Wheat phytase potentially protects HT-29 cells from inflammatory nucleotides-induced cytotoxicity.

Abstract

The aim of this study was to investigate the protective effect of wheat phytase as a structural decomposer of inflammatory nucleotides, extracellular adenosine triphosphate (ATP), and uridine diphosphate (UDP) on HT-29 cells. Phosphatase activities of wheat phytase against ATP and UDP was investigated in the presence or absence of inhibitors such as L-phenylalanine and L-homoarginine using a Pi Color Lock gold phosphate detection kit. Viability of HT-29 cells exposed to intact- or dephosphorylated-nucleotides was analyzed with an EZ-CYTOX kit. Secretion levels of pro-inflammatory cytokines (IL-6 and IL-8) in HT-29 cells exposed to substrate treated with or without wheat phytase were measured with enzyme-linked immunosorbent assay kits. Activation of caspase-3 in HT-29 cells treated with intact ATP or dephosphorylated-ATP was investigated using a colorimetric assay kit. Wheat phytase dephosphorylated both nucleotides, ATP and UDP, in a dosedependent manner. Regardless of the presence or absence of enzyme inhibitors (L-phenylalanine and L-homoarginine), wheat phytase dephosphorylated UDP. Only L-phenylalanine inhibited the dephosphorylation of ATP by wheat phytase. However, the level of inhibition was less than 10%. Wheat phytase significantly enhanced the viability of HT-29 cells against ATP- and UDP-induced cytotoxicity. Interleukin (IL)-8 released from HT-29 cells with nucleotides dephosphorylated by wheat phytase was higher than that released from HT-29 cells with intact nucleotides. Moreover, the release of IL-6 was strongly induced from HT-29 cells with UDP dephosphorylated by wheat phytase. HT-29 cells with ATP degraded by wheat phytase showed significantly (13%) lower activity of caspase-3 than HT-29 cells with intact ATP. Wheat phytase can be a candidate for veterinary medicine to prevent cell death in animals. In this context, wheat phytase beyond its nutritional aspects might be a novel and promising tool for promoting growth and function of intestinal epithelial cells under luminal ATP and UDP surge in the gut.