinflammatory cytokine production, gingival infiltration, and osteoclast differentiation, thus promoting alveolar bone resorption. HDAC5 inhibition is therefore a potentially promising therapeutic strategy for the treatment of periodontitis.

Correlation Analysis of miR-1246 Expression in Saliva of Patients with Chronic Periodontitis and Periodontal Indexes, Inflammatory Cytokines, and Protease Molecules.

Objective: The study aimed to investigate the correlation of miR-1246 in saliva with periodontal indicators, inflammatory cytokines, and protease molecules in patients with chronic periodontitis. Methods: Thirty-five patients with chronic periodontitis were included as the chronic periodontitis group, and 35 healthy individuals were selected as the healthy control group during the same period. The miR-1246 levels, inflammatory cytokine interleukin (IL)-1ß, IL-6, IL-17, tumor necrosis factor-α (TNF-α), matrix metalloproteinase (MMP-1), MMP-8, and MMP tissue inhibitor (TIMP-1) in saliva were determined, and periodontal indexes, including the plaque index (PLI), bleeding index (BI), periodontal probing depth (PD), and attachment loss (AL) were examined. Results: The salivary levels of miR-1246, IL-1ß, IL-6, IL-17, TNF-α, MMP-1, MMP-8, and TIMP-1 and the periodontal indexes PLI, GI, PD, and AL in the chronic periodontitis group were significantly higher than those in the healthy control (P < 0.05). Salivary levels of miR-1246 in patients with chronic periodontitis were positively correlated with the levels of IL-1ß, IL-6, IL-17, TNF-α, MMP-1, MMP-8, TIMP-1, PLI, GI, PD, and AL (P < 0.05). Conclusion: Abnormally elevated levels of miR-1246 in saliva of patients with chronic periodontitis correlate with levels of periodontal indices, inflammatory cytokines, and protease molecules.

Inhibition of nuclear factor kappa B inducing kinase suppresses inflammatory responses and the symptoms of chronic periodontitis in a mouse model.

Chronic periodontitis is an inflammatory disease that represents a major public health issue nowadays. Here, we investigated the protective role of nuclear factor kappa B (NF-κB) inducing kinase (NIK)-inhibitor on chronic periodontitis and revealed the underlying molecular mechanism. NIK-inhibitor was synthesized, and its functions were examined in primary osteoclasts and wild-type (WT) and NIK-/- chronic periodontitis mouse model. Lipopolysaccharides (LPS) or activator of NF-κB was applied to stimulate inflammatory response of osteoclasts. The qRT-PCR, ELISA and Western blot were used to measure the expression of pro-inflammatory and osteoclast-related genes, and the activation of NF-κB signaling. Osteoclastogenesis and bone damage were detected by TRAP staining and micro-CT. NIK knockdown mice had lower expression of osteoclast-related genes and improved CEJ-ABC damage. Similarly, NIK-inhibitor administration inhibited inflammatory responses and CEJ-ABC damage in chronic periodontitis models. NIK-inhibitor suppressed osteoclastogenesis and osteoclast-related genes expression through inhibiting the non-canonical NF-κB signaling. NIK plays important role in bone destruction of chronic periodontitis and NIK-inhibitor represents a promising therapeutic strategy for this disease.

Epigenetic silencing of KLF2 by long non-coding RNA SNHG1 inhibits periodontal ligament stem cell osteogenesis differentiation.

BACKGROUND: Exploring the effects of lncRNA SNHG1 in the process of osteogenic differentiation of periodontal ligament stem cells (PDLSCs) would provide novel therapeutic strategies for tissue regeneration. METHODS: Loss-of-function and gain-of-function assays were induced by lentivirus. The osteogenic differentiation of PDLSCs were assessed by ALP staining and Alizarin Red staining as well as the mRNA and protein levels of osteogenic marker genes osterix, osteocalcin, and alkaline phosphatase through qRT-PCR and western blot. RNA immunoprecipitation assay and chromatin immunoprecipitation assays were performed to uncover the interaction between SNHG1 and EZH2. RESULTS: Our analysis revealed that SNHG1 was downregulated and KLF2 was upregulated during the osteogenic induction differentiation of PDLSCs. SNHG1 inhibited while KLF2 promoted osteogenic differentiation of PDLSCs. SNHG1 directly interact with the histone methyltransferase enhancer of the zeste homolog 2 (EZH2) and modulate the histone methylation of promoter of Kruppel-like factor 2 (KLF2) and altered the progress osteogenic differentiation of PDLSCs. CONCLUSIONS: Taken together, SNHG1 inhibited the osteogenic differentiation of PDLSCs through EZH2-mediated H3K27me3 methylation of KLF2 promotor and provided a novel class of therapeutic targets for regenerate dental tissues.

