Mechanical unloading and exercise volume differentially modulate the gut microbiome and intestinal gene expression in mice

Mechanical unloading and physical activity can influence the gut microbiome and intestinal physiology, but the effects of exercise volume and prior hindlimb suspension remain incompletely understood. This study investigated how hindlimb suspension, different exercise volumes, and exercise following hindlimb suspension affect gut microbial composition and intestinal gene expression in mice. Mice were assigned to five groups: control (C), hindlimb suspension (HL), high-volume exercise (HE), low-volume exercise (LE), and hindlimb suspension followed by exercise (HLE). Cecal microbiota composition was characterized using 16S rRNA gene sequencing, followed by alpha- and beta-diversity analyses and LinDA differential-abundance analysis. Jejunal gene expression was assessed by quantitative PCR for markers related to oxidative stress, inflammation, metabolic signaling, and epithelial function. Hindlimb suspension reduced microbial richness, as indicated by the Observed, Chao1, and ACE indices, and was associated with a significant difference in overall microbial community composition compared with controls. It was also associated with reductions in Streptococcaceae/Streptococcus and Spirochaetota. Low-volume exercise produced the most extensive phylum-level differential-abundance findings, whereas high-volume exercise showed no significant phylum-level differences from controls. Both exercise groups (HE and LE) exhibited increased intestinal expression of GPX1, HMOX1, NFKB1, and SOD2. HE also increased IGF and MUC2 expression, whereas HLE reduced IGF expression compared with controls. Community composition did not differ significantly between HLE and HL, and LGR5 expression did not differ significantly among groups. In conclusion, hindlimb suspension was associated with reduced microbial richness and selective taxonomic changes, whereas low-volume exercise produced the most extensive phylum-level alterations in microbial composition. Exercise volume was also associated with distinct intestinal transcriptional responses, while exercise following mechanical unloading did not produce a microbial community composition significantly different from that observed following unloading alone. These findings highlight the context-dependent effects of mechanical unloading and exercise on gut microbial composition and intestinal gene expression.