利胆合剂抑制肝脏钙敏感受体相关细胞凋亡保护幼龄大鼠肝细胞改善胆汁淤积的关键机制研究

Infantile cholestasis is a common refractory disease which affects infant quality of life and increases infantile motality. Recent researches have shown that hepatic calcium-sensing receptor (CaSR) is a key toggle protein in the development of cholestasis–related hepatocyte apoptosis. Therefore targeting CaSR is a promising new approach to treat infantile cholestasis. Previous clinical evidence has demonstrated a Chinese herbal prescription, Li-dan-he-ji, is effective in the treatment of infant jaundice. Experimental study also found that Li-dan-he-ji attenuated intrahepatic cholestasis in rats via down-regulation hepatic CaSR expression.Thus we hypothesize that Li-dan-he-ji protects hepatocyte and improves infantile cholestasis through inhibiting CaSR related apoptosis. To test our hypothesis, we will establish animal models of intrahepatic cholestasis and hepatocytes model of cholestasis related apoptosis. For mechanistic study, we will examine CaSR expression in the liver, as well as subsequent changes in downstream molecules involved in intracellular mitochondria and extracellular MAPK apoptosis signaling pathway. Different experimental techniques including immunohistochemistry, Western blot, real time PCR will be used. The successful completion of the proposed research at integrative, cellular, and molecular levels will illustrate the mechanisms of CaSR inhibitor actions of Li-dan-he-ji, based on which we will be able to develop a new anti CaSR related hepatocyte apoptosis based treatment for infantile cholestasis. Additionally, the proposed research will provide foundations for the therapeutic value of Li-dan-he-ji on infantile cholestasis and promote its wide use in clinical practice.

婴儿胆汁淤积严重影响婴儿生存质量并可直接危及生命，而针对性治疗方法长期匮乏。新近研究显示，肝脏钙敏感受体（CaSR）在胆汁淤积性肝细胞凋亡发病环节中具有“开关效应”,以此为治疗靶点前景广阔。本课题在前期研究证实：自主研发中药复方“利胆合剂”能利胆退黄，改善婴儿胆汁淤积；减轻大鼠胆汁淤积，下调CaSR表达的基础上，提出“利胆合剂抑制肝脏CaSR相关细胞凋亡，保护肝细胞、改善婴儿胆汁淤积的关键机制”的假说，分别建立幼龄大鼠肝内胆汁淤积动物及胆汁淤积相关性肝细胞凋亡模型，采用免疫组化、Western-blot、RT-PCR等分子生物学技术，检测肝脏CaSR及下游内源性/外源性肝细胞凋亡途径相关信号转导分子活性和表达，从整体、细胞、分子水平探讨利胆合剂作为CaSR抑制剂的可能性，促生一种基于抗CaSR相关肝细胞凋亡治疗婴儿胆汁淤积的新方法，并为临床有效研发利胆合剂相关中药复方制剂提供实验依据。

婴儿胆汁淤积严重影响婴儿生存质量并可直接危及生命，而针对性治疗方法长期匮乏。新近研究显示，肝脏钙敏感受体（CaSR）在胆汁淤积性肝细胞凋亡发病环节中具有“开关效应”,以此为治疗靶点前景广阔。本课题在前期研究证实：自主研发中药复方“利胆合剂”能利胆退黄，改善婴儿胆汁淤积；减轻大鼠胆汁淤积，下调CaSR表达的基础上，提出“利胆合剂抑制肝脏CaSR相关细胞凋亡，保护肝细胞、改善婴儿胆汁淤积的关键机制”的假说，通过分别建立幼龄大鼠肝内胆汁淤积动物及胆汁淤积相关性肝细胞凋亡模型，采用免疫组化、Western-blot、RT -PCR等分子生物学技术，检测肝脏CaSR及下游内源性/外源性肝细胞凋亡途径相关信号转导分子活性和表达，从整体、细胞、分子水平探讨利胆合剂作为CaSR抑制剂的可能性，促生一种基于抗CaSR相关肝细胞凋亡治疗婴儿胆汁淤积的新方法，并为临床有效研发利胆合剂相关中药复方制剂提供实验依据。本研究分为二部分：第一部分建立肝内胆汁淤积性幼龄大鼠动物模型和胆汁淤积相关原代肝细胞凋亡模型，通过在体和离体相结合的方式，来观察胆汁淤积性肝细胞凋亡的发生时CaSR的表达变化。第二部分从抑制肝脏CaSR相关肝细胞凋亡探讨利胆合剂保护肝细胞、改善胆汁淤积的关键机制。研究结果：①证实了CaSR在胆汁淤积的发病作用，利胆合剂可以改善肝内外胆汁淤积，下调CaSR表达。②证实利胆合剂可以通过调控线粒体途径和MAPK途径抑制肝细胞凋亡。③利胆合剂君臣药物三个单体活性物质连翘酯苷A、大黄素、绿原酸，或具有CaSR抑制剂效能。通过此项研究的实验结果，对利胆合剂的主要药物和部分药物的剂量进行的调整，完善了全方。用于临床明显提高了临床疗效，也为有效研发利胆合剂相关中药复方制剂提供实验依据。