Interplay between metabolism and FXR activation in scoparone signaling

滨蒿内酯是一种香豆素衍生物，在结构上属于多酚类化合物超家族。这种化合物在Yin Chin（茵陈）中含量丰富，Yin Chin是一种数千年来用于治疗肝脏和肾脏疾病的中草药。最近，银琴制剂在世界范围内受到了广泛关注，并在北美作为保健品销售。滨蒿内酯被认为是一种主要的肝脏保护化合物，并已被证明可诱导小鼠尿苷二磷酸-5 <$-葡萄糖醛酸转移酶-1A1（UGT 1A 1），这是一种胆红素解毒酶。我们最近做了一个努力，以测试滨蒿内酯是否也诱导人类UGT 1A 1。用滨蒿内酯或沿着鹅去氧胆酸（CDCA）（法尼醇X受体（FXR）的激活剂）处理人原代肝细胞。令我们惊讶的是，滨蒿内酯并没有诱导人UGT 1A 1，而是显着增强CDCA诱导胆盐输出泵（BSEP）。这种胆汁转运蛋白是CDCA-FXR信号传导的靶标，在胆汁酸分泌中起重要作用。与BSEP诱导增强一致，滨蒿内酯增强CDCA激活FXR，并且FXR磷酸化缺陷突变体的增强作用大大降低。相反，在细胞色素P450 1A 2（CYP 1A 2），药物代谢酶转染的细胞中，增强显着增加。该项目的中心假设是滨蒿内酯代谢物通过有效调节FXR磷酸化而赋予针对胆汁淤积的有效肝保护。具体目标是：（1）确定作为滨蒿内酯代谢函数的BSEP诱导增强作用;（2）表征滨蒿内酯对FXR的磷酸化作用;（3）确定FXR和滨蒿内酯代谢相互作用在抗胆汁淤积中的作用。为了确定滨蒿内酯在一般人群中的代谢，将检测大量个体肝脏样本的滨蒿内酯代谢。将对主要代谢产物进行结构测定，并检测其对BSEP诱导的增强作用。为了测试滨蒿内酯是否是FXR磷酸化的调节剂，肝细胞将用滨蒿内酯单独处理或加CDCA处理，并分析FXR的磷酸化状态。为了确定滨蒿内酯的抗胆汁淤积潜力，将用或不用滨蒿内酯诱导小鼠胆汁淤积，并监测胆汁淤积标志物和滨蒿内酯的代谢。总之，拟定的研究将建立滨蒿内酯的代谢途径和代谢在胆汁酸消除中的意义。胆汁淤积是最常见的肝毒性形式，富含滨蒿内酯的草药茵陈蒿长期以来一直被用来恢复肝功能。这些研究将提供关于如何实现肝脏保护活性的重要信息。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，滨蒿内酯被发现发挥这些活动。因此，FXR和滨蒿内酯之间的功能连接的确认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.