基于AMPK-FXR-BSEP介导的齐墩果酸所致胆汁淤积性肝损伤作用机制研究

Drug induced liver injury is becoming an increasingly serious health problem. Oleanolic acid (OA) is used as a dietary supplement and an over-the-counter Chinese medicine for acute and chronic viral hepatitis. Some studies show that OA could produce cholestasis in the clinical practice. The mechanism is still not clear. Our previous study also demonstrated that OA could lead to cholestatic liver injury. AMPK, FXR and BSEP played a role in OA induced cholestatic liver injury, but as a signaling pathway for regulation of bile acid homeostasis need further study. We hypothesized that OA might increase AMPK Phosphorylation, suppress FXR transcriptional activity, and inhibit or suppress BSEP function, lead to the accumulation of bile acid and result in cholestatic liver injury. To confirm our hypothesis, we will exploit various murine and cell models to determine the profiles of bile acids composition and distribution using molecule biology and chemical analytical techniques in cell and animal level in OA induced cholestatic liver injury. And further explore the role of AMPK-FXR-BSEP signaling pathway in disruption of bile acids homeostasis induced by OA. We may provide the experimental basis for the early diagnosis and treatment of oleanolic acid and oleanolic acid-containing induced liver toxicity. It is of great significance for the clinical application of OA and the drug development of anti-cholestatic injury.

近年来药物性肝损伤已经成为严重的健康问题。齐墩果酸是临床用于治疗急性和慢性肝炎的非处方药，报道显示，临床使用含齐墩果酸的中药可致胆汁淤积，具体机制不明。我们研究也发现齐墩果酸可致胆汁淤积性肝损伤，AMPK、FXR和BSEP可能参与了齐墩果酸所致的胆汁淤积性肝损伤并由此假设：齐墩果酸增加了AMPK磷酸化，抑制了FXR转录活性，下调了BSEP的表达或功能，引起肝内胆汁积聚，有毒胆汁酸组分增加，最终导致胆汁淤积性肝损伤。因此，本项目采用AMPK低表达、FXR和Bsep敲除小鼠及多种细胞模型，在细胞和整体动物水平上观察在齐墩果酸所致胆汁淤积状态下，胆汁酸组分的构成及分布特点，并进一步探讨AMPK-FXR-BSEP在齐墩果酸所致胆汁酸平衡紊乱的调控作用。为齐墩果酸和含齐墩果酸组方肝毒性的早期诊断及治疗提供实验依据，对其临床用药安全和抗胆汁淤积性损伤的药物开发均具有重要意义。

近年来药物性肝损伤已经成为严重的健康问题。齐墩果酸是临床用于治疗急性和慢性肝炎的非处方药，临床使用含齐墩果酸的中药可致胆汁淤积。申请者根据肝脏代谢谱探讨齐墩果酸在高剂量或长期使用后引起的肝毒性。采用UHPLC-TSQ-MS技术获得肝脏代谢谱。研究发现，胆汁酸、氨基酸和能量代谢的改变可能导致肝毒性，表明共轭胆汁酸是齐墩果酸诱导的肝毒性的主要因素。为了进一步探索齐墩果酸诱导的胆汁淤积性肝损伤，研究者采用C57BL/6J (WT)小鼠和FXR敲除小鼠研究FXR在齐墩果酸诱导的胆汁淤积性肝损伤中的作用。发现在FXR-/-小鼠中，齐墩果酸诱导的胆汁淤积性肝损伤明显减轻，肝细胞坏死减少。胆汁酸的UPLC-MS分析显示，与WT小鼠相比，FXR-/-小鼠肝脏和血清中胆汁酸含量显著降低，胆汁中胆汁酸含量升高。齐墩果酸处理FXR-/-小鼠肝脏组织中肝转运蛋白、胆汁酸合成酶和胆汁酸解毒30种酶的mRNA表达均升高。与齐墩果酸处理的WT小鼠相比，FXR-/-小鼠肝脏中膜蛋白BSEP的表达显著升高。发现敲除FXR可以通过减少肝脏和血清中胆汁酸的积累，增加胆汁酸经胆汁输出，以及上调胆汁酸解毒酶来减轻齐墩果酸诱导的小鼠胆汁淤积性肝损伤。申请者为了研究FXR激动剂可以增强BSEP功能，加快积聚的胆汁酸排泄而抗胆汁淤积肝损伤的机制。采用野生型小鼠为研究对象，用齐墩果酸制备小鼠急性胆汁淤积型肝损伤模型，明确 FXR-BSEP 在齐墩果酸所致胆汁淤积中的调控作用。为了研究铁对FXR受体的影响，喂食富含铁的饮食的小鼠同时给予FXR激动剂GW4064。研究发现，铁抑制小鼠肝脏和肠道中FXR的表达和信号转导。通过本研究明确了齐墩果酸所致的胆汁淤积性肝损伤中胆汁酸组分的构成特点及分布特点，FXR-Bsep 路径在齐墩果酸所致胆汁淤积性肝损伤中的作用机制。本项目为齐墩果酸和含齐墩果酸组方肝毒性的早期诊断及治疗提供实验依据，对其临床用药安全和抗胆汁淤积性损伤的药物开发均具有重要意义。