In vitro Quantification of Collagen and Snail1 Gene Expression in Experimentally Induced Fibrosis by Arecoline and Commercial Smokeless Tobacco Products

INTRODUCTION: Extracellular matrix component derangement is the major event in pathogenesis of Oral submucous fibrosis. Many studies have elaborated the alteration of the matrix components at a cellular and genetic level. However elaborate quantification of the components with varying concentrations of Areca nut extract and commercial tobacco products have not been done so far. MATERIALS AND METHODS: Primary culture of tissues sourced during crown lengthening procedures were used for establishment of fibroblast monoculture and fibroblast / keratinocyte co-culture. Extracts of areca nut, commercial smokeless tobacco products (gutkha and haans) and control CCl4 were tested at concentrations ranging from 20 μL, 40 μL, 80 μL, 160 μL, 320 μL and time intervals of 12, 24, 48, 72 hours. Collagen quantification by spectrophotometry and SNAI1 gene expression study were done. RESULTS: Extract of areca nut was found to show increased collagen production than commercial tobacco products and closely similar values to CCL4. Kruskal Wallis test was used to analyse the difference in collagen obtained. The mean values of collagen obtained in co-culture were lesser than those obtained in the fibroblast monoculture. SNAI1 gene expression was negative in both the culture experiments. CONCLUSION: Areca nut extract was found to be more potent as an individual agent. Commercial smokeless tobacco products Gutka and Hans exhibited increased collagen production at higher concentration. These findings further steps up the persuasive ill effects of tobacco products. Negative SNAI1 gene expression was corroborated to lack of extracellular environment in the co coculture experiment.