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Background Deficiency of heparan sulfate proteoglycan is a risk factor for intestinal inflammation. Previous studies have reported that oral low-molecular-weight heparin (LMWH) ameliorated colitis by undefined mechanisms in UC patients. Our study aims to explore the mechanisms of LMWH on chronic colitis-associated intestinal fibrosis, focusing on gut microbiota and its metabolites.Methods A murine model of chronic colitis-associated intestinal fibrosis was established by repeated 1.5% DSS exposure with concurrent oral LMWH treatment. Intestinal fibrosis was evaluated using histological staining, WB, IHC, and IF. Safety was assessed through coagulation tests, biochemical analyses, and multi-organ histology. A pseudo-germ-free mouse model was used to determine whether the antifibrotic effect of LMWH depends on gut microbiota. Differential microbial metabolites were identified by targeted metabolomics and non-targeted metabolomics. The antifibrotic effect of N-Acetylglutamic acid (NAG) was verified in vitro and in vivo. Metagenomic and transcriptomic sequencing were conducted to elucidate underlying mechanisms.Results Oral administration of LMWH alleviated DSS-induced intestinal fibrosis in mice, as evidenced by reduced collagen deposition in the submucosa, decreased numbers of activated fibroblasts, and lower expression levels of fibrotic markers ( IDDF2026-ABS-0174 Figure 1(A-J) Oral administration of LMWH alleviated DSS-induced intestinal fibrosis in mice). LMWH exhibited a favorable safety profile following oral administration (IDDF2026-ABS-0174 Figure 1(K-M) Oral administration of LMWH alleviated DSS-induced intestinal fibrosis in mice). The protective effect of LMWH was abrogated following depletion of the gut microbiota, indicating that its anti-fibrotic action is dependent on the gut microbiota (IDDF2026-ABS-0174 Figure 2(A-L) The anti-fibrotic effect of LMWH is dependent on gut microbiota). Oral LMWH significantly elevated the fecal levels of N-acetylglutamic acid (NAG) in mice, while in patients with CD, fecal NAG levels were lower in the fibrotic stricture group than in the non-stricture group (IDDF2026-ABS-0174 Figure 2(M-S) The anti-fibrotic effect of LMWH is dependent on gut microbiota). In vitro experiments showed that NAG inhibited the activation and collagen production of human colonic fibroblasts (IDDF2026-ABS-0174 Figure 3(A-F) LMWH upregulates the level of N-acetylglutamic acid (NAG), a metabolite of intestinal bacteria, which can inhibit the activation of primary fibroblasts in vitro), and in vivo experiments confirmed that oral gavage of NAG ameliorated intestinal fibrosis (IDDF2026-ABS-0174 Figure 3(G-N) LMWH upregulates the level of N-acetylglutamic acid (NAG), a metabolite of intestinal bacteria, which can inhibit the activation of primary fibroblasts in vitro). Transcriptomic analysis revealed that upregulation of NRF2 downstream signaling pathways following NAG intervention, and NRF2 pathway activity in fibroblasts was negatively correlated with the expression of fibrotic genes (IDDF2026-ABS-0174 Figure 4(A-F) Oral administration of NAG alleviated DSS-induced intestinal fibrosis in mice). NAG is directly bound to NRF2, promotes its nuclear translocation, and downregulated glycolysis in fibroblasts, thereby inhibiting cell activation (IDDF2026-ABS-0174 Figure 4(G-K) Oral administration of NAG alleviated DSS-induced intestinal fibrosis in mice). Both in vitro and in vivo experiments confirmed that this protective effect was attenuated by the NRF2 inhibitor ML385 (IDDF2026-ABS-0174 Figure 4(L-O) Oral administration of NAG alleviated DSS-induced intestinal fibrosis in mice).Conclusions Oral LMWH demonstrated microbiota-dependent antifibrotic effects mediated by elevated NAG production, which inhibited fibroblast activation and collagen synthesis through enhancing NRF2 signal. These findings highlight the therapeutic potential of oral LMWH for intestinal fibrosis. ( IDDF2026-ABS-0174 Figure 5. Graphical abstract)Abstract IDDF2026-ABS-0174 Figure 1Abstract IDDF2026-ABS-0174 Figure 2Abstract IDDF2026-ABS-0174 Figure 3Abstract IDDF2026-ABS-0174 Figure 4Abstract IDDF2026-ABS-0174 Figure 5