The role of selenium in regulating turnover of bovine mammary epithelial cells.

摘要

This research is an investigation of the effect of nutritional amounts of selenium (Se) as selenomethionine (SeMet) on cell turnover of bovine mammary epithelial cells (BMEC) under normal and oxidative stress. Single mammary epithelial cells were collected from lactating bovine mammary parenchyma and cultured in a low-serum collagen gel system enriched with lactogenic hormones and 0, 10, 20 or 50 nM SeMet. Immunohistochemical and immunocytochemical staining revealed that both GPx1 and GPx3 are synthesized by BMEC. Up to 50 nM SeMet linearly increased BMEC number and viability over 5 d of culture. BMEC cultured in SeMet-supplemented media also exhibited markedly elevated GPx activity and linear increases in abundance of GPx1 and GPx3 proteins. When BMEC were exposed to 100 muM hydrogen peroxide (H2O2), cell viability decreased and the number of apoptotic cells increased with no effect on cell proliferation. SeMet supplementation increased GPx activity and restored intracellular H2O2 and cell viability to control levels. Apoptotic BMEC number was reduced to below control levels by SeMet and proliferating cell number was increased. On a molecular level, H2O2 induced cleavage of caspase-9 and -3, but not caspase-8 after 72 h of treatment. After 48 h of treatment, H2O2 decreased Bcl-2 protein expression while Bax, Bid and phosphorylated p53 abundances were similar to the control. Nonetheless, addition of 20 or 50 nM SeMet inhibited cleavage of caspase-9 and -3 for 72 h, and Bcl-2 levels increased for 48 h. These SeMet concentrations also reduced Bax abundance for 48 h and phosphorylation of p53 after 48 h. In general, these results suggest that SeMet enhances expression of GPx1 and GPx3 in vitro, and the growth and viability of BMEC. SeMet also protects BMEC from H2O2-induced apoptosis and increases proliferation and cell viability under oxidative stress. H2O2 in BMEC triggers the intrinsic apoptotic pathway, and Se may prevent apoptosis by up-regulation of Bcl-2 abundance and down-regulation of Bax abundance, caspase-9 and -3 cleavage, and phosphorylation of p53 at Ser-15.