Beneficial Effects of Glycyrrhizin in Chronic Periodontitis Through the Inhibition of Inflammatory Response.

Elevated inflammatory cytokines and high mobility group box 1 (HMGB1) production are associated with chronic periodontitis (CP). Glycyrrhizin is the major constituent of Glycyrrhiza glabra. L. (Fabaceae) root with anti-inflammation activities. This study evaluated the effects of glycyrrhizin on CP. TNF-α-treated human periodontal ligament stem cell (hPDLSC) model was established, and was administrated with 1, 2 or 5 mM glycyrrhizin for 24 h. After treatment, the expression of HMGB1and inflammatory cytokines was monitored. Significantly increased HMGB1 (median: 5646.4, range: 1918.2-8233.7 vs median: 204.5, range: 98.7-283.6, pg/mL), TNF-α (median: 345.5, range: 161.0-567.9 vs median: 93.5, range: 58.1-159.3, pg/mL), IL-1ß (median: 2014.6, range: 209.5-4308.1 vs median: 224.5, range: 48.8-335.8, pg/mL) and IL-6 (median: 1223.6, range: 398.2-2183.8 vs median: 240.4, range: 105.2-400.5, pg/mL) were detected in gingival crevicular fluid from CP patients. Glycyrrhizin significantly prevented TNF-α-induced expression of HMGB1 (691.5 ± 136.4 vs 142.8 ± 57.3 pg/mL), IL-6 (388.1 ± 85.2 vs 189.4 ± 61.2 pg/mL) and IL-1ß (176.3 ± 47.2 vs 53.9 ± 25.7 pg/mL) in hPDLSC. In CP rats, glycyrrhizin significantly decreased HMGB1 (5795.6 ± 1121.5 vs 586.4 ± 436.8 pg/mL), TNF-α (421.8 ± 93.7 vs 87.9 ± 21.6 pg/mL), IL-6 (1423.8 ± 235.2 vs 622.6 ± 176.1 pg/mL) and IL-1ß (1562.8 ± 334.3 vs 733.5 ± 265.1 pg/mL) in gingival crevicular fluid. Glycyrrhizin suppresses inflammatory activities in CP rats and represents a promising molecule for controlling CP.

Downregulation of miR-24-3p promotes osteogenic differentiation of human periodontal ligament stem cells by targeting SMAD family member 5.

Osteogenic differentiation is a complicated process that depends on various regulatory factors and signal pathways. In our research, the osteogenic differentiation capacity was analyzed by alizarin red staining, alkaline phosphatase activity, and protein levels of osteogenic differentiation markers including runt-related transcription factor 2, bone morphogenetic protein 2, and osteocalcin (OCN). We observed a notable decrease of miR-24-3p level in osteogenic-differentiated human periodontal ligament stem cells (hPDLSCs) by microarray analysis. In our gain- and loss-of-function experiments, we discovered that miR-24-3p has a suppression effect on hPDLSCs osteogenic differentiation. Moreover, SMAD family member 5 (Smad5), the critical osteogenic differentiation transcription factors, was predicted to be targets of miR-24-3p. In addition, luciferase reporter assay further proved that miR-24-3p directly targeted the 3'-untranslated region of Smad5. Similarly, we found that the overexpression of miR-24-3p significantly decreased the Smad5 messenger RNA level in hPDLSCs, which was detected by real-time quantitative polymerase chain reaction. Then hPDLSCs were transfected with miR-24-3p mimics to inhibit Smad5 expression; meanwhile, Smad5 RNA interference could significantly reverse the osteogenic differentiation inhibition effect of miR-24-3p. In brief, a series of data showed that miR-24-3p is a regulator of Smad5, playing an important role in osteogenic differentiation.