Effect of Human Breast Milk on the Expression of Proinflammatory Cytokines in Caco-2 Cells after Hypoxia/Re-Oxygenation

Wei-Yong Ruan, Jiangsu Traditional and Western Medicine Hospital, Nanjing, China
Ming-Yuan Bi, Nanjing University of Chinese Medicine, Nanjing, China
Wei-Wei Feng, Jiangsu Traditional and Western Medicine Hospital, Nanjing, China
Yu-Jun Wang, Jiangsu Traditional and Western Medicine Hospital, Nanjing, China
Wei-Quan Bu, Jiangsu Traditional and Western Medicine Hospital, Nanjing, China
Ling Lu, Nanjing University of Chinese Medicine, Nanjing, China

Background: Neonatal necrotizing enterocolitis is a common and often fatal gastrointestinal disease, especially in premature infants. To study potential mechanisms underlying the protective effect of breast milk on neonatal necrotizing enterocolitis, we induced intestinal inflammation in a Caco-2 cell model of neonatal necrotizing enterocolitis by hypoxia/re-oxygenation to investigate whether breast milk supernatant fluid inhibited the expression of proinflammatory cytokines interleukin-1β, interleukin-6, and tumor necrosis factor-α. Methods: Caco-2 cells were divided into normal (control) and neonatal necrotizing enterocolitis groups. Neonatal necrotizing enterocolitis was mimicked by exposing Caco-2 cells to hypoxia/re-oxygenation. Cells were independently maintained in minimal essential medium alone, minimal essential medium containing 5% breast milk supernatant, or 5% boiled breast milk supernatant. Production of interleukin-1β, interleukin-6, and tumor necrosis factor-α was investigated in cell culture supernatants by ELISA, reverse transcription polymerase chain reaction, and immunofluorescence. Results: Hypoxia/re-oxygenation significantly increased the expression of interleukin-1β, interleukin-6, and tumor necrosis factor-α. In the normal group, breast milk supernatant and boiled breast milk supernatant markedly downregulated the expression of interleukin-1β, interleukin-6, and tumor necrosis factor-α when compared with the minimal essential medium group, with the reduction in inter­leukin-1β expression being more pronounced in the breast milk group. In Caco-2 cells undergoing hypoxia/re-oxygenation, both breast milk supernatant and boiled breast milk supernatant significantly reduced the expression of interleukin-1β, interleukin-6, and tumor necrosis factor-α, where the decrease in interleukin-1β expression was greater in the breast milk group. Conclusions: Breast milk supernatant fluid inhibited the expression of proinflammatory cytokines interleukin-1β, interleukin-6, and tumor necrosis factor-α in Caco-2 cells, especially after hypoxia/re-oxygenation. This may be one of the mechanisms underlying the protective effect of breast milk on neonatal necrotizing enterocolitis.

Keywords: Necrotizing enterocolitis. Human milk. Interleukin-1. Interleukin-6. Tumor necrosis factor-α.