Effects Of Arecoline On Filament Actin And Collagen Phagocytosis Of Human Oral Mucous Fibroblasts In Vitro

Objectives: The role of arecoline in the pathogenesis of OSF wasexplored by investigating the effects of arecoline on proliferation,migration, collagen phagocytosis and filament actin (F-actin) of oralmucous fibroblast (OMFb) in vitro.Methods:1. OMFb were obtained from normal human oral mucosa andcultured in vitro. 3～10 passages of cell were used for all experiments.Arecoline was diluted in DMEM medium at the final concentrations of 5,10, 20, 40, 80μg/ml. OMFb were treated with arecoline at differentconcentration, and control group contained only medium.2. When treated with arecoline at different concentration for 12, 24,48, 72 hours, MTT assay was used to access the OMFb proliferation.3. When treated with arecoline at different concentration for 12hours,Cell migration was evaluated with the use of Scratch-Wound assay.4. When treated with arecoline at different concentration for 24hours,OMFb were starved for 24 hours with serum-free DMEM, and thenincubated with collagen-coated fluorescent beads for 4 hours. Thephagocytic activity of OMFb was analyzed by a flow cytometer.5. OMFb on glass coverslips were treated with arecoline at differentconcentration for 24 hours, F-actin were probed with immuno-fluorescence technique and scanned with laser scanning confocalmicroscopy.Results:1. There were no statistically difference in proliferation rate betweenthe experimental groups and the control group for 12 hours. However, theOMFb proliferation were enhanced by arecoline at 10, 20μg/ml for 24hours and at 5, 10μg/ml for 48 hours, while inhibited by arecoline at 40,80μg/ml for 24 hours and at 20, 40, 80μg/ml. The proliferation of all theOMFb treated by arecoline was inhibited for 72 hours.2. Arecoline at 5, 10μg/ml enhanced migration of OMFb, while at 20,40, 80μg/ml inhibited migration of OMFb significantly.3. Arecoline at 5, 10μg/ml enhanced collagen phagocytosis, while at 20, 40, 80μg/ml inhibited collagen phagocytosis of OMFb significantly.4. Arecoline at 5, 10μg/ml increased the content of OMFb F-actinand formed the stress fibers, but at 20, 40, 80μg/ml induced disruption ofOMFb F-actin and showed abundant staining aggregates in the cellcortex.Conclusions:1. Cell proliferation, migration and collagen phagocytosis of OMFbin vitro were enhanced by arecoline at lower concentrations, and inhibitedby arecoline at higher concentrations.2. The polymerization of OMFb F-actin was enhanced by arecoline atlower concentrations, and inhibited by arecoline at higher concentrations.3. Arecoline changed cell proliferation, migration and collagenphagocytosis of OMFb by affecting the shape in morphology anddistribution of F-actin. It was likely to be one of pathogenesis of OSF.