raw date Dahuang Gancao Dandelion Decoction regulates intestinal flora and inhibits NF-κB / ARA signaling pathway to alleviate ulcerative colitis

Objective: The aim of this study was to assess the therapeutic effects and underlying mechanisms of Dahuang Gancao Dandelion Decoction (DGD-D) on dextran sulfate sodium (DSS)-induced ulcerative colitis (UC) in a mice model, with an emphasis on modulation of gut microbiota and intestinal metabolites, maintenance of intestinal barrier integrity, and inhibition of inflammatory signaling pathways. Methods: Ultra-high-performance liquid chromatography with quadrupole electrospray ionization mass spectrometry (UHPLC-QE-MS) was used to examine the DGD-D, which was prepared from 40 g rhubarb, 10 g licorice, and 10 g dandelion. Potential routes and targets were found using network pharmacology. Six male C57BL/6 mice per group were randomized into control, DSS, DGD-D, and mesalazine (5-ASA) groups. UC was induced with 3% DSS for 7 days, with DGD-D administered prophylactically at 1950 mg/kg. Evaluated parameters included colon length, spleen index, and DAI, cytokine levels (ELISA, qRT-PCR), histological changes (H&E, PAS, AB-PAS), barrier proteins (IF, IHC, qRT-PCR), gut microbiota (16S rRNA sequencing), metabolites (LC-MS) and pathway validation. Results: UHPLC-QE-MS identified 418 components in DGD-D, including flavonoids (25.12%) and phenolic acids (9.33%). Network pharmacology highlighted NF-κB signaling as a key pathway. In comparison to DSS, DGD-D dramatically decreased spleen index, restored colon length, and decreased DAI scores (P < 0.05). It increased anti-inflammatory IL-4 and IL-10 while downregulating pro-inflammatory cytokines (IL-1β, IL-6, IL-17, TNF-α, and IFN-γ) and MPO (P < 0.01). Histologically, DGD-D attenuated intestinal epithelial damage, increased goblet cells and glycoproteins, and enhanced ZO-1, Occludin, and MUC2 expression (P < 0.01). Microbiota analysis showed increased α-diversity, elevated Firmicutes/Bacteroidetes ratio, enriched beneficial Lachnospiraceae and Ruminococcaceae, and reduced Proteobacteria and Alcaligenaceae (P < 0.001). Metabolomics revealed downregulation of arachidonic acid (ARA) pathway mediators (ARA, LTA4, LTB4, LTD4; P < 0.001). DGD-D inhibited TLR4/MyD88/NF-κB p65/NLRP3/5-LOX expression (P < 0.01 or P < 0.001), suppressing NF-κB/ARA signaling. Conclusion: DGD-D is a viable TCM-based treatment for UC since it improves DSS-induced UC by modifying gut microbiota and intestinal metabolites, reestablishing intestinal barrier function, and blocking the NF-κB/ARA pathway.

研究目的：本研究旨在评估大黄甘草蒲公英汤（Dahuang Gancao Dandelion Decoction, DGD-D）对硫酸葡聚糖钠（dextran sulfate sodium, DSS）诱导的小鼠溃疡性结肠炎（ulcerative colitis, UC）的治疗效果及其潜在作用机制，重点关注其对肠道菌群与肠道代谢物的调控、肠屏障完整性的维持，以及炎症信号通路的抑制作用。
实验方法：采用四极杆电喷雾电离质谱联用超高效液相色谱法（ultra-high-performance liquid chromatography with quadrupole electrospray ionization mass spectrometry, UHPLC-QE-MS）对由40g大黄、10g甘草及10g蒲公英制备的DGD-D进行成分表征。通过网络药理学预测其潜在作用通路与靶点。将6只雄性C57BL/6小鼠随机分为对照组、DSS模型组、DGD-D干预组及美沙拉嗪（5-ASA）组。采用3% DSS饮用7天以诱导UC模型，同时以1950 mg/kg剂量预防性给予DGD-D。检测指标包括结肠长度、脾脏指数、疾病活动指数（disease activity index, DAI）、细胞因子水平（酶联免疫吸附试验（enzyme-linked immunosorbent assay, ELISA）、实时荧光定量聚合酶链反应（quantitative real-time polymerase chain reaction, qRT-PCR））、组织病理学变化（苏木精-伊红染色（hematoxylin-eosin staining, H&E）、过碘酸-希夫染色（periodic acid-Schiff staining, PAS）、阿利新蓝-过碘酸-希夫染色（alcian blue-periodic acid-Schiff staining, AB-PAS））、屏障相关蛋白（免疫荧光（immunofluorescence, IF）、免疫组化（immunohistochemistry, IHC）、qRT-PCR）、肠道菌群（16S rRNA测序）、肠道代谢物（液相色谱-质谱联用（liquid chromatography-mass spectrometry, LC-MS））及信号通路验证。
实验结果：UHPLC-QE-MS在DGD-D中鉴定出418种成分，其中黄酮类占比25.12%、酚酸类占比9.33%。网络药理学分析显示核因子-κB（nuclear factor-κB, NF-κB）信号通路为核心调控通路。与DSS模型组相比，DGD-D可显著降低脾脏指数、恢复结肠长度并降低DAI评分（P < 0.05）。其可上调抗炎细胞因子IL-4与IL-10的表达，同时下调促炎细胞因子IL-1β、IL-6、IL-17、TNF-α、IFN-γ及髓过氧化物酶（myeloperoxidase, MPO）的水平（P < 0.01）。组织病理学观察显示，DGD-D可减轻肠上皮损伤，增加杯状细胞与糖蛋白含量，并上调紧密连接蛋白ZO-1、Occludin及黏蛋白MUC2的表达（P < 0.01）。肠道菌群分析表明，DGD-D可提高菌群α多样性、升高厚壁菌门（Firmicutes）/拟杆菌门（Bacteroidetes）比值，富集有益菌毛螺菌科（Lachnospiraceae）与瘤胃球菌科（Ruminococcaceae），减少变形菌门（Proteobacteria）与产碱杆菌科（Alcaligenaceae）的相对丰度（P < 0.001）。代谢组学分析显示，花生四烯酸（arachidonic acid, ARA）通路相关介质（ARA、LTA4、LTB4、LTD4）的表达显著下调（P < 0.001）。DGD-D可抑制Toll样受体4（Toll-like receptor 4, TLR4）、髓系分化因子88（myeloid differentiation primary response 88, MyD88）、NF-κB p65、核苷酸结合寡聚化结构域样受体蛋白3（NOD-like receptor family pyrin domain containing 3, NLRP3）及5-脂氧合酶（5-lipoxygenase, 5-LOX）的蛋白及基因表达（P < 0.01或P < 0.001），从而阻断NF-κB/ARA信号通路。
研究结论：DGD-D是一种极具潜力的中医药（traditional Chinese medicine, TCM）治疗UC的候选方案，其可通过调控肠道菌群与肠道代谢物、重建肠屏障功能、阻断NF-κB/ARA信号通路，从而改善DSS诱导的小鼠溃疡性结肠炎。