Effect of butyrate, a bacterial by-product, on the viability and ICAM-1 expression/production of human vascular endothelial cells: Role in infectious pulpal/periapical diseases.

Abstract

To investigate the effects of butyric acid (BA), a metabolic product generated by pulp and root canal pathogens, on the viability and intercellular adhesion molecule-1 (ICAM-1) production of endothelial cells, which are crucial to angiogenesis and pulpal/periapical wound healing. Endothelial cells were exposed to butyrate with/without inhibitors. Cell viability, apoptosis and reactive oxygen species (ROS) were evaluated using an MTT assay, PI/annexin V and DCF fluorescence flow cytometry respectively. RNA and protein expression was determined using a polymerase chain reaction assay and Western blotting or immunofluorescent staining. Soluble ICAM-1 (sICAM-1) was measured using an enzyme-linked immunosorbent assay. The quantitative results were expressed as mean±standard error (SE) of the mean. The data were analysed using a paired Student's t-test where necessary. A p-value ≤0.05 was considered to indicate a statistically significant difference between groups. Butyrate (>4mM) inhibited cell viability and induced cellular apoptosis and necrosis. It inhibited cyclin B1 but stimulated p21 and p27 expression. Butyrate stimulated ROS production and hemeoxygenase-1 (HO-1) expression as well as activated the Ac-H3, p-ATM, p-ATR, p-Chk1, p-Chk2, p-p38 and p-Akt expression of endothelial cells. Butyrate stimulated ICAM-1mRNA/protein expression and significant sICAM-1 production (p<.05). Superoxide dismutase, 5z-7oxozeaenol, SB203580 and compound C (p<.05), but not ZnPP, CGK733, AZD7762 or LY294002, attenuated butyrate cytotoxicity to endothelial cells. Notably, little effect on butyrate-stimulated sICAM-1secretion was found. Valproic acid, phenylbutyrate and trichostatin (three histone deacetylase inhibitors) significantly induced sICAM-1 production (p<.05). Butyric acid inhibited proliferation, induced apoptosis, stimulated ROS and HO-1 production and increased ICAM-1mRNA expression and protein synthesis in endothelial cells. Cell viability affected by BA was diminished by some inhibitors; however, the increased sICAM-1secretion by BA was not affected by any of the tested inhibitors. These results facilitate understanding of the pathogenesis, prevention and treatment of pulpal/periapical diseases.