Expression of deubiquitinases in human gingiva and cultured human gingival fibroblasts.

BACKGROUND: Although deubiquitinating enzymes (DUBs) such as CYLD, A20 and OTULIN are expressed in multiple tissues and thought to be linked with inflammatory diseases, their expression in periodontal tissues remains to be determined. This research was designed to assess the expression of CYLD, A20 and OTULIN in human gingiva, and to evaluate the regulation of these DUBs in human gingival fibroblasts (HGFs) upon different stimuli. METHODS: Immunohistochemistry assay was conducted to determine the expression of CYLD, A20 and OTULIN in human gingiva. Immunofluorescence assay was employed to observe the protein expression of CYLD, A20 and OTULIN in HGFs. RT-PCR and western blots were carried out to assess gene and protein expression changes of these DUBs in HGFs upon LPS or TNF-α. RESULTS: CYLD, A20 and OTULIN were found to be expressed in human gingiva and HGFs. The expression of CYLD, A20 and OTULIN was lower in the inflamed gingival tissue samples compared with the healthy gingival tissue samples. Further, the expression of CYLD, A20 and OTULIN in HGFs exhibited distinct regulation by different stimuli. TNF-α treatment markedly increased NF-κB activation in HGFs CONCLUSIONS: Our findings suggest that CYLD, A20 and OTULIN might play a role in the progression of periodontitis.

The inhibitory effect of the deubiquitinase cylindromatosis (CYLD) on inflammatory responses in human gingival fibroblasts.

OBJECTIVE: Experiments were performed to evaluate CYLD expression in human gingival tissue samples and to examine the effects of CYLD on inflammatory responses in lipopolysaccharide (LPS)- or TNF-α-stimulated human gingival fibroblasts (HGFs). METHODS: Immunohistochemistry for CYLD and p65 expression was performed with healthy and inflamed gingival tissue samples. siRNA was used to knock down the expression of CYLD in HGFs. Upon LPS or TNF-α stimulation, NF-κB activation was detected in control and CYLD-knockdown HGFs. RT-PCR was applied to determine gene expression. Western blot analyses were employed to assess protein expression. Immunofluorescence staining was carried out to evaluate the nuclear translocation of p65. RESULTS: Immunohistochemical staining showed the expression of CYLD in human gingival tissues. In addition, CYLD protein expression was reduced in inflamed gingival tissue samples compared with healthy tissue samples. CYLD knockdown greatly enhanced the mRNA expression of proinflammatory cytokines in LPS- or TNF-α-stimulated HGFs. Furthermore, knocking down CYLD expression increased LPS-stimulated NF-κB activation in HGFs. Unexpectedly, CYLD knockdown did not affect TNF-α-induced NF-κB activation. CONCLUSIONS: Our results suggest that CYLD participates in periodontal inflammatory responses by negatively regulating LPS-induced NF-κB signalling.

AGEs induces apoptosis and autophagy via reactive oxygen species in human periodontal ligament cells.

The apoptosis of human periodontal ligament cells (HPDLCs) may be an important factor of the negative effect of advanced glycation end products (AGEs) on the periodontal tissue of diabetic patients. However, the pathways or potential effects of apoptosis in AGEs-treated HPDLCs have not been fully elucidated. Autophagy is closely related to apoptosis. Herein, we investigated the potential mechanism of apoptosis and autophagy in HPDLCs treated with AGEs via an in vitro model. We found that AGEs-treated HPDLCs showed a time- and concentration-dependent reduction in the cell survival rate. The mitochondrial-dependent apoptosis was induced in AGEs-treated HPDLCs, as confirmed by the mitochondrial membrane potential depolarization, decreased Bcl-2 expression, increased Bax expression, and increased caspase-3 and PARP cleavage. Autophagy was also induced in AGEs-treated HPDLCs, as indicated by the conversion of LC3-II/LC3-I and the presence of autophagosomes. Interestingly, our study results suggested that apoptosis and autophagy were related to reactive oxygen species (ROS) production. In addition, AGEs-induced autophagy acted as a latent factor in decreasing the generation of ROS in HPDLCs and protecting against the AGEs-induced apoptosis. In summary, our study shows that ROS are essential in AGEs-induced HPDLCs apoptosis and autophagy, which may be a molecular mechanism for the repairment of ROS-induced damage in HPDLCs treated with AGEs to promote cell survival. The present study might provide new insights into the therapeutic targeting of HPDLCs autophagy, which could be an additional strategy for periodontitis in patients with diabetes mellitus.

