Combined polysaccharides from Angelica sinensis, Crataegus pinnatifida, Prunus persica, and Carthamus tinctorius attenuate cold exposure-induced bone loss by modulating the gut microbiota and fecal metabolites.

Chronic cold stress is a significant risk factor for skeletal deterioration; however, effective therapeutic strategies targeting the underlying environmental-metabolic interactions remain unclear. This study investigated the osteoprotective potential of Mixed Polysaccharides (MPs) and elucidated the mediating role of the gut microbiome. Cold exposure-induced bone loss was established in rats. Fecal microbiota transplantation (FMT), 16S rRNA gene sequencing, and untargeted metabolomics was employed to illustrate the positive effect of MPs on the improvement of cold-exposed bone loss. MPs treatment effectively reversed cold-induced trabecular microarchitecture deterioration and bone mass loss. In femoral tissue, MPs rebalanced skeletal turnover by upregulating osteogenic markers (Runx2, Osterix) and suppressing osteoclastogenic factors (TRAP, c-fos), concurrent with a marked reduction in the levels of pro-inflammatory cytokines TNF-α and IL-1β in femur. Furthermore, MPs restored intestinal barrier integrity by upregulating tight junction proteins (ZO-1, Occludin), thereby mitigating the intestinal barrier impairment driven by cold stress. FMT experiments demonstrated that the osteoprotective effects of MPs are microbiota-dependent, as the transplantation of MPs-modulated microbiota recapitulated the bone-preserving and barrier-restoring phenotypes in recipient mice. Multi-omics integration identified that MPs selectively promoted the expansion of Lactobacillus intestinalis and the accumulation of cholylhistidine. Correlation analysis further revealed a strong link between the enrichment of these microbial and metabolic signatures, reduced pro-inflammatory cytokine levels, and improved bone formation. Our findings indicate that MPs alleviate cold-stress-induced bone loss by remodeling the gut microbiota and metabolic profile, fortifying the intestinal barrier and decreasing pro-inflammatory cytokine.