[Xianglian Pills ameliorate ulcerative colitis by regulating ASS1-mediated arginine metabolism: a study based on...

This study integrated untargeted metabolomics with in vivo and in vitro molecular experiments to elucidate the targets of Xianglian Pills(XLP) in treating ulcerative colitis(UC). A mouse model of UC was established with 3% dextran sulfate sodium(DSS) in drinking water. C57BL/6J mice were randomized into six groups: control, model, mesalazine(positive control, 0.4 g·kg~(-1)), and low-, medium-, and high-dose(0.5, 1.0, and 2.0 g·kg~(-1), respectively) XLP. In parallel, an in vitro intestinal epithelial barrier injury model was induced in Caco-2 monolayers by combined stimulation with tumor necrosis factor(TNF)-α(100 ng·mL~(-1)) and interferon(IFN)-γ(100 ng·mL~(-1)). The results demonstrated that XLP alleviated DSS-induced weight loss, colon shortening, and elevated disease activity index(DAI) in a dose-dependent manner. XLP treatment markedly reduced the serum levels of pro-inflammatory cytokines TNF-α and interleukin(IL)-6 and restored the expression and localization of the tight junction proteins Zonula occludens-1(ZO-1) and Occludin in the colon tissue. Serum metabolomics analysis based on UPLC-MS/MS identified 24 differential metabolites, and the pathway enrichment analysis revealed that XLP predominantly regulated the arginine biosynthesis pathway. Mechanism investigation demonstrated that XLP significantly suppressed the inflammation-induced upregulation of argininosuccinate synthetase 1(ASS1) and inducible nitric oxide synthase(NOS2/iNOS). Concurrently, XLP upregulated the expression of arginase 1(ARG1) and argininosuccinate lyase(ASL), shifting the metabolic flux towards polyamine synthesis and tissue repair. In vitro assays confirmed that XLP at 50 μg·mL~(-1) significantly increased the transepithelial electrical resistance(TEER), reduced FITC-dextran permeability, and accelerated wound healing in Caco-2 cells. Plasmid-mediated overexpression of ASS1 significantly reversed the protective effect of XLP on barrier integrity and the inhibitory effect of XLP on the NOS2 pathway. In conclusion, XLP ameliorates UC by reprogramming the ASS1-NOS2 metabolic axis in intestinal epithelial cells to rebalance arginine metabolism, thereby mitigating mucosal inflammation and restoring the intestinal physical barrier.