长链非编码RNA H19在何首乌致肝损伤中的作用及机制研究

Polygonum multiflorum (PM) is commonly used in clinical practice due to the therapeutic effect of detoxification, eliminating carbuncle, defaecation, replenishing vital essence and blacking hair. Moreover, PM is also widely used as a tonic. Recently, an increasing number of adverse effect reports concerning PM induced liver injury attracted domestic and overseas attention, therefore, food and drug administration offices from different countries have issued either clinical warnings or regulatory measures regarding the risk of liver injury caused by PM and its preparation. However, the mechanism underlying PM associated liver injury remains poorly understood. Our preliminary experimental results showed that the gene expression of long non-coding RNA H19, which plays a crucial role in liver injury, was significantly up-regulated after oral administration of PM for 14 days. Based on the abovementioned findings, sandwich cultured mouse hepatocyte and C57BL/6 mouse models will be used to investigate the role of H19 in PM induced liver injury using gene silencing and knockout techniques. Also, specific biomarkers will be searched and identified as novel therapeutic targets. Moreover, RNA Pull-down and RNA binding protein immunoprecipitation assay methods will be used to explore the interaction between potential molecules and H19 as well as detailed mechanism of PM associated liver injury. In summary, the research results will not only provide a practical interpretation for PM induced liver injury, but also offer a theoretical understanding of hepatotoxicity caused by similar herbal medicine.

何首乌具有解毒、消痈、通便、益精血以及乌须发等功效，是临床常用中药，也是民众热爱的滋补品。近年来，有关何首乌致肝损伤不良事件的报道引起国内外高度关注，各国药监部门相继发布何首乌及其制剂肝损伤风险的警示。但是，何首乌致肝损伤机制尚不明确。我们前期研究发现，何首乌会上调小鼠体内长链非编码RNA H19（简称H19）的表达。据报道，H19是肝损伤过程中的重要信号分子。本项目拟采用“三明治”小鼠原代肝细胞与C57BL/6小鼠模型，分别沉默或敲除H19表达，在细胞和整体动物水平考察H19在何首乌肝损伤中的作用，寻找特异性生物标志物和新的治疗靶点。然后，利用RNA Pull-down和RNA结合蛋白免疫沉淀技术探究与H19相互作用的分子，考察何首乌对其作用的影响，阐明何首乌肝损伤的机制。本研究结果将为何首乌肝损伤研究提供理论基础，也为同类中药的肝毒性机制研究提供思路，具有重要的理论意义和临床实用价值。

何首乌是一种临床常用中药，也是民众热爱的滋补品。近年来，有关何首乌致肝损伤不良事件的报道引起国内外高度关注。本项目研究了何首乌提取物及其主要化学成分大黄素对胆汁酸稳态和长链非编码RNA H19（简称H19）的影响，并探讨其诱发药物性肝损伤的可能机制。.本研究考察何首乌提取物口服给药3天和14天后对小鼠肝损伤作用以及肝脏中H19表达水平调控。何首乌连续给药3天后，小鼠生化指标没有显著变化，但肝脏中胆汁酸浓度显著升高；连续给药14天后，肝脏中ALT，AST以及总胆汁酸水平都显著升高。切片结果显示连续药3天后，肝组织有少量炎性细胞浸润；给药14天后，肝组织出现炎性细胞浸润和胆小管增生。此外，连续给药14天后，肝脏中H19和S1pr2的基因表达水平显著升高。此外，本研究考察何首乌提取物及其主要蒽醌衍生物对小鼠肝脏肝脏毒性作用。实验结果表明何首乌提取物及其主要蒽醌衍生物通过调控胆汁酸主要外排转运体Bsep和Mrp2的表达扰乱胆汁酸稳态，进而诱导肝损伤，提示蒽醌类衍生物在何首乌肝损伤过程中的重要作用。.本研究进一步考察了何首乌的主要蒽醌衍生物大黄素和二苯乙烯苷的肝毒性作用及其机制。小鼠连续口服给与大黄素或者大黄素联合二苯乙烯苷14天或28天后发现大黄素可以增加小鼠血浆中AST或ALT水平，导致肝脏出现病理损伤和肝细胞凋亡，二苯乙烯苷可以增强大黄素的致肝损伤作用。小鼠给药7天或14天后，主要胆汁酸外排转运体蛋白表达下调，胆汁酸合成酶表达增加；连续给药28天后，胆汁酸外排转运体蛋白代偿性表达上调。此外，大黄素可以诱导凋亡执行蛋白cleaved caspase-3的表达，二苯乙烯苷可以增强其诱导作用。体外实验表明大黄素浓度依赖性地诱导L-02细胞凋亡，二苯乙烯苷增强大黄素促凋亡作用。此外，大黄素以浓度依赖性和时间依赖性方式诱导H19在L-02中的表达，沉默H19表达可以降低大黄素对L-02的毒性作用。.综上，本项目考察了何首乌及其主要化学成分大黄素对胆汁酸稳态以及H19的表达调控作用，并进一步验证了H19在大黄素诱导肝损伤过程中的作用。本研究为何首乌的肝毒性机制提供了理论依据，为临床安全用药提供指导意义。