Interplay between metabolism and FXR activation in scoparone signaling

天冬Scoparone是香豆素衍生物，在结构上属于多酚类化合物超家族。这种化合物在阴琴（蒿属）中含量丰富，阴琴是一种数千年来用于治疗肝脏和肾脏疾病的中草药。阴金制剂最近在世界范围内受到广泛关注，并在北美作为保健品销售。天冬Scoparone被认为是一种主要的肝脏保护化合物，并已被证明可以诱导小鼠尿苷二磷酸-5¿-葡萄糖醛酸基转移酶1a1 (UGT1A1)，一种胆红素解毒酶。我们最近努力测试scoparone是否也能诱导人类UGT1A1。人原代肝细胞用scoparone或与鹅去氧胆酸（CDCA）一起处理，CDCA是farnesoid X受体（FXR）的激活剂。令我们惊讶的是，scoparone没有诱导人UGT1A1，而是显著增强CDCA诱导胆汁盐输出泵（BSEP）。这种胆道转运蛋白是CDCA-FXR信号的靶标，在胆汁酸的分泌中起重要作用。与BSEP诱导的增强作用一致，scoparone增强了激活FXR的CDCA，而在FXR磷酸化缺陷突变体中，这种增强作用大大减弱。相比之下，转染细胞色素P450 1A2 (CYP1A2)（一种药物代谢酶）的细胞中，这种增强显著增强。该项目的中心假设是，天scopone代谢物通过有效调节FXR磷酸化，赋予有效的肝脏保护，防止胆汁淤积。具体目的是：(1)确定BSEP诱导的增强与天scoparone代谢的关系；(2)考察天scoparone对FXR的磷酸化作用；(3)明确FXR和scoparone代谢在抗胆汁淤积中的相互作用。为了确定天scopone在一般人群中的代谢情况，将对大量个体肝脏样本进行天scopone代谢检测。主要代谢物将在结构上确定并测试BSEP诱导的增强作用。为了检验天scopone是否是FXR磷酸化的调节剂，我们将肝细胞单独或加CDCA处理，并分析FXR的磷酸化状态。为了确定天scopone的抗淤胆潜能，我们对小鼠进行了含或不含天scopone的淤胆诱导，并监测了天scopone的淤胆标志物和代谢。综上所述，本研究将确立天scoparone的代谢途径及其代谢在胆汁酸消除中的意义。胆汁淤积是最常见的肝毒性，而富含天冬scoparone的中药阴饮长期以来一直被用于使肝功能正常化。这些研究将为肝保护作用如何实现提供重要信息。此外，FXR被认为是一种重要的代谢调节剂，具有抗炎作用。有趣的是，scoparone已经被发现发挥这些活动。因此，确认FXR和scoparone之间的功能联系将具有广泛的机制和治疗意义。

Scoparone is a coumarin derivative and structurally belongs to the superfamily of polyphenolic compounds. This compound is abundant in Yin Chin (artemisiae scopariae), a Chinese herb that has been used for thousands of years to treat liver and kidney diseases. Yin Chin preparations have received much attention worldwide lately and are marketed as health supplements in North America. Scoparone is recognized as a major hepatic protective compound and has been shown to induce mouse uridine diphosphate-5¿-glucuronosyltransferase-1A1 (UGT1A1), a bilirubin detoxification enzyme. We recently made an effort to test whether scoparone also induces human UGT1A1. Human primary hepatocytes were treated with scoparone or along with chenodeoxycholic acid (CDCA), an activator of the farnesoid X receptor (FXR). To our surprise, scoparone did not induce human UGT1A1, but instead significantly potentiated CDCA in inducing the bile salt export pump (BSEP). This biliary transporter is a target of CDCA-FXR signaling and plays an essential role in the secretion of bile acids. Consistent with the potentiation of BSEP induction, scoparone enhanced CDCA in activating FXR and the enhancement was much reduced with FXR phosphorylation-deficient mutants. In contrast, the enhancement was significantly increased in cells transfected with cytochrome P450 1A2 (CYP1A2), a drug-metabolizing enzyme. The central hypothesis of this project is that scoparone metabolites confer potent hepatic protection against cholestasis by efficaciously modulating FXR phosphorylation. The specific aims are: (1) to determine the potentiation of BSEP induction as a function of scoparone metabolism; (2) to characterize the phosphorylation of FXR by scoparone; and (3) to define the role of the FXR and scoparone metabolism interplay in anti-cholestasis. To determine the metabolism of scoparone in general population, a large number of individual liver samples will be tested for the metabolism of scoparone. The major metabolites will be structurally determined and tested for the potentiation of BSEP induction. To test whether scoparone is a modulator of the phosphorylation of FXR, hepatocytes will be treated with scoparone alone or plus CDCA, and the phosphorylation status of FXR will be analyzed. To ascertain the anti-cholestatic potential of scoparone, mice will be subjected to inducing cholestasis with or without scoparone, and cholestatic markers and the metabolism of scoparone will be monitored. Overall, the proposed studies will establish the metabolic pathway of scoparone and the significance of the metabolism in bile acid elimination. Cholestasis is the most common form of hepatotoxicity, and the scoparone-rich herb Yin Chin has long been used to normalize liver functions. These studies will provide important information on how the hepatic protective activity is achieved. In addition, FXR is recognized as an important metabolic regulator and has anti-inflammatory effect. Interestingly, scoparone has been found to exert these very activities. Thus, a confirmation on the functional connection between FXR and scoparone will have broad mechanistic and therapeutic implications.