Asbt(Slc10a2)抑制效应在栀子调控的回肠-粪便利胆途径中的作用研究

Traditional Chinese medicine science (TEMS) holds that Fructus Gardenia could unchoke Triple Burner, Dispel Dampness and Heat via urine excretion, and we investigated the mechanism of this TEMS in the early National Natural Science Fundation, and we found the key point of Fructus Gardenia increased the excretion of bile acids in urine is that Fructus Gardenia regulated the expression of liver and kidney bile acids associated transporters. However，we found that Fructus Gardenia could increase more bile acids into feces, which might relate with Apical Sodium-Dependent Bile Acid Transporter (Asbt/Slc10a2), but the mechnism remains unclear.Scholars both at home and abroad have confirmed inhibition of ileal Asbt protein can block bile acids reabsorption, and increase the excretion of bile acids in feces, and achieve the cholagogic and hepatoprotective effect. Therefore, our project intend to use animal experiment, in situ intestine circulation experiment, in vitro everted gut sac experiment, cell experiment, to demonstrate the Asbt inhibition effect which regulated by Fructus Gardenia, and base on FXR/VDR path or direct inhibition to clarify the regulating mechanism of Fructus Gardenia. To provide new ideas and methods for the treatment of cholestatic disease of Fructus Gardenia.

中医理论认为栀子可“清热燥湿，通利三焦，引湿热自小便而出”，课题组前期国基金针对该理论展开探究，并发现栀子对胆汁淤积大鼠肝脏及肾脏胆酸盐相关转运体表达的调控是其促进胆酸盐的尿液排泄而利胆的关键。但课题组同时发现栀子也可促进更多的胆酸盐经粪便排泄，且这一现象可能与回肠顶端钠依赖性胆盐转运体（Apical Sodium-Dependent Bile Acid Transporter, Asbt/Slc10a2）的抑制效应有关，但其具体的机制尚不清楚。国内外学者已研究证实抑制回肠Asbt蛋白可阻断胆酸盐的重吸收，促进胆酸盐的粪便排泄而利胆保肝。为此，本项目拟采用体内动物实验、原位肠循环实验、离体外翻肠囊实验及细胞实验，证实栀子调控的回肠Asbt抑制效应并阐明栀子是直接调控还是通过FXR/VDR信号路径调控的机制，为栀子治疗胆汁淤积疾病提供新的思路和策略。

本项目探讨了Na+依赖性胆酸盐转运体(Asbt/Slc10a2)的抑制效应在栀子调控的回肠-粪便利胆途径中的作用，并基于FXR/VDR信号通路探究了栀子水提物调控ASBT/Asbt表达及功能的分子机制。结果表明，1）栀子水提物显著降低了α-异硫氰酸酯（ANIT）所致的小鼠血清生化指标ALT、AST、ALP及TBIL的升高，缓解了ANIT所致的肝脏病理学变化；2）与ANIT组相比，栀子水提物可显著降低小鼠血清、肝脏及回肠组织的胆酸盐含量，显著增加粪便中胆酸盐的含量；3）与ANIT组相比，栀子水提物可显著减少小鼠回肠Asbt的表达，增加回肠Fxr受体的表达，对Vdr受体的表达无显著影响；4）回肠原位循环实验结果显示，与ANIT组相比，栀子水提物可显著降低回肠组织对Asbt底物TCA的摄取，减少TCA进入肝门静脉血中的含量；5）外翻肠囊实验结果显示，与ANIT组相比，栀子水提物可显著减少回肠组织及肠内液中TCA的含量；6）MODE-K细胞实验结果显示，栀子水提物对ANIT所致的细胞毒性具有明显的保护作用，且可显著减少Asbt的表达，增加Fxr的表达，对Vdr无显著影响；7）Caco-2细胞实验结果显示，栀子水提物能显著减少ASBT的表达，且能减少其对底物TCA的摄取；而当Caco-2细胞中FXR受体沉默后，对ASBT的表达及其底物的摄取均无显著影响。因此，栀子水提物通过激活回肠Fxr受体而抑制Asbt的表达和功能减少胆酸盐的肠道重吸收，促进粪便胆酸盐的排泄，是其缓解ANIT诱导的小鼠肝内胆汁淤积的机制之一。本研究为栀子治疗胆汁淤积相关疾病提供了新的思路和策略，同时为治疗胆汁淤积的药物开发提供了一定的理论及基础依据。