苦参甘草组方通过肝脏核受体LXR/RXR双向调节肝药酶的作用及其机理研究

The regulation of hepatic drug - metabolizing enzymes mediated by the nuclear receptors LXR/RXR in liver, especially the interactions between phase Ⅰ metabolizing enzymes and phase Ⅱ metabolizing enzymes or hepatic drug - metabolizing enzymes and phase Ⅲ transporters, plays an important role for the balance of hepatic microenvironment. This finding provides a new molecular target for the study of hepatoma susceptibility, prevention and treatment of chronic liver diseases and liver cancer. Recently, we found that the flavescen and liquorice prescription biphasically affected the activity of hepatic drug - metabolizing enzymes in the mice, and we speculate that its molecular mechanism is involved in a negative feedback regulation by 18β-glycyrrhizin glucuronate, a metabolite from liquorice. In this study, our aim is to investigate the molecular mechanism by which the flavescen and liquorice prescription biphasically regulates the activity of hepatic drug - metabolizing enzymes mediated by LXR/RXR in the mice. The experiment methods and techniques used in this investigation include the C57BL/6 mice of LXR or RXR knock-out, analysis of nuclear receptor transcription activity, gene and protein chip. In addition, the metabolite of liquorice, 18β-glycyrrhizin glucuronate, will be extracted by liver perfusion technique in this study. We shall pay close attention to the protection and its molecular targets of flavescen and liquorice prescription from hepatocarcinogenesis in DEN mouse model. The effects of 18β-glycyrrhizin glucuronate on nuclear receptor transcription activity will be analyzed in Hep G2 cell line. We expect that the mechnism, by which the flavescen and liquorice prescription biphasically regulates the phase Ⅰ metabolizing enzymes, phase Ⅱ metabolizing enzymes and phase Ⅲ transporters, will be disclosed in mRNA and protein levels. We hope that our studies can provide a new idea for the investigation of interaction on the Chinese medicinal herbs. Based on the studies, the flavescen and liquorice prescription will be further applied to clinic practice.

肝脏核受体LXR/RXR属于孤核受体家族成员，其介导的肝药酶调节作用，对维持肝脏微环境平衡至关重要。这一发现为研究原发性肝癌形成的机理和慢性肝脏疾病的防治，提供了新的分子靶点。最近，我们的研究表明，苦参甘草组方具有保肝、抑制肝癌形成的作用，该作用与肝脏核受体LXR/RXR对肝药酶的调控密切相关，但分子机制不详。本课题拟采用LXR和RXR基因敲除小鼠，研究苦参甘草组方保肝、抑制肝癌形成作用的分子位点；采用核受体转录活性分析法，研究甘草的代谢产物18β甘草甜素-葡萄糖醛酸双醛酯，对上游靶分子的负反馈调节作用；采用基因芯片和蛋白芯片技术，研究LXR/RXR激活后，其下游相关靶分子mRNA表达及蛋白质含量的变化；该研究旨在：阐明苦参甘草组方双向调节肝药酶作用的分子机制，为拓展苦参甘草组方的临床应用，提供实验依据；为中西医结合模式研究中药方剂“相辅相成、相生相克”的作用特点，提供一种新的思路。

研究背景：肝脏核受体LXR/RXR介导的肝药酶调节作用，对维持肝脏微环境平衡至关重要。苦参甘草方具有保肝、抑制肝癌形成的作用，该作用与肝脏核受体LXR/RXR密切相关，但分子机制不详。.主要研究内容：采用LXR和RXR基因敲除小鼠，研究了苦参甘草方保肝、抑制肝癌形成作用的分子位点；采用核受体转录活性分析法，研究了18β甘草甜素-葡萄糖醛酸双醛酯，对上游靶分子的负反馈调节作用；采用基因芯片和蛋白印记分析，研究了LXR/RXR下游相关靶分子mRNA及蛋白含量的变化。.重要结果和关键数据：LXR/RXR被活化后，肝细胞转录组基因表达变化在10倍以上的基因共73个，与肝脏疾病密切相关的有：Prtn3，Saa1-3，Prom1，Pnpla3，Olfr969，HNF-a。18β甘草甜素-葡萄糖醛酸双醛酯对其的抑制率高达57.8%，曲线下面积 = 1：0.422。该抑制作用与剂量呈正相关，峰值效应在7 min，持续时间为20 min左右。与对照组比，CYP 1A2 mRNA增加了55倍（55.7±119.4），Gly 2E1 mRNA增加了1442倍（1442.4±3326.7）。对照组CYP 1A2蛋白含量为1.0±0.07，处理组为1.06±0.08。对照组Gly 2E1蛋白含量为1.0±0.08，处理组为0.96±0.09。小鼠接受醋氨酚300mg/kg腹腔注射后24小时，阳性对照组小鼠的肝脏损伤，明显被改善。ALT、AST、透射电镜及组织病理等检测结果显示，该损伤与肝脏标本所见一致。与野生型小鼠比，苦参甘草方对LXR基因敲除小鼠肝脏损伤的保护作用消失。组织病理学检测结果表明，在肝脏核受体RXR基因敲除小鼠，苦参甘草方抑制肝癌形成作用明显减弱。.科学意义：该研究表明，肝脏核受体LXR是苦参甘草方保肝作用的关键分子；肝脏核受体RXR是苦参甘草方抑制肝癌形成作用的重要信号通路之一。