Enzymatic-fermentation-derived milk protein hydrolysate alleviates dexamethasone-induced osteoporosis in association with gut microbiota and metabolite alterations.

Osteoporosis is a prevalent skeletal disorder, and current pharmacological treatments are often associated with adverse effects. The present study aimed to investigate the protective effects and underlying mechanisms of fermented milk protein hydrolysate (FMPH), prepared via combined enzymatic hydrolysis and microbial fermentation, against dexamethasone (Dex)-induced osteoporosis. Compared with enzymatic hydrolysate alone, FMPH contained a higher proportion of low-molecular-weight peptides and amino acids associated with bone metabolism and significantly promoted osteoblast proliferation while attenuating Dex-induced cytotoxicity in vitro. In vivo, FMPH administration markedly improved bone microarchitecture, suppressed excessive osteoclast activation, and restored serum bone turnover markers. At the molecular level, FMPH promoted bone formation by upregulating osteogenic markers including runt-related transcription factor 2 (Runx2), Osterix, alkaline phosphatase (ALP), osteocalcin (OCN), and type I collagen alpha 1 (Col1α1), while inhibiting bone resorption through modulation of the receptor activator of nuclear factor-κB ligand (RANKL)/osteoprotegerin (OPG) signaling pathway. In addition, FMPH altered gut microbiota composition, including a reduced Firmicutes/Bacteroidota ratio and altered abundances of key genera such as Lactobacillus, Bifidobacterium, and Desulfovibrio, accompanied by significant changes in gut metabolite profiles. Correlation analysis revealed significant associations among gut microbiota, metabolites, and bone-related biochemical indices. Taken together, these findings suggest that FMPH exerts bone-protective effects by promoting bone formation and inhibiting bone resorption. Notably, these effects were associated with alterations in gut microbiota and metabolites. These findings provide a theoretical basis for the development of functional foods.