基于基因表达谱分析蒙古山萝卜花单、复方抗大鼠肝纤维化作用研究

Mongolian scabious flower is applicated exclusively and frequently in traditional Mongolian medicines and it has a long pharmaceutical history. Clinical datas illustrated that its mono and compound recipes such as Taosongtaoerma-7 has pharmaceutical values in treating of liver diseases, especially in anti-liver-fibrosis treatments. Based on the values of Mongolian scabious flower in the treatment of anti-liver-fibrosis in traditional Mongolian medicines, the program will not initiate from the relevant adaptation diseases in traditional Mongolian medicines, but to adapt the means of modern pharmacological experiments, as the object of study to liver fibrosis models of rat, probe into the effects of mono and compound recipe of Mongolian scabious flower to the indexes of liver fibrosis models from the aspects of pathomorphism , biochemical functions, and the hydroxylproline(Hyp) content of hepatic tissue, to define the pharmaceutical effects of the Mongolian medicine in curing liver fibrosis. The program will adopt entire rat gene expression profile chip technology, sybr green real time PCR technology, Western blot technology, etc. to examine the gene level variations among the normal group, fibrosis model group and the Mongolian-medicine-treated groups and undertake the research in association with the analytical technologies introduced from systems biology, in order to obtain the essential information such as the genetic and molecular marks, the important pathways as well as the regulatory mechanism that are in relation to the liver fibrosis . All of these will contribute to the data basis of the research of Mongolian scabious flower’s therapeutic mechanism, the development of these medicines and gene therapy of liver fibrosis, etc.

蒙古山萝卜花，为蒙医专用、常用药物之一，具有悠久的用药历史。临床数据表明，其单方及复方如陶松桃尔玛7味等在肝脏疾病特别是抗肝纤维化治疗中具有确切疗效。基于蒙古山萝卜花单、复方在蒙医临床抗肝纤维化治疗的良好应用价值，本项目从不同于传统蒙医适应症入手，采用现代药理实验方法，以肝纤维化大鼠模型为研究对象，研究该蒙药单、复方对肝纤维化模型的肝脏病理形态学、肝脏生化功能、肝组织羟脯氨酸含量等指标的影响，以明确该蒙药单、复方在肝纤维化治疗中的作用；运用大鼠全基因组表达谱芯片技术、sybr green荧光定量PCR技术、Western blot技术等，检测正常组、纤维化模型组、蒙药治疗组之间的基因表达水平变化情况，结合系统生物学分析技术，以获取肝细胞纤维化有关的基因、分子标记、重要通路以及调控机制等重要信息，为该药治疗机制研究、药物开发，以及肝纤维化基因工具治疗等提供一定的实验数据基础。

肝纤维化严重危害人类健康，传统医药因其多层次、多靶点的特点对肝纤维化治疗具有优势。蒙古山萝卜花为蒙医专用药物，常用于蒙医肝病治疗。动物实验从病理形态学、肝生化指标、肝纤维化四项、羟脯氨酸及肝纤特征性蛋白指标α-SMA，CollagenⅠ等多个方面表明，蒙古山萝卜花总黄酮提取物及以蒙古山萝卜花为主药的额力根-7（陶松桃尔玛-7）复方具有较好的抗肝纤维化作用。为进一步明确其作用机制，课题组采用全转录组测序，比较空白组、模型组、治疗组基因表达谱（mRNA）的差异变化，发现蒙古山萝卜花治疗组和模型组相比，FPKM值差异大于2倍，且qvalue<0.05的差异mRNA共有139个；额力根-7治疗组和模型组相比，差异mRNA共有189个。通过GO和KEGG富集，并通过结合空白组比较，均发现多条有显著变化的信号转导通路，我们选择和肝纤维化关系最为密切的几条通路加以细胞实验验证，并寻找通路中的关键靶点。其中TGF-β/Smads通路是目前已知和肝纤维化关系最为密切的通路，课题组通过一系列实验证明，蒙古山萝卜花治疗组可通过下调该通路发挥作用，进而发现Smad3是其作用的主要靶标之一。另外我们还选择了蒙古山萝卜花治疗组的整合素通路及额力根-7治疗组的JAK/STAT作为研究对象，通过q-PCR、WB、免疫组化、免疫荧光、细胞凋亡等实验予以明确。结果显示，药物可通过下调此二通路发挥药效，ITGB4，FAK，p38是蒙古山萝卜花作用于整合素通路的靶点；JAK2、STAT3是额力根-7作用于JAK/STAT通路的靶点。药物作用的药效物质基础及上述两条通路与TGF-β/Smads通路的交互关系（cross talk）将是我们后续的研究内容。此外，在本基金和课题组获批其他经费支持下，我们分析发现了治疗组和模型组的差异非编码RNA，药物通过非编码RNA调控基因表达进而产生抗肝纤维化作用，也是我们后续的研究方向之一。本资助项目研究结果发表论文8篇，其中SCI收录论文2篇。