Butyrate induces reactive oxygen species production and affects cell cycle progression in human gingival fibroblasts

• Published in: Journal of periodontal research Vol. 48; no. 1; pp. 66 - 73
• Main Authors: Chang, M.-C., Tsai, Y.-L., Chen, Y.-W., Chan, C.-P., Huang, C.-F., Lan, W.-C., Lin, C.-C., Lan, W.-H., Jeng, J.-H.
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Publishing Ltd 01-02-2013; Blackwell
• Subjects: Apoptosis - drug effects; Biological and medical sciences; butyrate; Butyrates - pharmacology; Butyrates - toxicity; CDC2 Protein Kinase; cdc25 Phosphatases - drug effects; Cell Culture Techniques; cell cycle; Cell Cycle - drug effects; Cell Proliferation - drug effects; Cell Shape - drug effects; Cell Survival - drug effects; Coloring Agents; Cyclin B - drug effects; Cyclin B1 - drug effects; Cyclin-Dependent Kinases; Facial bones, jaws, teeth, parodontium: diseases, semeiology; Fibroblasts - cytology; Fibroblasts - drug effects; Flow Cytometry; Fluorescein-5-isothiocyanate; Fluoresceins; Fluorescent Dyes; G1 Phase Cell Cycle Checkpoints - drug effects; G2 Phase Cell Cycle Checkpoints - drug effects; Gingiva - cytology; Gingiva - drug effects; Humans; M Phase Cell Cycle Checkpoints - drug effects; Medical sciences; Non tumoral diseases; Otorhinolaryngology. Stomatology; periodontal pathogen; periodontitis; Porphyromonas gingivalis; Propidium; reactive oxygen species; Reactive Oxygen Species - metabolism; Resting Phase, Cell Cycle - drug effects; short-chain fatty acid; Human; short-chain fatty acid; Short chain; Oxygen; periodontal pathogen; Stomatology; Fatty acids; Butyrate; Periodontal disease; Periodontitis; Cell cycle; reactive oxygen species; Gingiva; Fibroblast
• ISSN: 0022-3484; 1600-0765
• DOI: 10.1111/j.1600-0765.2012.01504.x

Background and Objective:  Short‐chain fatty acids, such as butyric acid and propionic acid, are metabolic by‐products generated by periodontal microflora such as Porphyromonas gingivalis, and contribute to the pathogenesis of periodontitis. However, the effects of butyrate on the biological activities of gingival fibroblasts (GFs) are not well elucidated.
Material and Methods:  Human GFs were exposed to various concentrations of butyrate (0.5–16 mm) for 24 h. Viable cells that excluded trypan blue were counted. Cell cycle distribution of GFs was analyzed by propidium iodide‐staining flow cytometry. Cellular reactive oxygen species (ROS) production was measured by flow cytometry using 2’,7’‐dichlorofluorescein (DCF). Total RNA and protein lysates were isolated and subjected to RT‐PCR using specific primers or to western blotting using specific antibodies, respectively.
Results:  Butyrate inhibited the growth of GFs, as indicated by a decrease in the number of viable cells. This event was associated with an induction of G0/G1 and G2/M cell cycle arrest by butyrate (4–16 mm) in GFs. However, no marked apoptosis of GFs was noted in this experimental condition. Butyrate (> 2 mm) inhibited the expression of cdc2, cdc25C and cyclinB1 mRNAs and reduced the levels of Cdc2, Cdc25C and cyclinB1 proteins in GFs, as determined using RT‐PCR and western blotting, respectively. This toxic effect of butyrate was associated with the production of ROS.
Conclusion:  These results suggest that butyrate generated by periodontal pathogens may be involved in the pathogenesis of periodontal diseases via the induction of ROS production and the impairment of cell growth, cell cycle progression and expression of cell cycle‐related genes in GFs. These events are important in the initiation and prolongation of inflammatory processes in periodontal diseases.