补虚化瘀法全方及有效组分调控miRNA223抑制胆管上皮细胞凋亡在PBC治疗中的机制研究

The BuXuHuaYu recipe is effective to cure PBC, which has been proved in the recent study funded by NSFC. This study is aimed to confirm that miRNA223 can decrease the apoptosis of bile duct epithelial cells, which could be the pathogenesis of PBC. We will utilize the slow virus mediated RNA interference skill on the bile duct epithelial cell lines and the patients live tissue fluorescence in situ hybridization(FISH) method to verify the hypothesis, which is based on the previous miRNA chip screen results of PBC patients. On the other hand, we will found the fingerprint of BuXuHuaYu recipe using HPLC-MS method, and find its effective components by the way of orthogonal test method comparing with the standard products. Further more, the vitro and vivo experiment will be carried on the bile duct epithelial cell line and 2-OA induced mice models, and the express of miRNA223-3p, FOXO1 and the apoptosis index will be observed by real-time RT-PCR ,Western Blot, and TUNEL before and after the treatment by BuXuHuaYu recipe and its effective components. So we are about to clarify that miRNA223 regulating target gene FOXO1 to inhibit the apoptosis of bile duct epithelial cells might be the potential drug target of BuXuHuaYu recipe and its effective components to remedy PBC effectively.

补虚化瘀法组方的中药是治疗PBC的有效药物，通过前期的自然科学基金资助已经证实，此次研究将在以往临床和细胞研究同时，结合miRNA芯片筛选结果，将通过胆管上皮细胞慢病毒干扰试验以及PBC患者肝脏组织荧光原位杂交技术标记，来证实miRNA223调控胆管上皮细胞凋亡是PBC发病机制之一。同时利用二维液相色谱——质谱联用技术建立补虚化瘀法全方的药物指纹图谱，与组方中的单药标准品比对，并正交优化确定验证组分。通过2-OA导致胆管上皮细胞株免疫损伤的体外实验，以及对2-OA PBC模型小鼠的体内实验，观察补虚化瘀法全方以及组分干预治疗前后miRNA223-3p、FOXO1基因和蛋白表达，胆管上皮细胞凋亡情况，以及体内实验小鼠肝脏组织miRNA223和FOXO1标记，从细胞、基因、蛋白层面，证实“miRNA223调控FOXO1从而抑制胆管上皮细胞凋亡”是补虚化瘀法全方以及有效组分发挥作用的药物靶点。

本课题在前期研究的基础上，旨在阐明PBC发病机制，以及补虚化瘀法组方及其有效组分的作用机制。发病机制研究通过miRNA芯片筛选找到目标基因并通过PBC患者血清学验证锁定miRNA223-3p、miRNA150-5p，然后模拟PBC患者细胞凋亡的GCDC造模的细胞模型经过慢病毒转染以及mimics实验进一步证实miRNA150-5p抑制了AKT的磷酸化，从而促进了HiBEC细胞的凋亡可能是PBC发病机制之一。并结合2-OA-BSA小鼠动物模型验证和人的病理标记佐证进一步阐明胆管上皮细胞凋亡是PBC发病机制之一、同时利用网络药理学筛选出补虚化瘀法组方的有效组分，通过临床全方验证，胆管上皮细胞全方及其有效组分验证以及动物实验验证补虚化瘀法一定程度上提高气虚血瘀型PBC患者的UDCA应答进程，并促进肝功能ALP、GGT等的恢复，调节免疫，改善患者的临床症状体征，细胞上也证实补虚化瘀法中药对miRNA150-5p具有很好的调节作用，能够通过调控miRNA150-5p，促进AKT的磷酸化，从而抑制HiBEC凋亡，证实miRNA150-5p是补虚化瘀法中药治疗PBC的潜在药靶。此作用机制研究亦处于PBC发病机制前沿。