Study of TNF-α, IL-1ß and LPS levels in the gingival crevicular fluid of a rat model of diabetes mellitus and periodontitis.

OBJECTIVE: In this study, we sought to investigate the dynamic changes in the levels of TNF-α, IL-1ß and LPS in the gingival crevicular fluid (GCF) in a rat model of diabetes mellitus (DM) and periodontitis (PD). Additionally, we evaluated alveolar bone loss and the histopathological response associated with experimental diabetes mellitus and experimental periodontitis. METHODS: DM and PD were induced together in 15 rats (group 1) by streptozotocin injection and ligature induction. Periodontitis alone was produced by ligature induction in 15 rats (group 2), diabetes alone was produced by streptozotocin injection in 15 rats (group 3), and fifteen systemically and periodontally healthy rats were used as controls (group 4). The gingival TNF-α, IL-1ß and LPS levels were measured by using ELISA method. Periodontal destruction was assessed by measuring the alveolar bone loss. Periodontal inflammation was quantified by histopathological grading in H&E stained samples. RESULTS: Higher levels of TNF-α, IL1-ß and LPS, increased alveolar bone loss and more serve histopathology were found in group 1 compared with group 2, group 3 and group 4 (p< 0.05). The quantities of TNF-α, IL1-ß and LPS, the amount of alveolar bone loss and the severity of the histopathological finding were greater in group 2 than group 3 and group 4 (p< 0.05). Group 3 demonstrated higher levels of TNF-α, IL1-ß and LPS, increased alveolar bone loss and more serve histopathology than group 4 (p< 0.05). Statistically significant differences were noted between all of the groups. CONCLUSIONS: These data indicate that DM may lead to enhanced TNF-α, IL1-ß and LPS production in the periodontal tissues. The resorption values of alveolar bone and the histological inflammation were more severe in rats with periodontitis and diabetes mellitus than in those with periodontitis alone, diabetes mellitus alone and control rats. Our findings are consistent with the hypothesis that hyperglycemia contributes to the heightened inflammatory response associated with periodontitis.

Sonic hedgehog alleviates the inhibitory effects of high glucose on the osteoblastic differentiation of bone marrow stromal cells.

To assess the influence of high extracellular glucose levels on the osteogenic differentiation of bone marrow stromal cells (BMSCs) and to determine if Sonic hedgehog (Shh) protein can alleviate those effects. BMSCs were incubated with NG (normal glucose), NG+Shh (200 ng/ml Shh in normal glucose), NG+Shh+Gan (200 ng/ml Shh and 5 micromol/L GANT61 in normal glucose), HG (high glucose), HG+Shh (200 ng/ml Shh in high glucose), and HG+Shh+Gan (200 ng/ml Shh and 5 micromol/L GANT61 in high glucose). The expression levels of Shh signaling pathway genes Patched 1 (PTCH1) and osteogenesis-related genes were tested, which included bone morphogenetic protein 4 (BMP4), runt-related transcription factor 2 (Runx2), and osteopontin (OPN). Alkaline phosphatase (ALPase) activity and mineralized matrix formation were also investigated. Immunofluorescent staining of Gli1 was tested for Shh signaling activation. We found that recombinant Shh in normal-glucose medium could promote osteogenic differentiation of BMSCs, while inhibiting Shh signaling by GANT61 could antagonize this differentiation. Besides that high glucose impaired the Shh signaling as well as osteogenic differentiation of BMSCs, reactivation of Shh signal pathway by addition of Shh protein could mitigate the inhibition while further deactivation by Shh inhibitor GANT61 could retain their osteogenic inhibitions. The above data suggest that Shh pathway activity is involved in the HG condition mediated osteoblastic differentiation deficiency for BMSCs and that recombinant Shh could alleviate this inhibitory effect.

A20 inhibits human salivary adenoid cystic carcinoma cells invasion via blocking nuclear factor-kappaB activation.

BACKGROUND: A20, also known as tumor necrosis factor alpha induced protein 3 (TNFaip3), is a cytoplasmic zinc finger protein that inhibits nuclear factor kappa-B (NF-kappaB) activity and prevents tumor necrosis factor (TNF)-mediated programmed cell death. NF-kappaB is a transcription factor that regulates expression of genes involved in cell proliferation, cell survival and anti-apoptosis. Several studies have implicated that the NF-kappaB signal pathway is associated with angiogenesis and clinico-pathological process of adenoid cystic carcinoma (ACC) of the salivary glands. METHODS: The ability of overexpression of A20 to influence the biological behavior and invasion of ACC cells was examined. The cells were stably transfected with full-length A20 cDNA. Stable gene transfer was verified by realtime-polymerase chain reaction (PCR) and Western blot analysis. The change of cell biological behavior was examined by methyl thiazolyl tetrazolium (MTT) and NF-kappaB luciferase reporter assay and the invasion of the cells was examined by a Matrigel invasion chamber. RESULTS: pEGPFN3-A20 gene was stably transferred into ACC-2 cells and overexpressed. When cells were treated with TNFalpha, the NF-kappaB activity of ACC-2-A20 cells could be down-regulated about 46.32% in contrast to ACC-2-GFP cells (P < 0.05). A20 potently inhibited growth of A20 transfectant ACC-2-A20 compared with control vector transfected groups and the ACC-2 empty control group (P < 0.05). The ACC-2-A20 cells showed significantly reduced ability to invade through Matrigei-coated filters compared to ACC-2-GFP and ACC-2 cells. The inhibition rate was up to 71.05% (P < 0.05). CONCLUSIONS: A20 gene transfer is associated with decreased tumor invasion, in part via the down-regulation of NF-kappaB expression, providing evidence for a potential application of A20 in designing a treatment modality for salivary gland cancers such as ACC.