Abstract：
BACKGROUND: 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.
OBJECTIVE: 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.
METHODS: Healthy and HCC rats were treated with or without 1,4-GL (40.0 mg/kg) and 1HNMR-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.
RESULTS: 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.
CONCLUSIONS: 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.
OBJECTIVE: 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.
METHODS: Healthy and HCC rats were treated with or without 1,4-GL (40.0 mg/kg) and 1HNMR-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.
RESULTS: 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.
CONCLUSIONS: 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.
摘要：
背景：六味地黄丸是一种具有多种抗癌特性的著名中药。中国有50多家制药商生产六味地黄丸，全世界每天都有数百万人口服。六味地黄丸中的D-glucaro-1,4-内酯（1,4-GL）定量为约12.0mg/g，是其主要生物活性成分，可在体内抑制β-葡萄糖醛酸苷酶的活性。1,4-GL可以预防和有效抑制各种类型的癌症。然而,其确切的作用机制仍然未知。这项研究将证明六味地黄丸对癌症的传统用法是合理的。
目标：1,4-GL，来自六味地黄丸的生物活性成分，著名的中药，可以延缓二乙基亚硝胺（DEN）诱导的大鼠肝细胞癌（HCC）的进展。支撑这种效果的机制，然而,仍然知之甚少。
方法：用或不用1,4-GL（40.0mg/kg）治疗健康和HCC大鼠，并采用基于1HNMR的代谢组学分析。通过UPLC-MS/MS定量测定尿酸途径中的10种代谢产物。黄嘌呤脱氢酶(XDH)的表达,SLC2A9mRNA,使用RT-qPCR和Western印迹测定SLC2A9蛋白。体外验证了1,4-GL对HCC-LM3细胞的作用。ROS活性的改变，在1,4-GL处理后脂多糖（LPS）诱导的NCTC-1469细胞中观察到SLC2A9和XDH基因水平。
结果：1,4-GL干预后，改善病理形态学，肝癌大鼠肝脏病变观察到血清AFP水平恢复，AST,ALP,γ-GGT和费希尔比。肝脏代谢组学显示，肝癌大鼠的1,4-GL可显著调节肝脏代谢。尿酸,通过UPLC-MS/MS定量黄嘌呤和次黄嘌呤水平，发现在HCC大鼠中1,4-GL治疗后几乎恢复到对照水平。黄嘌呤氧化酶活性的变化，XDHmRNA表达，SLC2A9mRNA和蛋白表达也发生逆转。LM3HCC细胞中的1,4-GL处理与体内结果一致。此外，氧化应激指标，如T-SOD，GSH,用1,4-GL处理的HCC大鼠血清和肝脏中的CAT和MDA均得到改善。体外，观察到1,4-GL降低了NCTC-1469细胞中脂多糖诱导的ROS水平，增强了SLC2A9的mRNA和蛋白表达，并降低了XDH的mRNA水平。
结论：由于尿酸产生下调和SLC2A9表达上调，1,4-GL通过降低尿酸和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。
目标：1,4-GL，来自六味地黄丸的生物活性成分，著名的中药，可以延缓二乙基亚硝胺（DEN）诱导的大鼠肝细胞癌（HCC）的进展。支撑这种效果的机制，然而,仍然知之甚少。
方法：用或不用1,4-GL（40.0mg/kg）治疗健康和HCC大鼠，并采用基于1HNMR的代谢组学分析。通过UPLC-MS/MS定量测定尿酸途径中的10种代谢产物。黄嘌呤脱氢酶(XDH)的表达,SLC2A9mRNA,使用RT-qPCR和Western印迹测定SLC2A9蛋白。体外验证了1,4-GL对HCC-LM3细胞的作用。ROS活性的改变，在1,4-GL处理后脂多糖（LPS）诱导的NCTC-1469细胞中观察到SLC2A9和XDH基因水平。
结果：1,4-GL干预后，改善病理形态学，肝癌大鼠肝脏病变观察到血清AFP水平恢复，AST,ALP,γ-GGT和费希尔比。肝脏代谢组学显示，肝癌大鼠的1,4-GL可显著调节肝脏代谢。尿酸,通过UPLC-MS/MS定量黄嘌呤和次黄嘌呤水平，发现在HCC大鼠中1,4-GL治疗后几乎恢复到对照水平。黄嘌呤氧化酶活性的变化，XDHmRNA表达，SLC2A9mRNA和蛋白表达也发生逆转。LM3HCC细胞中的1,4-GL处理与体内结果一致。此外，氧化应激指标，如T-SOD，GSH,用1,4-GL处理的HCC大鼠血清和肝脏中的CAT和MDA均得到改善。体外，观察到1,4-GL降低了NCTC-1469细胞中脂多糖诱导的ROS水平，增强了SLC2A9的mRNA和蛋白表达，并降低了XDH的mRNA水平。
结论：由于尿酸产生下调和SLC2A9表达上调，1,4-GL通过降低尿酸和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。
