Background: Lactoferrin (Lf) is a multifunctional protein and one of the most abundant proteins in human milk. Various factors may affect its concentration in human milk, such as stage of lactation, ethnicity, and diet. Objectives: The objectives of the present study were to examine the dynamic change in milk Lf throughout the course of lactation and explore factors associated with milk Lf concentrations in various Chinese populations. Methods: This investigation was a part of a large cross-sectional study conducted in 11 provinces/autonomous regions/municipalities (Beijing, Gansu, Guangdong, Guangxi, Heilongjiang, Inner Mongolia, Shandong, Shanghai, Xinjiang, Yunnan, and Zhejiang) across China between 2011 and 2013. Lactating women (n = 6481) within 0–330 days postpartum were recruited in the original study. A sub-sample of 824 women was randomly selected, and milk Lf concentrations were determined by UPLC/MS. Results: The Lf concentration in milk from women delivering at term was 3.16 g/L, 1.73 g/L and 0.90 g/L for colostrum, transitional milk, and mature milk, respectively. Lf concentrations differed significantly between stages of lactation (colostrum vs. transitional milk, colostrum vs. mature milk, transitional milk vs. mature milk, all p < 0.001). Maternal BMI, age, mode of delivery, parturition, protein intake, and serum albumin concentration were not correlated with milk Lf concentration. However, milk Lf concentrations varied among different geographical regions (Guangdong (1.91 g/L) vs. Heilongjiang (1.44 g/L), p = 0.037; Guangdong (1.91 g/L) vs. Gansu (1.43 g/L), p = 0.041) and ethnicities (Dai (1.80 g/L) vs. Tibetan (0.99 g/L), p = 0.007; Han (1.62 g/L) vs. Tibetan (0.99 g/L), p = 0.002) in China. Conclusions: The concentration of Lf in human milk changes dynamically throughout lactation. Few maternal characteristics affect the milk Lf concentration, but it varies across different geographical regions and ethnicities in China.

Osteopontin (OPN) is a pleiotropic protein involved in numerous biological processes such as cell proliferation and differentiation. Since OPN is abundantly present in milk and is known to be relatively resistant to in vitro gastrointestinal digestion, the current study aimed to investigate the roles of oral intake of milk OPN in intestinal development using an established OPN knockout (KO, OPN ^−/− ) mouse model, in which wild‐type (WT, OPN ^+/+ ) mouse pups were nursed by either WT (OPN ^+/+ OPN+ group) or OPN KO dams (OPN ^+/+ OPN− group; +/+ indicates genotype and − indicates milk without OPN), receiving milk with or without OPN from postnatal days 0 to 21 (P0−P21). Our results showed that milk OPN is resistant to in vivo digestion. Compared to OPN ^+/+ OPN− pups, OPN ^+/+ OPN+ pups at P4 and P6 had significantly longer small intestines, at P10 and P20 had larger inner jejunum surfaces, and at P30 exhibited more mature/differentiated intestines, as revealed by higher activities of alkaline phosphatase in brush border and more goblet cells, enteroendocrine cells, and Paneth cells. qRT‐PCR and immunoblotting results showed that milk OPN increased the expression of integrin αv, integrin β3, and CD44 in jejunum of mouse pups (P10, P20, and P30). Immunohistochemistry analysis showed that both integrin αvβ3 and CD44 are localized in jejunum crypts. In addition, milk OPN increased the phosphorylation/activation of the ERK, PI3K/Akt, Wnt, and FAK signaling pathways. In summary, oral intake of milk OPN in early life promotes intestinal proliferation and differentiation by upregulating the expression of integrin αvβ3 and CD44 and thus regulates OPN‐integrin αvβ3 and OPN‐CD44 mediated cellular signaling pathways.

MicroRNA (miRNA) is small non-coding RNA involved in gene silencing and post-transcriptional regulation of gene expression. Milk exosomes are microvesicles containing microRNAs (miRNAs). miR-22-3p (miR-22) is plentiful in human milk exosomes and may contribute to intestinal development since milk exosomes and microRNAs are resistant to gastrointestinal digestion in infants. After miR-22 mimics were transfected to human intestinal crypt-like epithelial cells (HIECs) using Lipofectamine for 24 h, RNA was isolated for microarray assay. Microarray results show that miR-22 markedly regulates gene expression, and the roles of miR-22 include promotion of proliferation, regulation of immune functions, and inhibition of apoptosis. Based on the microarray results and miR-22 predicted target genes, CCAAT/enhancer-binding protein δ (C/EBPδ) may be an important direct target of miR-22. C/EBPδ is a transcription factor that regulates numerous biological processes including cell proliferation. In miR-22 transfected HIECs, expression of the C/EBPδ gene was significantly inhibited. Silencing of the C/EBPδ gene by siRNA resulted in increased proliferation of HIECs. A luciferase assay showed that miR-22 specifically binds to the 3′-untranslated region of C/EBPδ mRNA. In summary, milk-derived miR-22 promotes intestinal proliferation by modifying gene expression, and C/EBPδ may be an important target for miR-22 involved in this effect.