Interplay between metabolism and FXR activation in scoparone signaling

滨藜内酯是一种香豆素衍生物，在结构上属于多酚类化合物的超家族。这种化合物在银杏中含量很高，银杏是一种几千年来一直用于治疗肝肾疾病的中草药。银杏叶制剂最近在世界范围内受到了极大的关注，并作为健康补充剂在北美销售。滨蒿内酯被认为是一种主要的肝脏保护化合物，并已被证明能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。我们最近做了一项努力，以测试司马铃酮是否也能诱导人类UGT1A1。人的原代肝细胞分别用滨蒿内酯或鹅去氧胆酸(CDCA)处理，鹅去氧胆酸是法尼醇X受体(FXR)的激活剂。令我们惊讶的是，滨蒿内酯没有诱导人UGT1A1，而是显著增强了CDCA诱导胆盐输出泵(BSEP)的作用。这种胆汁转运蛋白是CDCA-FXR信号转运蛋白的靶标，在胆汁酸的分泌中起着至关重要的作用。与BSEP诱导的增强作用一致，滨藜内酯增强CDCA激活FXR的作用，而FXR磷酸化缺陷突变体的这种增强作用明显减弱。相反，在细胞色素P4501A2(一种药物代谢酶)的转染组，这种增强作用显著增强。该项目的中心假说是，滨蒿内酯代谢产物通过有效地调节FXR的磷酸化，对胆汁淤积提供了有效的肝脏保护作用。其具体目的是：(1)确定BSEP诱导的增强作用与滨藜内酯代谢的关系；(2)表征滨蒿内酯对FXR的磷酸化作用；以及(3)明确FXR和滨蒿内酯代谢在抗胆汁淤积中的相互作用。为了确定东莨菪内酯在一般人群中的代谢情况，我们将对大量的个体肝脏样本进行东莨菪内酯的代谢检测。将对主要代谢物进行结构测定和测试，以增强BSEP的诱导作用。为了验证滨蒿内酯是否是FXR磷酸化的调节剂，我们将用滨蒿内酯单独或与CDCA共同处理肝细胞，并分析FXR的磷酸化状态。为了确定东泻内酯的抗胆汁淤积作用，我们将用或不加东莨菪内酯的方法诱导小鼠胆汁淤积，并监测胆汁淤积标志物和东泻内酯的代谢。总体而言，拟议的研究将建立东五味子内酯的代谢途径以及该代谢在胆汁酸消除中的意义。胆汁淤积症是最常见的肝毒性形式，富含东菱藤内酯的草本植物银杏叶长期以来一直被用来恢复肝功能。这些研究将为如何实现肝脏保护活性提供重要信息。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，已经发现滨藜内酯发挥了这些作用。因此，确认FXR和司马藤酮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.