Arecoline

Dulbecco’s modified Eagles’ medium (DMEM), penicillin/streptomycin, trypsin/EDTA, fetal bovine serum (FBS), KGM-SFM medium, and phosphate-buffered saline (PBS) were from Gibco (Life Technologies, USA). Arecoline, melatonin and MTT were from Sigma (Sigma-Aldrich Chemical Company, St. Louis, MO, USA). ELISA kits for MMP-9 were from PeproTech (PeproTech, Inc., Rocky Hill, NJ, USA). Catalase, SB431542, 5Z-7-Oxozeaenol, PD153035, AG490, U0126, LY294002, and aspirin were purchased from Tocris. Phospho-TAK1 (p-TAK1) antibody was from Abcam (ab79583). Antibodies for TGF-β1, and GAPDH were from Santa Cruz. P-Smad2 antibody was from Cell Signaling Technology. PBL extract and ANE were prepared as before [8 (link), 12 (link), 13 (link), 21 (link)]. HC was synthesized as previously [22 (link)].

Twenty-four pathogen-free female mice (BALB/c, six weeks old) were purchased from the State Key Laboratory of Oral Diseases, Sichuan University. After two weeks of normal feeding in the animal laboratory of the West China Hospital of Stomatology, the mice were equally divided into four groups. The three groups of mice were treated with saline (PBS, 1 mg/ml), arecoline (ARE, Sigma-Aldrich, 31593, 1 mg/ml), and bleomycin (BLE, 1 mg/ml). Every 2 days, we used a 29-gauge insulin syringe (Ultra-Fine, BD) to administer 50 μl of drug treatment to the bilateral buccal mucosa of the mouse for 14 weeks. After the mice were sacrificed, the obtained buccal mucosa of the oral cavity was fixed (4% paraformaldehyde), dehydrated, and embedded for subsequent experiments.

Arecoline and collagen solution from bovine skin were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Arecoline and collagen solution from bovine skin were purchased from Sigma‐Aldrich (St. Louis, MO, USA). TGFβ type I receptor inhibitor, SB431542, was purchased from EMD Millipore (Billerica, MA, USA). Human TGFβ1 was obtained from R&D Systems (Minneapolis, MN, USA). All methods applied in this study were carried out in accordance with the approved guidelines from the Institutional Review Board of Chung Shan Medical University Hospital and informed written consent was obtained from each individual prior to commencing the study. Tissue specimens from areca quid chewers were collected from Department of Dentistry, Chung Shan Medical University Hospital, Taichung, Taiwan. Biopsy specimens were taken from the histologically normal oral mucosa and fibrotic mucosa at the time of surgical third molar extraction. Fibroblast cultures were grown and maintained by using the explant method as described previously.28 Cell cultures between the third and eighth passages were used in this study.

To determine cytotoxicity of arecoline, 4 x 10⁴ cells of ORL-48(T) or ORL-136(T) cells were seeded into each well of 96-well tissue culture plates, and maintained in complete medium for 24 hours. The cells were treated with arecoline (Sigma-Aldrich, St. Louis, MO, USA) at 0, 25, 50, 100, 200, 400, 800 and 1,200 μg/ml in triplicate for 24 hours. Cell viability was determined by MTT assay.
To determine the effect of arecoline on cell proliferation, 5 x 10³ cells of ORL-48(T) and ORL-136(T) cells in serum-starved DMEM/F12 medium were seeded into each well of 96-well tissue culture plates for 24 hours. The cells were treated with arecoline at 0, 0.025, 0.25, 2.5 and 25 μg/ml in serum-starved DMEM/F12 medium in triplicate for a further 24 hours. Cell proliferation was determined by the MTT assay.
By MTT assay, 10 μl MTT (5 mg/ml) was added to each well. After 4 hours, the medium was removed and the water-insoluble purple formazan particles were dissolved in 100 μl DMSO solution. The absorbance was read at 570 nm with a Microplate Reader (TECAN, Salzburg, Austria).

Arecoline, SB431542 (a specific inhibitor of the TGF-β type I receptor), and collagen solution from bovine skin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Arecoline stimulation was applied for the induction of myofibroblast transdifferentiation [2 (link)] and a collagen solution was used for collagen gel contraction analysis.