Human milk oligosaccharides 3'-sialyllactose and lacto-N-neotetraose ameliorate intestinal decline in naturally aged male mice in association with microbiome-metabolome reprogramming.

Intestinal epithelial homeostasis undergoes progressive functional decline during aging and contributes to systemic age-related dysfunction. Human milk oligosaccharides (HMOs) have well-documented prebiotic and immunomodulatory properties; however, their effects on the aging gut and functional differences among distinct structures remain largely unexplored. Here, we show that structurally distinct HMOs differentially regulate intestinal epithelial homeostasis in naturally aged male mice, with 3'-sialyllactose (3'-SL) and lacto-N-neotetraose (LNnT) showing broader effects than 2'-fucosyllactose (2'-FL). 3'-SL and LNnT supplementation improved barrier-related markers, including Muc2 and ZO-1, and attenuated age-associated epithelial hyperproliferation while maintaining intestinal stem cell (ISC)-related function. Antibiotic-mediated microbiota depletion markedly weakened these effects, supporting an important role of the gut microbiota. Multi-omics analyses further revealed distinct microbiome-metabolome alterations associated with 3'-SL and LNnT supplementation. Specifically, 3'-SL increased the relative abundance of Alistipes and was accompanied by higher levels of metabolites such as spermine. LNnT was associated with increased Allobaculum abundance and elevated acetylcholine levels in colonic contents. Collectively, our findings indicate that specific HMO structures ameliorate age-related decline of the intestinal epithelium in association with alterations in the microbiota-metabolite axis, providing a basis for further investigation of 3'-SL and LNnT as candidate dietary strategies for intestinal aging.