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.
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。