**Background:** Inflammatory bowel disease (IBD) is a chronic condition affecting millions worldwide, with gut microbiota dysbiosis implicated in its pathogenesis. The endocannabinoidome (eCBome) is a complex system of bioactive lipid mediators involved in inflammation and immune responses. A bidirectional interaction between the eCBome and gut microbiome (the eCBome-mBIome axis) has been proposed, but its role in colitis development was not well understood. This study aimed to investigate whether manipulation of gut microbiota affects DNBS-induced colitis severity and whether these effects correlate with alterations in colonic eCBome lipid mediators.
**Methods:** Colitis was induced in conventionally raised (CR), antibiotic-treated (ABX; ampicillin 1 mg/mL, streptomycin 1 mg/mL, clindamycin 1 mg/mL in drinking water for 2 weeks), and germ-free (GF) Balb/c male mice using intracolonic DNBS (120 mg/kg, 100 µL/mouse). After 3 days, mice were euthanized. Inflammation was assessed by Disease Activity Index (DAI) score, body weight change, colon weight-length ratio, myeloperoxidase (MPO) activity, and gene expression of cytokines (Il1b, Il6, Tnfa, Il10) and neutrophil markers (Cxcl1, Elane, Ly6g, Arg1) by qPCR. Colonic eCBome lipid mediators (NAEs, 2-MAGs, and 15-lipoxygenase derivatives) were quantified by HPLC-MS/MS. Statistical analysis used two-way ANOVA with Fisher's LSD test; correlations used Pearson's r. Sample sizes were 6-8 mice per group.
**Key Results:** GF mice showed significantly lower colon weight/length ratios after DNBS treatment compared to DNBS-treated CR and ABX mice (p<0.05). DAI scores in DNBS-treated GF mice were lower in the first days, reaching similar values only on day 3. Body weight loss was comparable across all DNBS-treated groups. DNBS treatment significantly increased Il1b and Il6 expression in ABX mice (Il1b: p<0.05; Il6: p<0.05) but not in GF mice. Tnfa expression increased after DNBS in CR and ABX mice but not in GF mice. Il10 expression was significantly lower in GF mice at baseline compared to CR and ABX mice (p<0.05) and was unaffected by DNBS. Basal MPO activity was significantly higher in GF mice than in CR and ABX mice (p<0.05). DNBS increased MPO activity only in CR mice (p<0.05). Cxcl1 expression was strongly upregulated by DNBS in CR and ABX mice but not in GF mice. Ly6g was significantly increased by DNBS in CR mice only (p<0.05). Arg1 was massively upregulated in CR and ABX mice after DNBS (significant only in ABX, p<0.05) with no effect in GF mice. At baseline, GF mice had significantly higher levels of anti-inflammatory NAEs: OEA, LEA, DHEA (all p<0.05 vs. CR and ABX), and 13-HODE-EA (p<0.05 vs. CR). DNBS treatment significantly reduced AEA, OEA, LEA, and DHEA levels in GF mice (p<0.05). In inflamed tissues, LEA and DHEA remained significantly higher in GF than CR mice, and 13-HODE-EA was significantly higher in GF mice than both DNBS-treated CR and ABX mice (p<0.05). Correlation analysis showed that AEA, PEA, OEA, LEA, DHEA, 2-OG, and 2-LG were all significantly negatively associated with colon weight/length ratio. Cxcl1 and Ly6g were significantly negatively correlated with PEA, LEA, DHEA, OEA, and 13-HODE-EA.
**Clinical Implications:** This study demonstrates that complete absence of gut microbiota in GF mice is associated with reduced DNBS-induced colonic inflammation and concurrent elevation of anti-inflammatory eCBome lipid mediators. The negative correlations between these lipid levels and inflammatory markers suggest that the eCBome-mBIome axis may represent a therapeutic target for IBD. However, the differences between GF and ABX mice indicate that developmental immune alterations from congenital microbiota absence, rather than acute bacterial depletion alone, contribute to this phenotype. The findings support further investigation of eCBome-targeted therapies, including N-acylethanolamines and their 15-lipoxygenase derivatives, as potential treatments for colitis. Limitations include the short 3-day observation period, use of a single mouse strain (Balb/c), and the unclear biological roles of 13-HODE compounds.