胆淤方通过PDC-E2调控丙酮酸代谢改善PBC胆管上皮细胞损伤的作用和机制

The pathological mechanism of Primary Biliary Cholangitis (PBC) is controversial. Some PBC patients have no biochemical response to the first-line treatment drug ursodeoxycholic acid (UDCA). Pyruvate Dehydrogenase Complex-E2 (PDC-E2) is highly expressed in Bile Duct Epithelial Cells of PBC patients. The specific antibody of PEC-E2 is Anti-Mitochondrial Antibody-M2 (AMA-M2). The high level of AMA-M2 is an important serological feature of PBC and is correlated with the disease progression. However, the pathogenesis of PDC-E2 on PBC has not been elucidated. Our previous studies revealed the distinct pyruvate metabolism dysfunction of PBC patients. Pyruvate Dehydrogenase (PDC) is the key irreversible enzyme in pyruvate metabolism. Our clinical practice validated that Chinese medicine Danyu Decoction, when combined with ursodeoxycholic acid, could significantly improve the biochemical responses for PBC patients compared with ursodeoxycholic acid monotherapy. In vivo experiments showed that Danyu Decoction could protect Bile Duct Epithelial Cells, alleviate intrahepatic cholestasis, and reduce the expression of CK19 in portal area of model mice. Thus, we hypothesizes that Danyu Decoction can attenuate the bile duct epithelial cells impairment via PDC-E2 mediated pyruvate metabolism and thus can be used for the treatment of PBC. We intends to design a series of in vitro and in vivo experiments, from molecular, cell and tissue levles, to elucidate the following issues. ① Effect of PDC-E2 mediated pyruvate metabolism dysfunction on the injure of bile duct epithelial cells. ② The protective effect of Danyu Decoction on bile duct epithelial cells and the regulation of Danyu Decoction on PDC-E2 mediated pyruvate metabolism. ③ Pharmacological mechanism of Danyu Decoction for the treatment PBC. The project can not only clarify the non-immune pathological mechanism of PBC and expand our understanding of PBC from Chinese medical pathogenesis, but also reveal the pharmacological mechanism of Danyu Decoction for the treatment of PBC. The study could promote the research and development of integrated traditional Chinese Medicine and Western medicine for the treatment of PBC through the pharmacological action of Danyu Decoction.

原发性胆汁性胆管炎（PBC）病理机制复杂，部分患者对熊去氧胆酸无生化应答。患者胆管上皮细胞线粒体高表达丙酮酸脱氢酶复合物-E2（PDC-E2），产生特征性抗体AMA-M2，且与疾病进展相关。我们既往发现PBC患者存在特异性丙酮酸代谢异常。而丙酮酸脱氢酶（PDC）是丙酮酸代谢关键不可逆酶。中药胆淤方可增强熊去氧胆酸对PBC患者的生化应答；改善模型小鼠肝内胆汁淤积与保护胆管上皮细胞。据此提出假说：胆淤方通过PDC-E2调控丙酮酸代谢改善PBC胆管上皮细胞损伤。拟通过体内外实验，从分子、细胞和组织水平，①分析PDC-E2介导丙酮酸代谢异常促进胆管上皮细胞损伤的作用；②研究胆淤方对胆管上皮细胞保护作用和对PDC-E2介导丙酮酸代谢调控作用，③明确胆淤方防治PBC的药理机制。以期阐明PBC非免疫途径的病理机制和中医脾肾两虚的病机，揭示胆淤方防治PBC药理作用机制，促进中西医结合防治PBC研究发展。

基于前期工作基础，我们提出假说：胆淤方通过线粒体PDC-E2调控丙酮酸代谢改善PBC胆管上皮细胞损伤。PBC患者的重要病理特征为胆管上皮细胞损伤及由此引起的胆汁淤积。DDC诱导动物模型能较为准确模拟PBC的这种病理特征。使用胆淤方干预DDC诱导胆汁淤积小鼠，不仅生化得以改善，而且肝内汇管区炎症反应、细胆管增生及纤维化均明显减轻。通过PCR和Western blot方法证实DDC诱导胆汁淤积小鼠Pdk1基因及蛋白表达水平均显著增高，表明DDC诱导胆汁淤积与Pdk1基因过表达有关，而胆淤方通过抑制Pdk1基因和蛋白表达，发挥减轻肝内胆汁淤积的作用。通过代谢组学研究发现DDC小鼠存在显著的胆汁酸代谢紊乱，胆淤方干预后胆汁酸代谢谱出现了广泛下降。通过腺病毒干扰和化学抑制剂干扰DDC小鼠肝脏Pdk1基因表达，结果显示DDC小鼠生化指标、肝内炎症反应、细胆管增生及纤维化均有所减轻。由此进一步证实DDC诱导的胆汁淤积与Pdk1介导PDC活性改变有关。进一步研究胆淤方的有效成分-龙胆苦苷对DDC诱导胆汁淤积小鼠的作用，结果发现龙胆苦苷具有改善生化、减轻肝内汇管区炎症反应、纤维化和细胆管增生等作用，且具有一定的剂量依赖性。通过PCR实验发现龙胆苦苷对胆汁酸合成、排泄、摄取及代谢相关基因表达具有调控作用。流式细胞术分析发现龙胆苦苷可显著改善DDC诱导的肝脏免疫细胞浸润，特别是淋巴细胞和巨噬细胞的过度浸润。以上结果提示胆淤方通过抑制Pdk1基因和蛋白表达介导PDC活性改变，调控丙酮酸代谢紊乱，发挥改善肝内胆汁淤积的作用，而龙胆苦苷是其发挥作用的有效成分之一。