Inflammatory bowel disease (IBD) is a chronic inflammatory disorder of the colon characterized by persistent mucosal inflammation and abnormal immune cell infiltration, leading to ulceration, erosion, and fibrosis [1]. Clinical manifestations include diarrhea, abdominal pain, hematochezia, and systemic inflammation, significantly impairing quality of life and posing life-threatening risks. Although the etiology remains elusive, IBD may arise from immune dysregulation, gut microbiota imbalance, environmental triggers, and genetic susceptibility, collectively compromising intestinal barrier integrity and promoting inflammatory and oxidative tissue damage [2,3]. Immunological imbalance plays a central role, particularly dysfunctional differentiation and activity of Th17 and Treg cells [4]. Overactive Th17 responses coupled with impaired Treg function exacerbate intestinal inflammation, correlating with elevated IL-17 levels and reduced Treg populations in patients [5,6]. The NF-κB pathway is pivotal in IBD pathogenesis: upon barrier disruption, Lipopolysaccharide (LPS) activates NF-κB via TLR4/MyD88, driving nuclear translocation and transcription of pro-inflammatory cytokines (e.g., TNF-α, IL-6, IL-1β) [7,8]. This pathway also disrupts tight junctions, promotes epithelial apoptosis, and facilitates microbial translocation [9,10]. Concurrently, oxidative stress amplifies damage through reactive oxygen and nitrogen species (ROS/RNS), which impair lipids, proteins, and DNA while synergizing with inflammatory cascades [11,12]. Dysregulation of the Keap1/Nrf2 antioxidant system further accelerates disease progression by failing to suppress inflammation and activate cytoprotective genes [13,14].

Furthermore, gut dysbiosis plays a critical role in the pathogenesis and progression of IBD, with clinical, genetic, and animal model studies consistently demonstrating its involvement in immune modulation [[15], [16], [17]]. IBD patients exhibit significantly reduced microbial diversity, characterized by an expansion of pathobionts such as adherent-invasive Escherichia coli (AIEC) and a decline in beneficial probiotics including Bifidobacterium and Lactobacillus. This ecological imbalance disrupts intestinal immune homeostasis, promoting Th17 hyperactivation and impairing Treg cell function, thereby perpetuating chronic inflammation [18]. Alterations in specific bacterial microbes are correlated with increased gut permeability, and functional metabolic pathways derived from dysbiotic microbiota further contribute to barrier dysfunction. For instance, AIEC invades intestinal epithelial cells, activates the TLR4/NF-κB signaling pathway, and stimulates the release of pro-inflammatory cytokines, ultimately compromising barrier integrity. Additionally, AIEC may secrete enzymes or toxins that degrade the mucosal layer, exacerbating epithelial damage [19].

Current management of IBD primarily relies on pharmacological interventions, with surgical resection reserved for medically refractory cases or severe complications. Commonly used drugs include aminosalicylates, corticosteroids, immunomodulators, and biologics; however, these are associated with long-term adverse effects such as hepatic and renal impairment and osteoporosis, in addition to imposing substantial economic burdens [20]. Consequently, there is an urgent need to develop simple, feasible, and cost-effective preventive and therapeutic strategies. Molecular hydrogen (H₂), an endogenous gaseous molecule characterized by its low molecular weight, colorlessness, odorlessness, and physiological inertness, has demonstrated therapeutic potential across various disease contexts via mechanisms encompassing antioxidative stress, anti-inflammation, and anti-apoptosis [[21], [22], [23], [24], [25]]. Its efficacy has also been observed in IBD models [26]. Mechanistically, H₂ exerts protective effects by: (i) selectively scavenging hydroxyl radicals (·OH) and peroxynitrite (ONOO−), thereby alleviating oxidative stress through reduction of malondialdehyde (MDA) and enhancement of superoxide dismutase (SOD) and glutathione (GSH) activities [27]; (ii) downregulating the TLR4/MyD88 signaling pathway and inhibiting the release of pro-inflammatory cytokines [28]; (iii) suppressing the unfolded protein response via reduction of endoplasmic reticulum stress proteins (p-eIF2α/ATF4/CHOP) [29]; (iv) upregulating tight junction protein expression, reducing intestinal permeability, and increasing goblet cell numbers and mucus secretion, collectively promoting barrier restoration [26]; and (v) maintaining an anaerobic microenvironment conducive to probiotic proliferation and microbiota reconstitution [28,30].

In both clinical and animal studies, the primary administration routes for H2 encompass inhalation, hydrogen-rich water (HRW) ingestion/injection, and hydrogen baths. Although HRW is frequently utilized due to its portability and safety [31], it faces significant limitations: low aqueous solubility (≈0.8 mM at ambient temperature), poor stability (<12-h maintenance) [32], short metabolic half-life (peak blood concentration sustained <10 min) [33], coupled with inconsistencies in preparation protocols and dosing standards. These factors compromise data comparability, posing challenges for clinical standardization. In contrast, H₂ inhalation capitalizes on its minimal molecular size to achieve unimpeded transmembrane diffusion, enabling precise targeting of subcellular structures such as mitochondria. This approach offers higher delivered concentrations, prolonged retention, and procedural simplicity, thus effectively addressing the limitations of HRW.

Although H₂ demonstrates therapeutic potential in anti-oxidative and anti-inflammatory applications, its mechanisms for intestinal barrier restoration and immune homeostasis modulation via gut microbiota remain unclear. Our study revealed that H₂ inhalation significantly ameliorated dextran sulfate sodium (DSS)-induced murine colitis. H2 upregulated tight junction proteins (ZO-1 and occludin), stimulated goblet cell proliferation, and enhanced mucin synthesis—collectively restoring intestinal barrier integrity. Consequently, this process suppressed LPS/TLR4/NF-κB-mediated inflammatory cascades, activated the Keap1/Nrf2 anti-oxidative signaling pathway, reinstated Treg/Th17 immune homeostasis, and effectively attenuated intestinal inflammatory infiltration and oxidative stress damage. Through analysis of changes in fecal gut microbiota and fecal microbiota transplantation (FMT) experiments, it was further evaluated and confirmed that H₂ alleviates IBD primarily by modulating the gut microbiota.