Hydroxyproline alleviates 4-hydroxy-2-nonenal-induced DNA damage and apoptosis in porcine intestinal epithelial cells

Oxidative stress has been confirmed in relation to intestinal mucosa damage and multiple bowel diseases. Hydroxyproline (Hyp) is an imino acid abundant in sow's milk. Compelling evidence has been gathered showing the potential antioxidative properties of Hyp. However, the role and mechanism of Hyp in porcine intestinal epithelial cells in response to oxidative stress remains unknown. In this study, small intestinal epithelial cell lines of piglets (IPEC-1) were used to evaluate the protective effects of Hyp on 4-hydroxy-2-nonenal (4-HNE)-induced oxidative DNA damage and apoptosis. IPEC-1 pretreated with 0.5 to 5 mmol/L Hyp were exposed to 4-HNE (40 μmol/L) in the presence or absence of Hyp. Thereafter, the cells were subjected to apoptosis detection by Hoechst staining, flow cytometry, and Western blot or DNA damage analysis by comet assay, immunofluorescence, and reverse-transcription quantitative PCR (RT-qPCR). Cell apoptosis and the upregulation of cleaved-caspase-3 induced by 4-HNE (40 μmol/L) were inhibited by 5 mmol/L of Hyp. In addition, 5 mmol/L Hyp attenuated 4-HNE-induced reactive oxygen species (ROS) accumulation, glutathione (GSH) deprivation and DNA damage. The elevation in transcription of GADD45a (growth arrest and DNA-damage-inducible protein 45 alpha) and GADD45b (growth arrest and DNA-damage-inducible protein 45 beta), as well as the phosphorylation of H2AX (H2A histone family, member X), p38 MAPK (mitogen-activated protein kinase), and JNK (c-Jun N-terminal kinase) in cells treated with 4-HNE were alleviated by 5 mmol/L Hyp. Furthermore, Hyp supplementation increased the protein abundance of Krüppel like factor 4 (KLF4) in cells exposed to 4-HNE. Suppression of KLF4 expression by kenpaulone impeded the resistance of Hyp-treated cells to DNA damage and apoptosis induced by 4-HNE. Collectively, our results indicated that Hyp serves to protect against 4-HNE-induced apoptosis and DNA damage in IPEC-1 cells, which is partially pertinent with the enhanced expression of KLF4. Our data provides an updated explanation for the nutritional values of Hyp-containing animal products.