Human milk oligosaccharide 2’-fucosyllactose and lacto-n-neo-tetraose attenuate early-weaning-induced intestinal barrier dysfunction in mice

Breastfeeding is universally acknowledged as pivotal for optimal infant development, yet early weaning remains a common choice among young mothers. Accumulating evidence indicates that early weaning disrupts intestinal development and induces gut dysbiosis; however, the impact of human milk oligosaccharides (HMOs) on intestinal function remains unclear. In this study, we aimed to investigate the effects of two major HMOs, 2’-fucosyllactose (2’-FL) and lacto-N-neotetraose (LNnT), on intestinal barrier function and their underlying mechanisms. Two-week-old mice were weaned and supplemented with 2’-FL, LNnT, or a combination of both (referred to as 2’-FL/ LNnT) for one week while being maintained on a liquid diet to support normal growth. We observed that both 2’-FL and LNnT, individually and in combination, alleviated early-weaning-induced gut barrier damage in the jejunum. RNA sequencing and subsequent validation revealed that the 2’-FL/LNnT combination activated the AMPK/SREBPs signaling pathway more robustly than either one alone. Interestingly, in vitro experiments demonstrated that HMOs did not directly affect the AMPK/SREBPs signaling. By 16S rDNA sequencing, we found that early weaning significantly reduced the microbial diversity, which was restored only in the 2’-FL/LNnT group. Finally, through antibiotic treatment and fecal microbiome transplantation (FMT) experiments, we confirmed that the protective effects of 2’-FL/LNnT on intestinal barrier function were gut microbiota-dependent. Collectively, these findings suggest that 2’-FL and LNnT protect against gut barrier dysfunction in early-weaned mice and may offer novel therapeutic strategies.