ABSTRACT
With an increasing aging society, China is the world's fastest growing markets for oral implants. Compared with traditional oral implants, immediate implants cause marginal bone resorption and increase the failure rate of osseointegration, but the mechanism is still unknown. Therefore, it is important to further study mechanisms of tension stimulus on osteoblasts and osteoclasts at the early stage of osseointegration to promote rapid osseointegration around oral implants. The results showed that exosomes containing circ_0008542 from MC3T3-E1 cells with prolonged tensile stimulation promoted osteoclast differentiation and bone resorption. Circ_0008542 upregulated Tnfrsf11a (RANK) gene expression by acting as a miR-185-5p sponge. Meanwhile, the circ_0008542 1916-1992 bp segment exhibited increased m6A methylation levels. Inhibiting the RNA methyltransferase METTL3 or overexpressing the RNA demethylase ALKBH5 reversed osteoclast differentiation and bone resorption induced by circ_0008542. Injection of circ_0008542 + ALKBH5 into the tail vein of mice reversed the same effects in vivo. Site-directed mutagenesis study demonstrated that 1956 bp on circ_0008542 is the m6A functional site with the abovementioned biological functions. In conclusion, the RNA methylase METTL3 acts on the m6A functional site of 1956 bp in circ_0008542, promoting competitive binding of miRNA-185-5p by circ_0008542, and leading to an increase in the target gene RANK and the initiation of osteoclast bone absorption. In contrast, the RNA demethylase ALKBH5 inhibits the binding of circ_0008542 with miRNA-185-5p to correct the bone resorption process. The potential value of this study provides methods to enhance the resistance of immediate implants through use of exosomes releasing ALKBH5.
Subject(s)
Bone Resorption/metabolism , Cell Communication , Cell Differentiation , Exosomes/metabolism , Osteoblasts/metabolism , Osteoclasts/metabolism , Osteogenesis , RNA, Circular/metabolism , 3T3 Cells , AlkB Homolog 5, RNA Demethylase/genetics , AlkB Homolog 5, RNA Demethylase/metabolism , Animals , Bone Resorption/genetics , Bone Resorption/pathology , Cellular Microenvironment , Exosomes/transplantation , Female , Mechanotransduction, Cellular , Methylation , Methyltransferases/genetics , Methyltransferases/metabolism , Mice , Mice, Inbred BALB C , MicroRNAs/genetics , MicroRNAs/metabolism , Osseointegration , Osteoblasts/transplantation , Osteoclasts/pathology , RAW 264.7 Cells , RNA, Circular/genetics , Rats , Receptor Activator of Nuclear Factor-kappa B/genetics , Receptor Activator of Nuclear Factor-kappa B/metabolism , Stress, MechanicalABSTRACT
The aim of this study was to investigate the protective effect of sulforaphane (SFN) on 1methyl4phenyl pyridine ion (MPP+)induced cytotoxicity and to investigate its possible mechanisms. METHODS: PC12 cell toxicity induced by MPP+ served as a cell model of Parkinson's diseases. The cell culture + experiments were divided into four groups based on the different treatments, namely, vehicle control, SFN, MPP+ and SFN pretreatment plus MPP+. Cell viability and apoptosis were examined by MTT assay and flow cytometry, respectively. Expressions of nuclear factor erythroid 2related factor 2 (Nrf2), heme oxygenase 1 (HO1) and nicotinamide quinone oxidoreductase 1 (NQO1) were detected using western blotting. RESULTS: MPP+ reduced the survival rate of PC12 cells in a dose and timedependent manner. After 24h treatment with 500 µmol/l MPP+, the survival rate of PC12 cells decreased to 58.2±0.03% of that in the control groups. Under the same conditions MPP+ resulted in significant apoptosis of PC12 cells (apoptosis rate: 30.4±0.6%). However, SFN pretreatment significantly attenuated the cell damage induced by MPP+. Furthermore, it was demonstrated that SFN reversed the reduction of Nrf2, HO1 and NQO1 expression induced by MPP+. CONCLUSION: SFN may protect PC12 cells from MPP+induced damage via activating the Nrf2ARE (antioxidant responsive element) pathway.
Subject(s)
Isothiocyanates/pharmacology , NF-E2-Related Factor 2/metabolism , Oxidative Stress/drug effects , PC12 Cells/drug effects , Protective Agents/pharmacology , 1-Methyl-4-phenylpyridinium/toxicity , Animals , Antioxidant Response Elements , Antioxidants/pharmacology , Antiparkinson Agents/pharmacology , Apoptosis/drug effects , Cell Survival/drug effects , Heme Oxygenase-1/metabolism , Isothiocyanates/administration & dosage , NAD(P)H Dehydrogenase (Quinone)/metabolism , Parkinson Disease/drug therapy , Rats , Reactive Oxygen Species/metabolism , Signal Transduction/drug effects , Sulfoxides , Survival Rate , Time FactorsABSTRACT
OBJECTIVE: To investigate the effects of basic fibroblast growth factor (bFGF) on the proliferation of human salivary adenoid cystic carcinoma (ACC) cell line ACC-2 in vitro. RESULTS: The effect of ectogenic bFGF on proliferation of ACC-2 was observed by MTT assay. Extracellular signal-regulated kinase (ERK) activity was measured by immuno-precipitation. p-ERK1/2, Cyclin D1 and p21waf/cip1 expression were assessed by Western blot. RESULTS: bFGF could enhance the proliferation of ACC-2. Stimulated by bFGF, the proliferation ratio increased significantly. The intracellular ERK activity, p-ERK1/2 and Cyclin D1 expression were increased, while p21waf/cip1 expression was inhibited by different concentrations of bFGF. The above effects of bFGF could be attenuated by MEK inhibitor U0126. CONCLUSION: bFGF stimulates the proliferation of ACC-2 in a dose dependent manner. The proliferation effect of bFGF may be due to up-regulating ERK, Cyclin D1 and p21waf/cip1 signaling pathway. This research can help us to explore a new pathogenesis and therapy of the ACC.
