背景:慢性炎症是肿瘤发生的致病因素之一。天麻素是从天麻中分离出的主要活性成分,具有悠久食用历史的著名药草。
目的:本研究旨在探讨天麻素对小鼠结肠炎相关癌变(CRC)的影响,并阐明其潜在的分子机制。
方法:用偶氮甲烷(AOM)和葡聚糖硫酸钠(DSS)诱导Balb/c小鼠12周。天麻素(50mg/kg)通过口服管饲法每周施用三次,直至实验结束。疾病指数,包括体重,血性腹泻,结肠长度,组织病理学评分,和肿瘤大小,被测量。通过BrdU掺入测定评估肿瘤细胞增殖,并通过细胞计数试剂盒(CCK-8)评估肿瘤细胞的细胞毒性。Toll样受体4(TLR4)/核因子κB(NF-κB)信号分子的表达水平,NF-κB荧光素酶,实时荧光定量聚合酶链反应(RT-qPCR)检测促炎细胞因子,免疫印迹,免疫组织化学(IHC),酶联免疫吸附测定(ELISA),或报告基因测定。通过分子对接和细胞热转移测定(CETSA)分析天麻素与骨髓分化蛋白2(MD2)之间的结合亲和力。
结果:天麻素被证明可以减轻小鼠的各种CRC相关症状,包括减肥,腹泻,和组织异常。值得注意的是,天麻素抑制了结肠炎相关肿瘤发生过程中的肿瘤细胞生长,导致结肠腺瘤减少和减少。不像伊立替康,广谱抗肿瘤药物,天麻素在各种结直肠腺癌细胞系中均未表现出明显的细胞毒性。此外,天麻素下调小鼠和巨噬细胞中的TLR4/NF-κB信号分子和促炎介质。分子对接和CETSA实验表明天麻素与MD2蛋白结合,可能干扰TLR4对脂多糖(LPS)的识别,导致NF-κB途径抑制。
结论:这项研究首次提供了证据,证明天麻素可以减轻小鼠结肠炎并预防与结肠炎相关的癌变,至少部分地,通过中断TLR4/MD2/NF-κB信号转导来减少促进肿瘤的细胞因子。
BACKGROUND: Chronic inflammation is one of the causative factors for tumorigenesis. Gastrodin is a main active ingredient isolated from Gastrodia elata Blume, a famous medicinal herb with a long edible history.
OBJECTIVE: This study aimed to explore the effects of gastrodin on colitis-associated carcinogenesis (CRC) in mice and to elucidate its potential molecular mechanisms.
METHODS: Balb/c mice were induced with azoxymethane (AOM) and dextran sulfate sodium (DSS) for 12 weeks. Gastrodin (50 mg/kg) was administered via oral gavage three times per week until the end of the experiment. Disease indexes, including body weight, bloody diarrhea, colon length, histopathological score, and tumor size, were measured. Tumor cell proliferation was evaluated by BrdU incorporation assay and tumor cell cytotoxicity was assessed by cell counting kit (CCK-8). The expression levels of toll-like receptor 4 (TLR4)/nuclear factor kappa-B (NF-κB) signaling molecules, NF-κB luciferase, and pro-inflammatory cytokines were determined by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR), immunoblotting, immunohistochemistry (IHC), enzyme-linked immunosorbent assay (ELISA), or reporter gene assays. The binding affinity between gastrodin and myeloid differentiation protein-2 (MD2) was analyzed by molecular docking and cellular thermal shift assay (CETSA).
RESULTS: Gastrodin administration was demonstrated to mitigate various CRC-related symptoms in mice, including weight loss, diarrhea, and tissue abnormalities. Notably, gastrodin suppressed tumor cell growth during colitis- associated tumorigenesis, resulting in fewer and smaller adenomas in the colon. Unlike irinotecan, a broadspectrum antitumor drug, gastrodin did not exhibit apparent cytotoxicity in various colorectal adenocarcinoma cell lines. Additionally, gastrodin downregulated TLR4/NF-κB signaling molecules and pro-inflammatory mediators in mice and macrophages. Molecular docking and CETSA experiments suggested that gastrodin binds to the MD2 protein, potentially interfering with the recognition of lipopolysaccharide (LPS) by TLR4, leading to NF-κB pathway inhibition.
CONCLUSIONS: This study provides evidence for the first time that gastrodin attenuated colitis and prevented colitisrelated carcinogenesis in mice, at least partially, by diminishing tumor-promoting cytokines through the interruption of TLR4/MD2/NF-κB signaling transduction.