Paper Title
Effect Of Butyrate On Viability Of Vascular Endothelial Cells

Abstract
Butyric acid is a co-productgeneratedby metabolism of fibrous food by intestinal, periodontal and root canal microorganisms.Butyric acid has been shown to have anti-carcinogenic effect, prevent obesity, and insulin resistance etc. But localized high concentration of butyric acid in periodontal pocket is suggested to impair periodontal/periapical wound healing responses. However, limited information is known about the effect of butyric acid on the viability of vascular endothelial cells, that plays important role in angiogenesis, wound healing and cardiovascular diseases. In this study, effect of butyrate on proliferation of EAHY endothelial cells was analyzed by MTT assay. Morphologic changes were taken by photographs. Propidium iodide and annexin V dual fluorescent flow cytometry was used to analyze cellular apoptosis. Cellular reactive oxygen species (ROS) production was determined by DCF fluorescence flow cytometry.In some experiments, EAHY cells were pretreated by superoxide dismutase or catalase 30 min before the addition of butyrate and further co-incubated for 3 days for MTT assay. We found that at concentrations higher than 4 mM, butyrate markedly suppressed the proliferation of EAHY endothelial cells as analyzed by MTT assay. EAHY endothelial cells are cuboid or polygonal-like in appearance. After exposure to butyrate (> 2 mM) for 3 days, cells became fibroblast-like with marked thin extended cellular processes. Exposure to butyrate (> 4 mM) further increased the cellular apoptosis and necrosis as shown by PI + annexin V flow cytometry analysis. Butyrate (> 4 mM) evidently stimulated ROS production of EAHY cells, as indicated by increase in cellular DCF fluorescence. Superoxide dismutase (SOD) partly attenuated the suppressive effect of butyrate on EAHY cell proliferation. But catalase showed little preventive effect on butyrate toxicity. These results reveal that butyrate may potentially affect angiogenesis via inhibition of endothelial cell proliferation. These events are associated with production of superoxide radicals. These results indicate that butyric acid may have potential anti-carcinogenic effect via inhibition of angiogenesis. But high butyric acid production in localized periodontal/periapical region may potentially impair regional wound healing response. (This study is supported by grants from Ministry of Science and Technology, Taiwan, ROC; Chang Gung Memorial Hospital, CMRP, Taiwan).