基于NGF信号通路研究一贯煎促进骨髓间充质干细胞逆转肝纤维化的机制

Liver fibrosis is the common pathologic change of all kinds of chronic liver diseases. liver fibrosis reversion is the key for treating chronic liver diseases . Yin deficiency of liver and kidney is the important pathogenesis of liver fibrosis, Yiguanjian has good effect on liver fibrosis reversion. TCM consider, kidney govers bone and marrow, Yin of liver and kidney can moist and nourish the zangfu and make tissue structure stabile. Modern study shows, mesenchymal stem cells(MSCs) can reverse liver fibrosis, apoptosis of hepatic stellate cell(HSCs) is the key factor. In this study, we used liver fibrosis rat model, on the whole animals in vivo and cells in vitro, proliferation and transference of MSCs was observed, the apoptosis of HSCs was detected with TUNEL. The expression of mRNA and protein of typeⅠ/Ⅲ collagen protein、MMP-1/-13、TIMP-1、NGF、P75、NF-kB、Bcl-xl were determined by Real-time PCR and western blotting respectively. We will explore the mechanism of Yiguanjian to reverse the liver fibrosis. This research has important meaning for explaining microcosmic pathologic basis of yin deficiency of liver and kidney and pharmacological mechanism of Yiguanjian.

肝纤维化是多数慢性肝病的共同病理变化，逆转肝纤维化是治疗慢性肝病的关键。肝肾阴虚是肝纤维化的重要病机。滋补肝肾中药一贯煎抗肝纤维化疗效显著。中医理论认为，肾主骨生髓，肝肾之阴具有润养脏腑组织、维持组织结构稳定的作用。现代研究说明，骨髓间充质干细胞(MSCs)能够逆转肝纤维化，通过NGF信号通路诱导肝星状细胞(HSCs)凋亡是其作用的关键。课题制备肝纤维化大鼠模型，体外培养肝纤维化恢复期MSCs和HSCs，观察一贯煎促进MSCs增生并迁移到肝脏的作用，实时荧光定量PCR、蛋白质印迹技术检测该药对MSCs诱导HSCs凋亡的NGF信号通路关键基因NGF、P75等mRNA转录水平及蛋白表达的改变，同时观察HSCs凋亡的变化，结合细胞微环境中主要胶原和胶原代谢关键酶的检测，从整体、细胞、分子等多层次探讨一贯煎逆转肝纤维化的分子机制。课题的完成对阐释肝肾阴虚的微观病理基础和一贯煎的药理机制有重要意义。

本项目在国家自然科学基金课题（No.81273668）的资助下，在肾主骨生髓、肝肾之阴润养脏腑组织、维持组织结构稳定这一理论指导下，研究一贯煎逆转肝纤维化的机理。研究发现：（1）一贯煎中剂量组在改善肝功能、减轻炎症、逆转纤维化方面作用较好，P＜0.05。（2）一贯煎可以促进肝纤维化的逆转恢复，其作用机制是诱导肝星状细胞凋亡、减少Ⅰ型胶原的蛋白表达而促进肝组织的修复。（3）制备大鼠骨髓移植+CCl4诱导的肝纤维化大鼠模型，证实一贯煎能够促进骨髓间充质干细胞迁移到肝脏，从而发挥逆转肝纤维化的作用， P＜0.05。总之，本研究提示，一贯煎逆转肝纤维化的作用机制之一是通过促进骨髓干细胞募集到肝脏而发挥修复肝脏的作用。对于临床合理应用一贯煎起到一定的指导作用。课题的完成对阐释肝肾阴虚的微观病理基础有重要意义。