Department of Gastroenterology, State Key Laboratory of Organ Failure Research, Guangdong Provincial Key Laboratory of Gastroenterology, Nanfang Hospital, Southern Medical University, Guangzhou, PR China.
Integrative Microecology Clinical Center, Shenzhen Key Laboratory of Gastrointestinal Microbiota and Disease, Shenzhen Clinical Research Center for Digestive Disease, Shenzhen Technology Research Center of Gut Microbiota Transplantation, Shenzhen Hospital, Southern Medical University, Shenzhen, PR China.
Phytomedicine. 2024 Jun;128:155425. doi: 10.1016/j.phymed.2024.155425. Epub 2024 Feb 7.
Intestinal barrier dysfunction caused by the disrupted balance of group 3 innate lymphoid cells (ILC3)/group 1 innate lymphoid cells (ILC1) is a significant feature in the pathogenesis of inflammatory bowel disease (IBD). Activation of aryl hydrocarbon receptor (AhR) signaling contributes to the maintenance of ILC3/ILC1 balance. Wogonin, a natural flavonoid from Scutellaria baicalensis Georgi, can repair intestinal mucosal damage of IBD. However, it remains unclear if wogonin can exert a therapeutic effect by activating the AhR pathway to regulate the plasticity of ILC3/ILC1.
In this study, we investigated the immunomodulatory effects of wogonin on IBD and its potential mechanisms in vitro and in vivo.
Chronic colitis was induced by four cycles of 2 % DSS treatment in mice. 20 mg kg/day wogonin was administrated by oral gavage and mice were treated intraperitoneally with 10 mg kg/2 days CH223191 to block the AhR pathway. Colon tissues were processed for histopathological examination and evaluation of the epithelial barrier function by immunohistochemistry. The activation of the AhR pathway and the plasticity of ILC3/ILC1 were determined by western blot and flow cytometry. Then, we also detected the intestinal microflora and their metabolites by 16 s sequencing and non-targeted Metabolomics analysis. Furthermore, an in vitro culture system consisting of MNK3 cells and NCM460 cells, and a CETSA assay were performed to confirm the molecular mechanism.
Wogonin ameliorated histological severity of the colon, decreased the secretion of inflammatory factors, and increased tight junction proteins in colitis mice. These effects are associated with the tendency of conversion from ILC3 to ILC1 prevented by wogonin, which was offset by AhR antagonist CH223191. In addition, wogonin exerted the curative effect by altering gut microbiota to produce metabolites such as Kynurenic acid, and 1H-Indole-3-carboxaldehyde as AhR endogenous ligands. In vitro data further verified that wogonin as an exogenous ligand directly binds to the structural domain of AhR by CETSA. Also, the supernatant of MNK-3 cells stimulated with wogonin enhanced expression of Occludin and Claudin1 in NCM460 cells induced by LPS.
Cumulatively, our study illustrated that wogonin improved the outcomes of DSS-induced chronic colitis via regulating the plasticity of ILC3/ILC1. Its specific mechanism is to binding to AhR directly, and to activate the AhR pathway indirectly by altering the tryptophan metabolisms of gut microbiota.
由 3 组固有淋巴细胞 (ILC3)/1 组固有淋巴细胞 (ILC1) 失衡引起的肠道屏障功能障碍是炎症性肠病 (IBD) 发病机制中的一个重要特征。芳香烃受体 (AhR) 信号的激活有助于维持 ILC3/ILC1 的平衡。来自黄芩的天然黄酮类化合物汉黄芩素可修复 IBD 的肠道黏膜损伤。然而，尚不清楚汉黄芩素是否通过激活 AhR 通路来调节 ILC3/ILC1 的可塑性，从而发挥治疗作用。
本研究旨在探讨汉黄芩素在体外和体内对 IBD 的免疫调节作用及其潜在机制。
通过给予 2% DSS 治疗进行 4 个周期的慢性结肠炎诱导小鼠，每天经口给予 20mg/kg 的汉黄芩素，并经腹腔给予 10mg/kg/2 天的 CH223191 以阻断 AhR 通路。通过免疫组织化学法检测结肠组织的组织病理学检查和上皮屏障功能评估。通过 Western blot 和流式细胞术检测 AhR 通路的激活和 ILC3/ILC1 的可塑性。然后，我们还通过 16s 测序和非靶向代谢组学分析检测肠道微生物群及其代谢产物。此外，还进行了 MNK3 细胞和 NCM460 细胞体外培养系统和 CETSA 测定，以证实分子机制。
汉黄芩素改善了结肠炎小鼠结肠的组织学严重程度，降低了炎症因子的分泌，并增加了紧密连接蛋白。这些作用与汉黄芩素阻止 ILC3 向 ILC1 转化的趋势有关，而 AhR 拮抗剂 CH223191 则抵消了这种作用。此外，汉黄芩素通过改变肠道微生物群产生犬尿氨酸和 1H-吲哚-3-甲醛等 AhR 内源性配体来发挥治疗作用。体外数据进一步证实，汉黄芩素作为外源性配体通过 CETSA 直接与 AhR 的结构域结合。此外，用汉黄芩素刺激 MNK-3 细胞后的上清液增强了 LPS 诱导的 NCM460 细胞中 Occludin 和 Claudin1 的表达。
总之，本研究表明，汉黄芩素通过调节 ILC3/ILC1 的可塑性改善了 DSS 诱导的慢性结肠炎的结局。其具体机制是通过直接与 AhR 结合，并通过改变肠道微生物群的色氨酸代谢间接激活 AhR 通路。
