圆叶冬青（Ilex rotunda Thunb.，以下简称IR）常被瑶族医生用于治疗胃肠疾病，在溃疡性结肠炎（UC）的临床治疗中具有较好的疗效。然而，IR治疗UC的主要活性成分和作用机制尚待阐明。本研究旨在探究IR治疗UC的主要活性成分和作用机制。采用超高效液相色谱-串联质谱（UPLC-MS/MS）法对IR的十种生物活性成分进行定量。体外实验中，利用脂多糖刺激Caco2细胞单层，并分别使用10种生物活性成分处理，以研究IR成分对粘膜屏障损伤的保护作用。体内实验中，采用硫酸葡萄糖苷复合物诱导小鼠UC模型，并给予IR的候选活性成分。在第8天，收集血清和结肠组织进行组织学和分子分析，以研究IR的主要活性成分和作用机制。结果发现，IR中的紫榆苷I、紫榆苷II、丁榄苷和蒲公英苷能减少粘膜屏障的酚红透过率，并抑制Caco2细胞中的癌细胞生成素M（oncostatin M）和癌细胞生成素M受体（oncostatin M receptor）的蛋白表达。值得注意的是，紫榆苷II和丁榄苷能降低细胞单层的经皮电阻，并促进Caco2细胞中Occludin、Claudin-1和紧密连接蛋白-1（ZO-1）的蛋白表达。在体内实验中，紫榆苷II和丁榄苷改善了UC小鼠的症状，包括体重、疾病活动评分、结肠长度缩短、酸性粘液层破坏、组织病理学变化以及Occludin、Claudin-1和ZO-1的蛋白表达。蒲公英苷能减少UC小鼠中的中性粒细胞和炎症反应。此外，当紫榆苷II、丁榄苷和蒲公英苷联合治疗UC时，丁榄苷和蒲公英苷能增强紫榆苷II的治疗效果。最后，RNA测序和RT-qPCR分析显示，紫榆苷II + 丁榄苷 + 蒲公英苷和IR共调控了高达42.7%的基因，主要降低了细胞因子相互作用途径中C-X-C基序化学因子1（CXCL1）、癌细胞生成素M受体（OSMR）、白细胞介素1受体I型（IL1R1）、肿瘤坏死因子受体超家族成员9（TNFRSF9）、C-X-C基序化学因子13（CXCL13）、癌细胞生成素M（OSM）和白细胞介素6（IL-6）的过度表达。紫榆苷II、丁榄苷和蒲公英苷的联合作用通过调节肠道粘膜屏障并抑制细胞因子相互作用途径，在治疗UC方面具有较好的保护作用，其效果与圆叶冬青的水提取物相当。版权所有 © 2023. Elsevier B.V. 发表。

Ilex rotunda Thunb. (IR) is widely used for gastrointestinal diseases by Yao physician, and it has a better clinical curative effect on ulcerative colitis (UC). However, the main active components and mechanism of IR in the treatment of UC remain to be clarified.To investigate the main active components and mechanism of IR in the treatment of UC.Ten biological active components of IR were quantified by UPLC-MS/MS. In vitro, Caco2 cell monolayers were stimulated by lipopolysaccharide, and were treated with 10 biologically active components individually to investigate the protective role of the components of IR in mucosal barrier damage. In vivo, a mouse model of UC was induced by dextran sulfate sodium and administered with the candidate active components of IR. On day 8, the serum and colon tissue were collected for histological and molecular analysis to investigate the main active components and mechanism of IR.Ziyuglycoside I, ziyuglycoside II, syringin, and pedunculoside in IR reduced phenol red transmission of the monolayer, and inhibited the protein expression of oncostatin M and oncostatin M receptor in Caco2 cells. Notably, ziyuglycoside II and syringin decreased the transepithelial electrical resistance of the monolayer, and promoted the protein expression of Occludin, Claudin-1 and zonula occludens-1 (ZO-1) in Caco2 cells. In vivo, ziyuglycoside II and syringin improved the symptoms of UC mice, including body weight, disease activity score, shortening of colon length, damaging of acidic mucus layer, histopathological changes, and protein expression of Occludin, Claudin-1, and ZO-1. Pedunculoside reduced the neutrophils and inflammatory response in the UC mice. Moreover, when the combination of ziyuglycoside II, syringin and pedunculoside was used for the treatment of UC, syringin and pedunculoside enhanced the therapeutic effect of ziyuglycoside II. Finally, RNA sequencing and RT-qPCR analysis revealed that ziyuglycoside II + syringin + pedunculoside and IR coregulated up to 42.7% of genes, and mainly reduced the overexpression of C-X-C motif ligand 1(CXCL1), oncostatin M receptor (OSMR), interleukin 1 receptor type I (IL1R1), tumor necrosis factor receptor superfamily member 9 (TNFRSF9), C-X-C motif chemokine 13 (CXCL13), oncostatin M (OSM), and interleukin 6 (IL-6) in the cytokine-cytokine interaction pathways.The combination of ziyuglycoside II, syringin, and pedunculoside protects against UC by modulating the intestinal mucosal barrier and inhibiting the cytokine-cytokine interaction pathways, and the effect is relatively equivalent to that of the water extract of Ilex rotunda Thunb.Copyright © 2023. Published by Elsevier B.V.