Butyric acid as a histone deacetylase (HDAC) inhibitor is produced by a number of periodontal and root canal microorganisms (such as Porphyromonas, Fusobacterium, etc.). Butyric acid may affect the biological activities of periodontal/periapical cells such as osteoblasts, periodontal ligament cells, etc., and thus affect periodontal/periapical tissue destruction and healing. The purposes of this study were to study the toxic effects of butyrate on the matrix and mineralization marker expression in MG-63 osteoblasts. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Cellular apoptosis and necrosis were analyzed by propidium iodide/annexin V flow cytometry. The protein and mRNA expression of osteoprotegerin (OPG) and receptor activator of nuclear factor kappa-B ligand (RANKL) were analyzed by Western blotting and reverse transcriptase-polymerase chain reaction (RT-PCR). OPG, soluble RANKL (sRANKL), 8-isoprostane, pro-collagen I, matrix metalloproteinase-2 (MMP-2), osteonectin (SPARC), osteocalcin and osteopontin (OPN) secretion into culture medium were measured by enzyme-linked immunosorbant assay. Alkaline phosphatase (ALP) activity was checked by ALP staining. Histone H3 acetylation levels were evaluated by immunofluorescent staining (IF) and Western blot. We found that butyrate activated the histone H3 acetylation of MG-63 cells. Exposure of MG-63 cells to butyrate partly decreased cell viability with no marked increase in apoptosis and necrosis. Twenty-four hours of exposure to butyrate stimulated RANKL protein expression, whereas it inhibited OPG protein expression. Butyrate also inhibited the secretion of OPG in MG-63 cells, whereas the sRANKL level was below the detection limit. However, 3 days of exposure to butyrate (1 to 8 mM) or other HDAC inhibitors such as phenylbutyrate, valproic acid and trichostatin stimulated OPG secretion. Butyrate stimulated 8-isoprostane, MMP-2 and OPN secretion, but not procollagen I, or osteocalcin in MG-63 cells. Exposure to butyrate (2–4 mM) for 3 days markedly stimulated osteonectin secretion and ALP activity. In conclusion, higher concentrations of butyric acid generated by periodontal and root canal microorganisms may potentially induce bone destruction and impair bone repair by the alteration of OPG/RANKL expression/secretion, 8-isoprostane, MMP-2 and OPN secretion, and affect cell viability. However, lower concentrations of butyrate (1–4 mM) may stimulate ALP, osteonectin and OPG. These effects are possibly related to increased histone acetylation. These events are important in the pathogenesis and repair of periodontal and periapical destruction.

Butyric acid as a histone deacetylase (HDAC) inhibitor was produced by a number of periodontal and root canal microorganisms (such as Porphyromonas, Fusobacterium etc.). Butyric acid may affect the biological activities of periodontal/periapical cells such as osteoblasts, periodontal ligament cells etc., and thus affect periodontal/periapical tissue destruction and healing. The purposes of this study were to study the toxic effects of butyrate on matrix and mineralization markers’ expression of MG-63 osteoblasts. Cell viability and proliferation were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Cellular apoptosis and necrosis were analyzed by propidium iodide/Annexin V flow cytometry. Protein and mRNA expression of OPG, and RANKL were analyzed by western blotting and RT-PCR. OPG, soluble RANKL (sRANKL), 8-isoprostane, pro-collagen I, MMP-2, osteonectin (SPARC), osteocalcin and osteopontin secretion into culture medium were measured by enzyme-linked immunosorbant assay. Histone H3 acetylation levels were evaluated by immunofluorescent staining (IF) and western blot. We found that butyrate induced morphologic changes of growing MG-63 cells, with bigger and flattened in appearance. Butyrate activated histone H3 acetylation of MG-63 cells. Exposure of MG-63 cells to butyrate partly decreased cell number with no marked increase in apoptosis and necrosis. Butyrate stimulated RANKL protein expression, whereas it inhibited OPG protein expression. Butyrate also inhibited the secretion of OPG in MG-63 cells, whereas sRANKL level was below detection limit. Butyrate stimulated 8-isoprostane, MMP-2 and osteopontin secretion, but not procollagen I, osteonectin, osteocalcin in MG-63 cells. In conclusion, butyric acid generated by periodontal and root canal microorganisms may potentially induce bony destruction and impair bone repair by alteration of OPG/RANKL expression/secretion, 8-isoprostane, MMP-2, and osteopontin secretion, and affect cell proliferation. These effects are possibly related to increased histone acetylation. These events are important in the pathogenesis of periodontal and periapical destruction.