School of Pharmaceutical Sciences, Guangzhou University of Chinese Medicine, Guangzhou, PR China.
The First Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou, PR China.
Phytomedicine. 2024 Jul;129:155571. doi: 10.1016/j.phymed.2024.155571. Epub 2024 Apr 6.
Repairing the intestinal mucosal barrier and reducing persistent inflammation is the key strategies for the treatment of ulcerative colitis (UC). Zhilining Formula (ZLN), composed of Andrographis herba (AH), Sophorae flavescentis radix (SFA), and Aucklandia radix (AR), is a well-tried formula for the clinical treatment of enteritis and dysentery in China, and its mechanism has not been clarified.
This study aims to investigate the effect of ZLN on UC and elucidate its underlying mechanism via metabolomics analysis and experimental verification.
The effect of ZLN on UC was evaluated in a 3.5 % dextran sulfate sodium (DSS)-induced mice model via the body weight, disease activity index (DAI), colon length, colonic histopathology, expression of inflammation factors, and intestinal barrier in mice. An UPLC-Q-TOF-MS/MS approach-based metabolomics analysis was performed to preliminary explore the mechanism of ZLN in colitis. Based on the results of metabolomics analysis, the expression of related protein or mRNA in AHR/NF-κBp65 axis was determined by qPCR and western blotting. Moreover, the potential interactions of active ingredients of ZLN with NF-κBp65 and AHR were investigated in vitro through using agonists and inhibitors of NF-κBp65 and AHR, respectively.
ZLN alleviated body weight loss and colonic shortening in colitis mice, and down-regulated the DAI and histopathological score as well. ZLN also decreased the levels of inflammatory factors (MPO, IL-1β, TNF-α and IL-18), protected goblet cell function and intestinal barrier in DSS-induced mice. Metabolomics results revealed that 36 metabolites that were significantly altered in mice after induction with DSS, which involved in 16 metabolic pathways, including biosynthesis of unsaturated fatty acid, phenylalanine metabolism, arachidonic acid (AA) metabolism, tryptophan (Trp) metabolism, retinol metabolism, and sphingolipid metabolism, etc. ZLN restored 26 different metabolites (DEMs) of them to normal-like levels, indicating ZLN regulated the AA metabolism and Trp-metabolism in UC mice, which hinted its potential pharmacological mechanism related to AHR/NF-κBp65 axis. We further confirmed that ZLN could restrain the activation of NF-κBp65 signaling pathway and then inhibit the expression of its mediated inflammatory cytokines, such as IL-1β, TNF-α, COX-2 and IL17A. Moreover, ZLN increased nuclear translocation of AHR and IL22 expression, which is an important regulatory signal for intestinal mucosal barrier repaired. Finally, we elucidated in vitro that the active ingredients of ZLN exerted anti-colitis effects by activating AHR and simultaneously inhibiting NF-κBp65.
ZLN relieved colitis by AHR/NF-κBp65 axis. This study highlighted the important role of AHR and NF-κBp65 in UC, and provided a theoretical basis for the application of ZLN.
修复肠道黏膜屏障和减轻持续性炎症是溃疡性结肠炎（UC）治疗的关键策略。智灵宁方（ZLN）由穿心莲（AH）、苦参（SFA）和广藿香（AR）组成，是中国治疗肠炎和痢疾的经验方，其机制尚未阐明。
本研究旨在通过代谢组学分析和实验验证，探讨 ZLN 对 UC 的作用及其潜在机制。
采用 3.5%葡聚糖硫酸钠（DSS）诱导的小鼠模型评价 ZLN 对 UC 的作用，通过小鼠的体重、疾病活动指数（DAI）、结肠长度、结肠组织病理学、炎症因子表达和肠道屏障来评估。采用 UPLC-Q-TOF-MS/MS 代谢组学分析方法初步探讨 ZLN 治疗结肠炎的机制。基于代谢组学分析的结果，通过 qPCR 和 Western blot 测定 AHR/NF-κBp65 轴相关蛋白或 mRNA 的表达。此外，通过使用 NF-κBp65 和 AHR 的激动剂和抑制剂，分别在体外研究 ZLN 活性成分与 NF-κBp65 和 AHR 的潜在相互作用。
ZLN 减轻了结肠炎小鼠的体重减轻和结肠缩短，并降低了 DAI 和组织病理学评分。ZLN 还降低了诱导型小鼠的炎症因子（MPO、IL-1β、TNF-α和 IL-18）水平，保护了杯状细胞功能和肠道屏障。代谢组学结果显示，36 种代谢物在 DSS 诱导后在小鼠体内发生显著变化，涉及 16 种代谢途径，包括不饱和脂肪酸的生物合成、苯丙氨酸代谢、花生四烯酸（AA）代谢、色氨酸（Trp）代谢、视黄醇代谢和鞘脂代谢等。ZLN 将其中 26 种不同的代谢物（DEMs）恢复到正常水平，表明 ZLN 调节了 UC 小鼠的 AA 代谢和 Trp 代谢，这暗示了其潜在的药理机制与 AHR/NF-κBp65 轴有关。我们进一步证实，ZLN 可以抑制 NF-κBp65 信号通路的激活，从而抑制其介导的炎症因子的表达，如 IL-1β、TNF-α、COX-2 和 IL17A。此外，ZLN 增加了 AHR 和 IL22 表达的核易位，这是肠道黏膜屏障修复的重要调节信号。最后，我们在体外阐明，ZLN 通过激活 AHR 并同时抑制 NF-κBp65 发挥抗结肠炎作用。
ZLN 通过 AHR/NF-κBp65 轴缓解结肠炎。本研究强调了 AHR 和 NF-κBp65 在 UC 中的重要作用，为 ZLN 的应用提供了理论依据。
