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.

氧化应激已被证实与肠黏膜损伤和多种肠道疾病有关。羟脯氨酸 (Hyp) 是母乳中含量丰富的亚氨基酸。已经收集了令人信服的证据，显示 Hyp 的潜在抗氧化特性。然而，Hyp 在猪肠上皮细胞对氧化应激反应中的作用和机制仍然未知。在这项研究中，使用仔猪小肠上皮细胞系 (IPEC-1) 来评估 Hyp 对 4-羟基-2-壬烯醛 (4-HNE) 诱导的氧化性 DNA 损伤和细胞凋亡的保护作用。在存在或不存在 Hyp 的情况下，将用 0.5 至 5 mmol/L Hyp 预处理的 IPEC-1 暴露于 4-HNE (40 μmol/L)。此后，通过Hoechst染色、流式细胞术和Western印迹或彗星试验的DNA损伤分析对细胞进行凋亡检测，免疫荧光和逆转录定量 PCR (RT-qPCR)。5 mmol/L Hyp 可抑制 4-HNE (40 μmol/L) 诱导的细胞凋亡和 cleaved-caspase-3 上调。此外，5 mmol/L Hyp 减弱了 4-HNE 诱导的活性氧 (ROS) 积累、谷胱甘肽 (GSH) 剥夺和 DNA 损伤。转录水平升高GADD45a（生长停滞和 DNA 损伤诱导蛋白 45 α）和GADD45b（生长停滞和 DNA 损伤诱导蛋白 45 β），以及 H2AX（H2A 组蛋白家族，成员 X）、p38 MAPK（丝裂原活化蛋白激酶）和 JNK（c-Jun N 末端激酶）的磷酸化) 在用 4-HNE 处理的细胞中，5 mmol/L Hyp 可减轻。此外，Hyp 补充剂增加了暴露于 4-HNE 的细胞中 Krüppel 样因子 4 (KLF4) 的蛋白质丰度。kenpaulone 对 KLF4 表达的抑制阻碍了 Hyp 处理的细胞对 4-HNE 诱导的 DNA 损伤和细胞凋亡的抵抗力。总的来说，我们的结果表明，Hyp 可以防止 4-HNE 诱导的 IPEC-1 细胞凋亡和 DNA 损伤，这与 KLF4 的增强表达部分相关。我们的数据为含有 Hyp 的动物产品的营养价值提供了更新的解释。