Medical College of Jiaxing University, Key Laboratory of Medical Electronics and Digital Health of Zhejiang Province, Jiaxing University, Jiaxing, China; School of Pharmaceutical Sciences, Zhejiang Chinese Medical University, Hangzhou, China.
School of Pharmacy, Jiangxi Medical College, Nanchang University, Nanchang, China.
J Ethnopharmacol. 2024 Nov 15;334:118569. doi: 10.1016/j.jep.2024.118569. Epub 2024 Jul 10.
Liuwei dihuang pills is a famous Traditional Chinese Medicine with various anti-cancer properties. Over 50 pharmaceutical manufacturers produce Liuwei dihuang pills in China and an estimated millions of people around the world orally take it every day. D-glucaro-1,4-lactone (1,4-GL) was quantified to be about 12.0 mg/g in Liuwei dihuang pills and a primary bioactive component of it inhibiting the activity of β-glucuronidase in vivo. 1,4-GL can prevent and effectively inhibit various types of cancer. However, its exact mechanism of action remains unknown. The study would justify the traditional usage of Liuwei dihuang pills against cancers.
1,4-GL, a bioactive ingredient derived from Liuwei dihuang pills, a famous Traditional Chinese Medicine, could delay the progression of diethylnitrosamine (DEN)-induced hepatocellular carcinoma (HCC) in rats. The mechanism underpinning the effect, however, remains poorly understood.
Healthy and HCC rats were treated with or without 1,4-GL (40.0 mg/kg) and HNMR-based metabonomic analysis was employed. 10 metabolites in uric acid pathway were quantitatively determined by UPLC-MS/MS. The expression of xanthine dehydrogenase (XDH), SLC2A9 mRNA, and SLC2A9 protein was determined using RT-qPCR and Western Blot. The effect of 1,4-GL on HCC-LM3 cells was verified in vitro. The alterations of ROS activity, SLC2A9 and XDH gene levels were observed in NCTC-1469 cells induced by lipopolysaccharide (LPS) after 1,4-GL treatment.
After the intervention of 1,4-GL, improved pathological morphology, liver lesions in HCC rats was observed with restored serum levels of AFP, AST, ALP, γ-GGT and Fisher's ratio. Hepatic metabonomics revealed that puring metabolism were significantly regulated by 1,4-GL in HCC rats. Uric acid, xanthine and hypoxanthine levels were quantified by UPLC-MS/MS and found to be nearly restored to control levels after 1,4-GL treatment in HCC rats. Changes in xanthine oxidase activity, XDH mRNA expression, and SLC2A9 mRNA and protein expression were also reversed. 1,4-GL treatment in LM3 HCC cells were consistent with the results in vivo. Furthermore, oxidative stress indicators such as T-SOD, GSH, CAT and MDA in serum and liver were improved after HCC rats treated with 1,4-GL. In vitro, 1,4-GL was observed to reduce lipopolysaccharide-induced ROS levels in NCTC-1469 cells with enhanced mRNA and protein expression of SLC2A9 and decreased mRNA level of XDH.
The protective effects of 1,4-GL against DEN-induced HCC by reducing uric acid and ROS levels due to down-regulation of uric acid production and up-regulation of SLC2A9 expressions. 1,4-GL may represent a novel treatment that improves recovery from HCC by targeting uric acid-ROS pathway.
伦理相关性：六味地黄丸是一种著名的中药，具有多种抗癌特性。中国有超过 50 家制药商生产六味地黄丸，估计全球有数百万人每天口服这种药物。研究发现，六味地黄丸中约含有 12.0mg/g 的 D-葡萄糖酸-1,4-内酯（1,4-GL），这是其体内抑制β-葡萄糖醛酸酶活性的主要生物活性成分之一。1,4-GL 可以预防和有效抑制各种类型的癌症。然而，其确切的作用机制尚不清楚。本研究旨在证明六味地黄丸在抗癌方面的传统应用。
研究目的：1,4-GL 是一种源自著名中药六味地黄丸的生物活性成分，可延缓二乙基亚硝胺（DEN）诱导的大鼠肝细胞癌（HCC）的进展。然而，其作用的潜在机制仍知之甚少。
材料和方法：健康和 HCC 大鼠分别用或不用 1,4-GL（40.0mg/kg）处理，采用基于 HNMR 的代谢组学分析。通过 UPLC-MS/MS 定量测定尿酸途径中的 10 种代谢物。使用 RT-qPCR 和 Western Blot 测定黄嘌呤脱氢酶（XDH）、SLC2A9 mRNA 和 SLC2A9 蛋白的表达。在体外验证 1,4-GL 对 HCC-LM3 细胞的作用。用 LPS 诱导 NCTC-1469 细胞后，观察 1,4-GL 对 ROS 活性、SLC2A9 和 XDH 基因水平的影响。
结果：干预后，HCC 大鼠的病理形态得到改善，肝损伤得到修复，血清 AFP、AST、ALP、γ-GGT 和 Fisher 比值恢复正常。肝代谢组学显示，1,4-GL 显著调节 HCC 大鼠的嘌呤代谢。通过 UPLC-MS/MS 定量测定尿酸、黄嘌呤和次黄嘌呤水平，发现 HCC 大鼠经 1,4-GL 治疗后，这些水平几乎恢复到对照水平。黄嘌呤氧化酶活性、XDH mRNA 表达以及 SLC2A9 mRNA 和蛋白表达的变化也得到了逆转。LM3 HCC 细胞的 1,4-GL 处理结果与体内结果一致。此外，HCC 大鼠经 1,4-GL 治疗后，血清和肝脏中的氧化应激指标如 T-SOD、GSH、CAT 和 MDA 得到改善。在体外，观察到 1,4-GL 降低 LPS 诱导的 NCTC-1469 细胞中的 ROS 水平，同时增强 SLC2A9 的 mRNA 和蛋白表达，并降低 XDH 的 mRNA 水平。
结论：1,4-GL 通过降低尿酸水平和降低尿酸生成、上调 SLC2A9 表达来减少活性氧（ROS）水平，对 DEN 诱导的 HCC 具有保护作用。1,4-GL 可能代表一种新的治疗方法，通过靶向尿酸-ROS 途径改善 HCC 的恢复。
