P0004Oral administration of low-molecular-weight heparin enhances microbiota-derived N-acetyl-glutamic acid level to alleviate chronic colitis-associated intestinal fibrosis through the NRF2 pathway

Background The deficiency of heparan sulfate proteoglycan is regarded as a risk factor for intestinal inflammation. Previous studies have reported that oral administration of low-molecular-weight heparin (LMWH) ameliorated colitis by undefined mechanisms in ulcerative colitis (UC) patients. Our study aims to explore the mechanisms of LMWH on chronic colitis-associated intestinal fibrosis from the perspective of 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 of oral LMWH 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, as evidenced by decreased collagen deposition and fibrotic marker expression. Toxicological evaluations confirmed its safety. Loss of antifibrotic efficacy in microbiota-depleted mice suggested microbiota-dependency. NAG, a metabolite involved in arginine biosynthesis, was elevated in feces following LMWH administration and validated via LC-MS/MS. Oral NAG administration also ameliorated fibrosis in vivo and inhibited primary intestinal fibroblast activation in vitro. Metagenomic analysis revealed enrichment of Limosilactobacillus reuteri, while transcriptomic data showed upregulation of arginine metabolism and reduced T cell infiltration in the lamina propria of LMWH-treated mice. Mechanistically, NAG inhibits fibroblast activation by up-regulating the NRF2 signaling pathway and the protective effects of NAG were reversed by the NRF2 inhibitor ML385 in vitro and in vivo. Conclusion 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. References: 1.Zheng D, Chen S, Feng H, et al. Oral administration of low-molecular-weight heparin ameliorates colitis by enhancing the gut mucus barrier via microbial tryptophan metabolites. Br J Pharmacol. Published online September 28, 2025. doi:10.1111/bph.70209 2.Gao G, Shen S, Zhang T, et al. Lacticaseibacillus rhamnosus Probio-M9 enhanced the antitumor response to anti-PD-1 therapy by modulating intestinal metabolites. EBioMedicine. 2023;91:104533. doi:10.1016/j.ebiom.2023.104533 3.Zhang H, Hu Q, Zhang Y, et al. Lachnospiraceae bacterium alleviates alcohol-associated liver disease by enhancing N-acetyl-glutamic acid levels and inhibiting ferroptosis through the KEAP1-NRF2 pathway. Gut Microbes. 2025;17(1):2517821. doi:10.1080/19490976.2025.2517821 4.Sarrazin S, Lamanna WC, Esko JD. Heparan sulfate proteoglycans. Cold Spring Harb Perspect Biol. 2011;3(7):a004952. Published 2011 Jul 1. doi:10.1101/cshperspect.a004952 Conflict of interest: Dr. Feng, Hanxiao: No conflict of interest Li, Linjie: No conflict of interest Chen, Shuze: No conflict of interest Chen, Ye: No conflict of interest