High-glucose concentration aggravates TNF-alpha-induced cell viability reduction in human CD146-positive periodontal ligament cells via TNFR-1 gene demethylation.

Periodontitis is a chronic inflammatory disease that results in the destruction of periodontal soft tissue and the resorption of alveolar bone. Evidence indicates that in diabetic patients, hyperglycemia suppresses periodontal ligament stem cell (PDLSC) functions and leads to difficulties in periodontal repair. The present study aimed to explore the mechanisms by which high-glucose concentrations aggravate cell viability reduction in human CD146-positive PDLCs (CD146+ PDLCs) under tumor necrosis factor-alpha (TNF-alpha) induction. CD146+ PDLCs were isolated from periodontal ligament tissues and treated in the absence or presence of 10 ng/ml of TNF-alpha and 30 mM glucose. Cell viability was detected using Cell Counting Kit-8 assays and Luminescent Cell Viability Assays. Western blotting and real-time polymerase chain reaction were performed to determine tumor necrosis factor-alpha receptor-1 (TNFR-1) protein and messenger RNA expression. Bisulfite and MassArray methylation analyses were used to analyze the methylation status of the TNFR-1 gene. Our results indicated that cell viability was reduced after treatment with a combination of both high-glucose concentration and TNF-alpha. Treatment with 30 mM glucose suppressed DNA methyltransferase (DNMT) activities and DNMT1 protein expression, and this was accompanied by the upregulation of TNFR-1. Additionally, we found that the CpG island located within the TNFR-1 gene was hypomethylated under 30 mM glucose conditions. S-adenosylmethionine, an established methyl donor, reversed TNFR-1 upregulation and restored cell viability against high-glucose concentration and TNF-alpha. In conclusion, the present findings suggest that high-glucose-induced CpG island hypomethylation within the TNFR-1 gene plays an essential role in TNFR-1 upregulation, and this further enhances the cell viability reduction of CD146+ PDLCs caused by TNF-alpha.

Platelet-rich plasma inhibits osteoblast apoptosis and actin cytoskeleton disruption induced by gingipains through upregulating integrin ß1.

The aim of this study was to explore the effects of platelet-rich plasma on gingipain-caused changes in cell morphology and apoptosis of osteoblasts. Mouse osteoblasts MC3T3-E1 cells were treated with gingipain extracts from Porphyromonas gingivalis in the presence or absence of platelet-rich plasma. Apoptosis was detected with terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining. F-actin was determined by phalloidin-fluorescent staining and observed under confocal microscopy. Western blot analysis was used to detect integrin ß1, F-actin, and G-actin protein expressions. A knocking down approach was used to determine the role of integrin ß1. The platelet-rich plasma protected osteoblasts from gingipain-induced apoptosis in a dose-dependent manner, accompanied by upregulation of integrin ß1. Platelet-rich plasma reversed the loss of F-actin integrity and decrease of F-actin/G-actin ratio in osteoblasts in the presence of gingipains. By contrast, the effects of platelet-rich plasma were abrogated by knockdown of integrin ß1. The platelet-rich plasma failed to reduce cell apoptosis and reorganize the cytoskeleton after knockdown of integrin ß1. In conclusion, platelet-rich plasma inhibits gingipain-induced osteoblast apoptosis and actin cytoskeleton disruption by upregulating integrin ß1 expression.

Gingipains promote RANKL-induced osteoclastogenesis through the enhancement of integrin ß3 in RAW264.7 cells.

As a crucial virulence factor of Porphyromonas gingivalis, gingipains play an important role in periodontal destruction. This study aimed to investigate the effect of gingipains on osteoclastogenesis. We used RAW264.7 cells as osteoclast precursors in our study. In experimental groups, cells were treated with gingipains and/or receptor activator of nuclear factor-κB ligand (RANKL). Tartrate-resistant acid phosphatase (TRAP) activity staining assay showed osteoclast precursors and RANKL-induced mature osteoclasts were increased in a gingipains dose-dependent manner. Real-time reverse transcription polymerase chain reaction analysis demonstrated that gingipains upregulated osteoclastic genes including the protease cathepsin K (Ctsk), matrix metalloprotein 9 (Mmp9), nuclear factor of activated T cells 1 (Nfatc1) and acid phosphatase 5, tartrate resistant (Acp5) in a time-dependent manner. Western blotting assays presented upregulated expressions of TNF receptor-activating factor 6 (TRAF6) and integrin ß3 induced by gingipains and RANKL compared to RANKL alone. Enhanced integrin-related signaling was also demonstrated by elevated phosphorylations of FAK and paxillin compared to control. Moreover, the pit resorption assays showed that gingipains augmented bone resorptive function of osteoclasts induced by RANKL. When we used Cilengitide to block integrin αvß3, gingipains reversed the reduction of formation and resorptive function in RANKL-induced osteoclasts, as they enhanced integrin αvß3 levels more than RANKL treatment alone. In conclusion, our data suggest that gingipains augmented the differentiation and function of mature osteoclasts induced by RANKL through the increase in integrin αvß3.