M1 macrophages regulate TLR4/AP1 via paracrine to promote alveolar bone destruction in periodontitis.

OBJECTIVE: Macrophages could be fully polarized and acquire specific phenotype like M1, which considered to be essential for the alveolar bone destruction during the development of periodontitis. However, the molecular mechanisms underlying the effects of M1 macrophages on the alveolar bone destruction are still not clear yet. METHODS: Mouse periodontitis model was established to determine the involvement of M1 macrophages in the pathogenic process. Condition medium of the M1 macrophages (M1-CM) was incubated with pre-osteoblasts to evaluate its effects on the osteoblastogenesis. Cells after treatment with CM were used for RNA-sequencing, quantitative PCR, Western blotting, and immunofluorescence staining to figure out pathways involved in the inhibition of osteoblastogenesis. RESULTS: Increased infiltration of M1 macrophages was associated with alveolar bone destruction in periodontitis. M1-CM markedly suppressed the generation of osteoblasts as evidenced by decreased expressions of Runx2 and Ocn, as well as reduced activity of ALP. Interestingly, RNA-sequencing indicated the activation of TLR4/AP1 signaling pathway in pre-osteoblasts treated with CM. Inhibition of TLR4 reduced the translocation of AP1 and rescued the osteoblastogenesis reduced by M1-CM. CONCLUSION: M1 macrophages induce TLR4/AP1 signaling of pre-osteoblasts to inhibit the osteoblastogenesis via paracrine, at least partially contributing to alveolar bone destruction in periodontitis.

Effects of periodontal therapy on serum lipid profile and proinflammatory cytokines in patients with hyperlipidemia: a randomized controlled trial.

OBJECTIVE: The aim of this study was to evaluate whether periodontal treatment in patients with periodontitis and hyperlipidemia may have any influence on plasma lipids and pro-inflammatory cytokine levels. MATERIAL AND METHODS: We randomly assigned 109 patients with hyperlipidemia and chronic periodontitis into group 1 (n = 55) and group 2 (n = 54). Patients in group 1 underwent a standard cycle of supragingival mechanical scaling and polishing. Patients in group 2 underwent the adjunctive full-mouth intensive removal of subgingival dental plaque biofilms with the use of scaling and root planning. Periodontal parameters, total cholesterol (TC), triglyceride (TRG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), tumor necrosis factor-alpha (TNF-α), interleukin(IL)-1ß(IL-1ß), and IL-6 were evaluated before treatment and 2 and 6 months after treatment. RESULTS: Two and 6 months after treatment, TRG levels were significantly lower in group 2 than in group 1 (P < 0.05), and the levels of HDL-C were significantly higher (P < 0.05). Two and 6 months after therapy, the levels of TNF-α were significantly lower in group 2 than in group 1 (P < 0.05), as were the levels of IL-1ß (P < 0.001) and IL-6 (P < 0.001). CONCLUSIONS: Intensive periodontal treatment of participants with hyperlipidemia and chronic periodontitis improved serum lipid levels and decreased circulating pro-inflammatory cytokine levels. CLINICAL RELEVANCE: This study showed that intensive treatment of periodontitis results in an improvement in serum lipid levels and a decrease in serum proinflammatory cytokine levels in patients with periodontitis and hyperlipidemia. These findings may contribute to present knowledge that periodontal therapy may be beneficial for individuals with hyperlipidemia.

[Valacyclovir as an adjunct to full-mouth scaling and root planing of advanced chronic periodontitis:a randomized clinical trail].

