Effects of Iron-Unsaturated Human Lactoferrin on Hydrogen Peroxide-Induced Oxidative Damage in Intestinal Epithelial Cells

Abstract

Human milk (HM) contains various bioactive antioxidants. Lactoferrin (Lf) has been assumed to be one of the major antioxidants in HM. We examined the antioxidative properties of iron-unsaturated human Lf (apo-hLf, the major form of Lf in HM) in two intestinal epithelial cell lines: (1) An intestinal epithelial cell line (IEC-6) were preincubated for 24 h with either 50 g/mL of apo-hLf, iron-saturated human Lf (holo-hLf), iron-unsaturated bovine transferrin (apo-bTf), or 800 ng/mL of the iron-chelating compound deferoxamine (DFX), followed by hydrogen peroxide (H 2 O 2 ) challenge to induce oxidative stress. Survival rates were significantly higher in the cells preincubated with apo-hLf and DFX than those preincubated with holo-hLf. (2) Caco-2 cells were preincubated with or without apo-hLf for 24 h, followed by an H 2 O 2 challenge. Intracellular oxidative stress was assessed by a fluorescent probe, 2=,7=-dichlorodihydrofluorescein diacetate (DCF-DA). Fluorescent intensity of cell images and cell homogenates was significantly lower in the cells preincubated with apo-hLF than those preincubated without apo-hLF. Our study indicates that apo-hLf alleviates H 2 O 2 -induced oxidative damage in intestinal cells due to the iron-chelating capacity. Therefore, Lf in HM may act as an antioxidant in the gastrointestinal tract (GIT). O xidative stress occurs when there is an imbalance between the amount of antioxidant properties and reactive oxygen species. In the neonatal period, especially in preterm infants injury to the GIT by oxidative stress is thought to be involved in the pathogenesis of serious diseases such as necrotizing enterocolitis (1,2).HM is the ideal food during infancy and is known to contain various types of bioactive substances, some of which are reported to be antioxidants (3). We demonstrated the antioxidative effects of HM in two previous studies. The level of urinary 8-hydroxy-2=-deoxyguanosine (a marker of oxidative DNA damage) excretions of breast-fed infants was significantly lower than that of formula-fed infants in 1 mo of age (4). HM-treated IEC-6 cells showed a significantly higher survival rate after H 2 O 2 -induced oxidative damage than cells treated with infant formula (5). Despite these findings, it is still unclear which components of HM play the most important antioxidative roles in the GIT of infants.Lf is an iron-binding glycoprotein that belongs to the transferrin (Tf) family. Although it has been identified in many secretions from various species, it mainly exists in mammalian milk and colostrum. The amino acid compositions of bovine Lf and human Lf show 69% sequence homology (6). Lf appears to play a role in many biologic processes, including the proliferation and differentiation of enterocytes (7), regulation of iron absorption (8), antimicrobial activities (9), immunomodulation and anti-inflammatory responses (10), and antivirus or anticancer activities (11). Lf is considered to be an antioxidant because of its ability to bind two atoms of iron, which is important in t...

Protective Effects of Lactoferrin against Intestinal Mucosal Damage Induced by Lipopolysaccharide in Human Intestinal Caco-2 Cells

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Maternal smoking decreases antioxidative status of human breast milk

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