title: Effect of Human Breast Milk on the Expression of Proinflammatory Cytokines in Caco-2 Cells after Hypoxia/Re-Oxygenation.
abstract: 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 inter-leukin-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.
authors.affiliation: Jiangsu Traditional and Western Medicine Hospital, Nanjing, China.
authors.forename: Wei-Yong
authors.initials: WY
authors.lastname: Ruan
authors.affiliation: Nanjing University of Chinese Medicine, Nanjing, China.
authors.forename: Ming-Yuan
authors.initials: MY
authors.lastname: Bi
authors.affiliation: Jiangsu Traditional and Western Medicine Hospital, Nanjing, China.
authors.forename: Wei-Wei
authors.initials: WW
authors.lastname: Feng
authors.affiliation: Jiangsu Traditional and Western Medicine Hospital, Nanjing, China.
authors.forename: Yu-Jun
authors.initials: YJ
authors.lastname: Wang
authors.affiliation: Jiangsu Traditional and Western Medicine Hospital, Nanjing, China.
authors.forename: Wei-Quan
authors.initials: WQ
authors.lastname: Bu
authors.affiliation: Nanjing University of Chinese Medicine, Nanjing, China.
authors.forename: Ling
authors.initials: L
authors.lastname: Lu
mesh_terms: D018938:Caco-2 Cells; D003296:Cooking; D016207:Cytokines / Q000378:metabolism*; D015536:Down-Regulation / Q000235:genetics; D020345:Enterocolitis, Necrotizing / Q000503:physiopathology / Q000517:prevention & control*; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005455:Fluorescent Antibody Technique; D006801:Humans; D000860:Hypoxia; D007249:Inflammation / Q000378:metabolism / Q000473:pathology; D018836:Inflammation Mediators / Q000378:metabolism*; D053583:Interleukin-1beta / Q000378:metabolism; D015850:Interleukin-6 / Q000378:metabolism; D008895:Milk, Human / Q000378:metabolism*; D010100:Oxygen / Q000008:administration & dosage; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
chemical_list: D016207:Cytokines; D018836:Inflammation Mediators; D053583:Interleukin-1beta; D015850:Interleukin-6; D014409:Tumor Necrosis Factor-alpha; D010100:Oxygen