PURPOSE: The aim of this study was to evaluate the clinical benefit of valacyclovir when performing full-mouth periodontal debridement in patients with advanced chronic periodontitis. METHODS: Fifty-nine patients with advanced chronic periodontitis were randomly assigned into control-treatment group(n=29) and intensive-treatment group(n=30). All patients were given instructions of basic oral hygiene and a standard cycle of supragingival mechanical scaling and polishing. Patients in the intensive-treatment group received oral valacyclovir for 1 week, while patients in the control-treatment group received placebo. Thereafter, patients in both groups underwent full-mouth intensive removal of subgingival dental plaque biofilms with the use of scaling and root planing within 48 hours. Periodontal parameters were evaluated before treatment and 2 or 6 months after treatment. The data was statistically analyzed using SPSS17.0 software package. RESULTS: No significant difference in clinical parameters was noted before treatment. 2 and 6 months after treatment, the mean percentage reduction of sites with BOP and PD≥4 mm were significantly higher in the intensive-treatment group than in the control-treatment group (P<0.05). Similarly, patients in the intensive-treatment group had higher mean PD reduction than those in the control-treatment group 2 months (P<0.05) and 6 months after therapy (P<0.05). However, the mean values of CAL reduction were slightly and not significantly higher in the intensive-treatment group than in the control-treatment group after therapy. CONCLUSIONS: It may be concluded that valacyclovir significantly improves clinical results of full-mouth non-surgical periodontal debridement in advanced chronic periodontitis.

Protamine sulfate-nanodiamond hybrid nanoparticles as a vector for MiR-203 restoration in esophageal carcinoma cells.

We report an innovative approach for miRNA-203 delivery in esophageal cancer cells using protamine sulphate (PS)-nanodiamond (ND) nanoparticles. The efficient delivery of miR-203 significantly suppressed the proliferation and migration of cancer cells through targeting Ran and ΔNp63, exhibiting a great potential for PS@ND nanoparticles in miRNA-based cancer therapy.

Apoptosis of periodontium cells in streptozototocin- and ligature-induced experimental diabetic periodontitis in rats.

OBJECTIVE: The aim of this study was to investigate the presence of apoptosis of periodontium cells in streptozototocin- and ligature-induced experimental diabetic periodontitis in rats. MATERIALS AND METHODS: Sixty-two 6-week-old male Sprague-Dawley (SD) rats were randomly divided into three groups: the diabetic periodontitis group (group DP; n = 22), periodontitis group (group P; n = 20) and normal control group (group N; n = 20). Diabetes was induced by intraperitoneal injection of streptozototocin (STZ). Periodontitis was initiated by ligating floss around maxillary second molars. The animals were sacrificed at 3 and 6 weeks after ligature placement in the P and DP groups. Maxillary dentoalveolar segments were isolated and were prepared for morphometric analysis of alveolar bone loss (ABL) and for histological analysis. RESULTS: ABL was significantly increased in group DP compared with group P (p < 0.05). The number of PDL fibroblasts, osteoblasts and osteocytes in group DP was decreased compared with group P (p < 0.05). Inter-group analysis revealed higher osteoclast numbers in the inflammatory area of group DP and group P when compared with group N (p < 0.05). Also, compared with group P, group DP had more higher osteoclast numbers (p < 0.05). Periodontitis and diabetic periodontitis also increased apoptosis of fibroblasts, osteoblasts and osteocytes. The percentage of these apoptotic cells was ∼ 2-fold higher in group DP vs group P. CONCLUSIONS: The results of these studies suggest that diabetes may have a negative effect on the periodontium by increasing the formation of osteoclasts and enhancing apoptosis of fibroblasts, osteoblasts and osteocytes in the periodontal tissue.

[Osteoblast apoptosis in experimental diabetic periodontitis in rats].

OBJECTIVE: To observe osteoblast apoptosis in diabetic periodontitis. METHODS: Sixty-two SD rats were randomly divided into three groups: Diabetic periodontitis group (DP), periodontitis group (P) and normal control group (N). Diabetes was induced by intraperitoneal injection of streptozotocin (STZ). Periodontitis was initiated by ligating floss around maxillary second molars and inoculation of Porphyromonas gingivalis (P. gingivalis). Three or six weeks later, the animals were sacrificed and the specimens were prepared for histological analysis. The percentage of apoptotic osteoblasts was examined. Statistical significance was determined by one-way ANOVA. RESULTS: The sequence of percentage of apoptotic osteoblasts among each group was group DP > group P > group N. Intergroup comparisons demonstrated that diabetic periodontitis and periodontitis might increase apoptosis of osteoblasts. The percentage of apoptotic osteoblasts in group DP was approximately two times higher than that in group P. CONCLUSION: The present study clearly demonstrates that diabetes can increase the apoptosis of osteoblasts in periodontitis.