一贯煎影响巨噬细胞极化调控肝祖细胞分化干预肝硬化的效应机制

Regulating the differentiation of endogenous hepatic progenitor cells into hepatic parenchymal cells is expected to become a new approach for reversing cirrhosis, so elucidating the regulatory mechanism underlying it is now become a key scientific issue of anti-cirrhotic study. Based on the rather good efficacy of Yiguanjian on cirrhosis in basic and clinical researches, and further mechanism study of our previous work that reveals Yiguanjian can regulate macrophage polarization, Wnt signaling activation and hepatic progenitor cell differentiation. We put forward the hypothesis that "in the pathological microenvironment of cirrhosis, Yiguanjian decoction might inhibit the differentiation of hepatic progenitor cells into myofibroblasts through inhibition of M1 polarization of macrophages, which further inhibit non-conanical Wnt signaling pathway activation in hepatic progenitor cells through reducing non-canonical Wnt ligands release; on the other hand, Yiguanjian decoction might promote the differentiation of hepatic progenitor cells into hepatic parenchymal cells through promoting M2 polarization of macrophages, which releasing canonical Wnt ligands to activate canonical Wnt pathway of hepatic progenitor cells." To verify our hypothesis, hepatic macrophage depletion, block bone marrow macrophage infiltration, polarized macrophage transplantation, bone marrow mesenchymal stem cells transplanted after co-culture with polarized macrophages, the secretion of Wnts from macrophages blockage, polarized macrophages co-culture with hepatic progenitor cells after gene modification, and blocking macrophage polarization are to be used to explore the mechanisms of macrophage on hepatic progenitor cell differentiation and to reveal the action mechanisms of Yiguanjian decoction on cirrhosis might be regulating the polarization of macrophages and the differentiation of hepatic progenitor cells. This study would provide a sound theoretical basis for the clinical application of Yiguanjian decoction in the treatment of cirrhosis.

调控内源性肝祖细胞向肝实质细胞分化有望成为肝硬化治疗的新途径，但明确其调控机制是目前面临的关键科学问题。基于一贯煎有效治疗肝硬化的基础与临床证据，及其调控巨噬细胞极化、Wnt信号通路活化及肝祖细胞分化取向等前期工作基础，提出“在肝硬化病理微环境，一贯煎可能下调巨噬细胞M1极化，减少非经典Wnt配体释放，抑制肝祖细胞非经典Wnt信号，从而抑制其向肌成纤维细胞分化；可能上调巨噬细胞M2极化，释放经典Wnt配体，激活肝祖细胞经典Wnt信号，促进其向肝实质细胞分化”假说。拟采用巨噬细胞耗竭、骨髓源巨噬细胞浸润阻断、巨噬细胞极化移植、骨髓间充质干细胞与极化巨噬细胞共培养后移植、巨噬细胞极化阻断、巨噬细胞Wnts分泌阻断、极化巨噬细胞与肝祖细胞基因改造共培养等方法，探析巨噬细胞影响肝祖细胞分化机制，揭示一贯煎调控巨噬细胞极化与肝祖细胞分化干预肝硬化的效应机制，为一贯煎治疗肝硬化的临床应用提供理论基础。

背景与目的：前期研究表明一贯煎具有良好的抗肝硬化作用，但其具体机制尚不明确。本研究旨在揭示一贯煎调控巨噬细胞极化影响肝祖细胞分化抗肝硬化的效应机制。.研究内容：（1）观察一贯煎对2-AAF/CCl4大鼠肝硬化肝脏巨噬细胞极化的影响。（2）观察骨髓巨噬细胞不同极化状态（BMDM-M1, BMDM-M2）对2-AAF/CCl4大鼠肝硬化的影响。（3）观察BMDM-M1 Wnt3A基因对2-AAF/CCl4肝硬化的干预作用以及一贯煎对其影响。（4）观察BMDM-M1 Wnt5A基因对2-AAF/CCl4肝硬化的干预作用以及一贯煎对其影响。（5）观察与BMDM-M1共培养后的骨髓间充质干细胞（BMSCM1）对2-AAF/CCl4大鼠肝硬化进展的影响以及一贯煎组分（iYGJ）对其影响。（6）观察BMSCM2对2-AAF/CCl4大鼠肝硬化进展的影响以及iYGJ对其影响。.结果：（1）一贯煎通过抑制巨噬细胞M1极化干预2-AAF/CCl4大鼠肝硬化（下文省略为“大鼠肝硬化”）的进展。（2）BMDM-M1和BMDM-M2移植均可抑制大鼠肝纤维化进展。（3）BMDM-M1或BMDM-M2移植均可抑制大鼠肝硬化的进展，一贯煎可提高BMDM-M2的干预效应，其机制与提高BMDM-M2对非经典Wnt信号的干预作用有关。（4）BMDM-M2Wnt3a-OE移植可抑制肝祖细胞向胆管细胞分化（即胆管反应）及大鼠肝硬化进展；而BMDM-M2Wnt3a-KD移植则促进胆管反应及肝硬化进展。（5）BMDM-M1Wnt5a-OE移植可促进胆管反应及肝硬化进展；而BMDM-M1Wnt5a-KD移植则可抑制胆管反应及肝硬化进展。（6）一贯煎可干预BMDM-M2Wnt3a-KD对大鼠肝硬化的促进作用，其机制与促进肝祖细胞经典LRP5/β-catenin信号活化、抑制胆管反应有关。（7）BMSCM1移植可抑制大鼠肝硬化的进展，iYGJ分可提高其干预效应。（8）BMSCM2移植也可抑制大鼠肝硬化的进展，但iYGJ对其干预效应无明显影响。.结论：BMDM-M1和BMDM-M2对大鼠肝硬化均具有良好的干预作用，两者的治疗效应分别受到Wnt5a和Wnt3a基因水平的影响。一贯煎可提高BMDM2、BMSCM1抗肝硬化作用。本研究为基于BMDM的终末期肝硬化的生物治疗奠定了科学基础，也为一贯煎的临床应用提供了科学依据。