School of Pharmaceutical Sciences, Guangzhou University of Chinese Medicine, Guangzhou 510006, PR China; School of Basic Medical Sciences, Guangzhou University of Chinese Medicine, Guangzhou 510006, PR China.
School of Pharmaceutical Sciences, Guangzhou University of Chinese Medicine, Guangzhou 510006, PR China.
Phytomedicine. 2021 Feb;82:153438. doi: 10.1016/j.phymed.2020.153438. Epub 2020 Dec 9.
5-Hydroxy-4-methoxycanthin-6-one (PQ-A) is the main active compound in Ramulus et Folium Picrasmae, a Chinese herbal medicine commonly used in colitis treatment.
To clarify PQ-A's role and mechanism in colitis treatment based on a non-targeted metabolomics study.
Rats with ulcerative colitis (UC) established with 4% dextran sulfate sodium (DSS) were orally treated with PQ-A. Body weight, disease activity index (DAI), colon length, biochemical parameters (MDA and SOD), and histopathological score in colon tissue were measured. A UPLC-Q-TOF-MS/MS approach-based metabolomics analysis was conducted to explore the underlying mechanisms of PQ-A in colitis treatment. Inflammatory cytokines (TNF-α, IL-1β, IL-6, and IL-10) concentrations in serum and their protein levels in the colon were determined. CD3 and NF-κB/p65 immunohistochemistry in the colon was semi-quantified. The related protein or mRNA in IKK-NF-κB/p65 signaling pathway was measured by Western blotting or RT-PCR, respectively. Potential molecular interactions between PQ-A and NF-κB/p65 was predicted using DS 2.5 software.
PQ-A significantly prevented body weight loss and colonic shortening in colitic rats, and reduced the DAI and histopathologic score as well. PQ-A decreased MDA levels in the UC rat serum and increased those of SOD. Metabolomics results revealed forty-nine differential metabolites as biomarkers of DSS-induced colitis, demonstrating that the path-mechanism of colitis involved the perturbation of eight metabolic pathways, including alpha-linolenic acid and linoleic acid metabolism, sphingolipid metabolism, retinol metabolism, bile acid metabolism, et al. Thirty-six biomarkers were especially reversed to normal-like levels by PQ-A via regulation of alpha-linolenic acid and linoleic acid metabolism, sphingolipid metabolism, and retinol metabolism, which effectively hinted the potential pharmacological mechanism of PQ-A related to NF-κB/p65 inflammatory signaling. Molecular docking results predicted high affinity interaction between PQ-A and NF-κB/p65, involving hydrogen-bond interactions at five amino acid residues, suggesting NF-κB/p65 as a target. PQ-A decreased TNF-α, IL-1β, and IL-6 concentrations in serum and their protein levels in colon tissue in colitic rats. CD3, MYD88, p-IκBα, NF-κB/p65, and p-NF-κB/p65 expression levels decreased, whereas those of IKKβ and IκBα increased in colitic tissue following PQ-A treatment. PQ-A strongly inhibited nuclear translocation of NF-κB/p65.
We provide an overview of PQ-A's possible mechanism of action in colitis treatment based on serum non-targeted metabolomics. PQ-A treatment can protect rats against DSS-induced colitis by suppressing the NF-κB/p65 signaling pathway.
5-羟基-4-甲氧基卡亭-6-酮（PQ-A）是一种中药千里光的主要活性成分，常用于结肠炎的治疗。
通过非靶向代谢组学研究阐明 PQ-A 在结肠炎治疗中的作用和机制。
采用 4%葡聚糖硫酸钠（DSS）诱导大鼠溃疡性结肠炎（UC）模型，给予 PQ-A 灌胃治疗。测量大鼠体重、疾病活动指数（DAI）、结肠长度、生化参数（MDA 和 SOD）以及结肠组织的组织病理学评分。采用 UPLC-Q-TOF-MS/MS 代谢组学分析方法探讨 PQ-A 治疗结肠炎的潜在机制。测定血清中炎性细胞因子（TNF-α、IL-1β、IL-6 和 IL-10）浓度及其在结肠组织中的蛋白水平。采用 CD3 和 NF-κB/p65 免疫组化半定量分析。采用 Western blot 或 RT-PCR 分别测定 IKK-NF-κB/p65 信号通路中的相关蛋白或 mRNA。利用 DS 2.5 软件预测 PQ-A 与 NF-κB/p65 的潜在分子相互作用。
PQ-A 可显著预防结肠炎大鼠体重下降和结肠缩短，并降低 DAI 和组织病理学评分。PQ-A 降低 UC 大鼠血清中 MDA 水平，增加 SOD 水平。代谢组学结果显示，49 种差异代谢物可作为 DSS 诱导的结肠炎生物标志物，表明结肠炎的发病机制涉及到包括α-亚麻酸和亚油酸代谢、鞘脂代谢、视黄醇代谢、胆汁酸代谢等在内的 8 种代谢途径的紊乱。PQ-A 通过调节α-亚麻酸和亚油酸代谢、鞘脂代谢和视黄醇代谢，使 36 种生物标志物恢复正常水平，这有效提示了 PQ-A 与 NF-κB/p65 炎症信号相关的潜在药理学机制。分子对接结果预测 PQ-A 与 NF-κB/p65 具有高亲和力相互作用，涉及五个氨基酸残基的氢键相互作用，表明 NF-κB/p65 可能是 PQ-A 的作用靶点。PQ-A 降低结肠炎大鼠血清中 TNF-α、IL-1β 和 IL-6 浓度及其在结肠组织中的蛋白水平。PQ-A 治疗后，结肠炎组织中 CD3、MYD88、p-IκBα、NF-κB/p65 和 p-NF-κB/p65 表达水平降低，而 IKKβ 和 IκBα 表达水平升高。PQ-A 强烈抑制 NF-κB/p65 的核转位。
本研究基于血清非靶向代谢组学，为 PQ-A 在结肠炎治疗中的作用机制提供了新的认识。PQ-A 通过抑制 NF-κB/p65 信号通路，对 DSS 诱导的结肠炎大鼠具有保护作用。
