Gut microbiota as a central mediator in hydrogen gas-induced alleviation of colitis via TLR4/NF-κB and Nrf2 pathway regulation.

Inflammatory bowel disease (IBD) is a chronic and relapsing autoimmune disorder of the gastrointestinal tract with incompletely elucidated pathogenesis and limited therapeutic options. Although hydrogen gas (H₂) has demonstrated therapeutic efficacy in various diseases including IBD, its mechanisms of action, particularly its interaction with the gut microbiota, remain poorly characterized. This study reveals that H₂ inhalation effectively reversed dextran sulfate sodium (DSS)-induced dysbiosis by suppressing the expansion of potential pathogenic bacteria (e.g., Enterobacteriaceae and Escherichia-Shigella) and promoting potential beneficial microbes (e.g., Bacteroides and Lactobacillaceae), thereby restoring microbial homeostasis. Furthermore, H₂ inhalation enhanced goblet cell density and mucus production, upregulated tight junction proteins (ZO-1 and occludin), and repaired intestinal barrier integrity. It also rebalanced the Treg/Th17 cell ratio, correcting immune dysregulation. At the molecular level, H₂ inhalation suppressed the TLR4/NF-κB signaling pathway and activated the Keap1/Nrf2 antioxidant axis, leading to reduced production of pro-inflammatory cytokines and oxidative stress markers, alongside elevated antioxidant enzymes, collectively ameliorating colonic injury. In brief, the ameliorative effects of H₂ are likely mediated through remodeling of the gut microbiota, restoration of the epithelial barrier, suppression of inflammatory signaling, and activation of antioxidant pathways. These findings were further validated by fecal microbiota transplantation (FMT) experiments. Collectively, this study links the therapeutic effects of H₂ to structural and functional reprogramming of the gut microbiome, indicating that microbial ecological restoration is a central mechanism through which H₂ alleviates colitis, thereby providing a mechanistic foundation for the therapeutic application of H₂ inhalation in IBD.