结合型胆汁酸/未结合胆红素比值升高在胆汁淤积中的致病作用及茵陈蒿汤干预机制研究

It is reported that there exists a contradictory phenomenon that serum bilirubin increased while intrahepatocellular bilirubin decreased in cholestasis liver disease and there is significant elevation of the bile acids/unconjugated bilirubin (BA/UCB) ratio in hepatocytes in cholestasis liver injury. Based on analyzing the complex relationship between the antioxidant effects of bilirubin and the toxic effects of bile acids and according to our previous studies in the treatment of cholestasis with Yinchenhaotang, we propose a hypothesis that elevation of the conjugated bile acids/unconjugated bilirubin (CBA/UCB) ratio in liver is an important risk factor increased cholestasis progress, while the main effect mechanism of Yinchenhaotang in the treatment of cholestasis could be through regulating in bile acids metabolism, and reducing the CBA/UCB ratio in liver. This project proposes to conduct studies systematically in animal models to test the hypothesis, aiming to provide evidence to use the CBA/UCB ratio in clinic and develop anti-cholestasis Chinese compound herbal formulae. We will clarify the scientific significance of the CBA/UCB ratio in cholestasis progress through measuring the CBA and UCB in serum and liver by UPLC-TQ-MS in an ANIT-induced cholestasis liver injury rat model. We will determine how Yinchenhaotang regulates the bile acid profile and the CBA/UCB ratio and reveals the intervention mechanism of bile acid metabolism using Yinchenhaotang and its effective component to treat the model rats.

本课题从胆汁淤积血清胆红素升高，肝细胞胆红素降低这一矛盾现象出发，通过剖析胆红素抗氧化作用和胆汁酸毒性作用的复杂关系，明确肝细胞存在BA/UCB比值升高的病理机制。结合前期茵陈蒿汤及其专利组分复方的工作基础，提出“肝组织CBA/UCB比值升高是胆汁淤积进展的重要致病因素，调控胆汁酸代谢，降低肝组织CBA/UCB比值，可能是茵陈蒿汤治疗胆汁淤积的主要效应机制”假说。拟采用ANIT诱导慢性胆汁淤积模型，使用代谢组学技术测定不同阶段血清、肝脏CBA和UCB含量，揭示CBA/UCB比值变化规律，阐明该比值用于判断胆汁淤积进展的科学意义；并以茵陈蒿汤和专利组分复方为对象，通过代谢组学和分子生物学技术检测胆汁酸谱、各胆红素组分浓度，调控胆汁酸代谢的核受体、TGR5、转运体的基因和蛋白表达，解析茵陈蒿汤调控CBA/UCB比值和胆汁酸代谢的干预机制。为该比值的临床应用及开发抗胆汁淤积中药组分复方提供依据。

本研究从血清和肝脏胆汁酸谱、胆红素谱，血清肝功能，肝组织病理学，胆汁酸转运体、胆汁酸核激素受体角度，探索胆汁淤积疾病进展的病理生理规律。采用雄性Wistar大鼠作为研究对象，使用ANIT诱导慢性胆汁淤积性肝病模型。造模时间40周，设11次动态采集标本时间点。每一标本采集时间点设正常对照组和模型组。使用常规方法观察基本病理生化改变，检测肝组织病理形态。使用代谢组学技术检测外周血、肝组织胆汁酸谱和胆红素谱。使用分子生物学方法检测肝组织、小肠组织胆汁酸配体（核激素受体、膜受体TGR5）表达。发现模型大鼠血清和肝组织结合型胆汁酸/未结合胆红素比值分别于造模24小时、4周、12周显著升高。揭示出ANIT诱导的大鼠胆汁淤积疾病进展可分为急性期、亚急性期、慢性期三个阶段，明确了亚急性期是胆汁淤积疾病进展的关键时期。.在此基础上，探索茵陈蒿汤及其组分治疗胆汁淤积的调控效应机制。复制亚急性期胆汁淤积性肝损伤大鼠模型。将实验动物分为8组，即正常组、模型组、茵陈蒿汤原方组、组分复方组、绵茵陈绿原酸组、栀子苷组、制大黄多糖组和制大黄鞣质组。使用常规方法观察基本病理生化改变。使用代谢组学方法检测血液、肝脏胆汁酸谱、胆红素谱。使用分子生物学技术检测胆汁酸转运体、胆汁酸配体（核激素受体、膜受体TGR5）mRNA表达。揭示出调控胆汁酸代谢，改善胆汁酸核激素受体、转运体表达，是茵陈蒿汤治疗胆汁淤积的主要效应机制。绿原酸、没食子酸、大黄多糖是茵陈蒿汤调控胆汁酸转运体、胆汁酸配体（核激素受体FXR、PXR、VDR，膜受体TGR5）表达，改善胆汁酸肠肝循环，治疗亚急性期胆汁淤积的主要效应组分。栀子苷具有降酶和降低血清未结合型胆红素作用，但对降低结合型胆红素、胆汁酸未见显著治疗作用，其机制在于抑制肝细胞胆汁酸转运体（NCTP、OATP1a1）表达，减少肝细胞从肝血窦摄取胆汁酸、胆红素，减少肝细胞向毛细胆管和肝血窦排泌胆汁。栀子苷阻断胆汁酸肠肝循环，降低了绿原酸、没食子酸、大黄多糖合用治疗胆汁淤积疗效。