基于肝窦内皮细胞(LSECs)的窗孔调控探究开通玄府法抗肝纤维化的分子机制

Liu Wansu created the “Xuanfu Theory” and considered “Micropore closure is the root cause of disease.” Later generations of doctors based on this theory proposed that “Opening the pore is the fundamental cure for disease.” Sanjiasan is one of the classic prescriptions of Opening the Micropore therapy, and its clinical effect is significant.Our research group used the therapy of Opening the Micropore to treat hepatic fibrosis in the early stage get good results. It can inhibit the activation of hepatic stellate cells, reduce pathological angiogenesis and improve the pathological grading of hepatic fibrosis in model rats. Recent studies have shown that the hepatic sinus endothelial cell (LSECs) surface fenestral differentiation closely related to the progression of hepatic fibrosis.Therefore, this project proposed a hypothesis thatSanjiasan may maintain normal LSECs phenotype by regulating the fenestrations differentiation of LSECs, thereby reducing pathological angiogenesis and blocking liver fibrosis. In this study, immunomagnetic bead separation, primary culture, scanning electron microscopy, immunofluorescence staining, qrt-pcr and Western blot were used to observe the effect of sanjiasan on fenestral differentiation of hepatic sinus endothelial cells in rat model of hepatic fibrosis and cultured in vitro. And to further explore the molecular mechanism of Sanjiasan in the treatment of liver fibrosis. It will provide some empirical evidence for the rationality of the treatment in liver fibrosis with Opening the Micropore therapy and expand the therapeutic principle and therapy of liver fibrosis.

刘完素首创玄府学说，提出“玄府郁闭为百病之根”。基于此，后世医家以“开通玄府”为治病之纲。三甲散为开通玄府法经典名方，临床运用三甲散治疗肝纤维化疗效显著。课题组前期研究发现，三甲散可抑制肝星状细胞激活，减少病理性血管新生，改善大鼠肝纤维化病理分级。新近研究表明，肝窦内皮细胞(LSECs)表型分化与肝纤维化发展密切相关。LSECs失窗孔化与玄府郁闭似有一定契合度，受此启发，课题组提出本项目研究假说：三甲散可能通过调控LSECs表面窗孔，维持LSECs正常分化表型，减少血管新生，阻断肝纤维化。本研究拟采用免疫磁珠分选、扫描电镜、免疫荧光染色、qRT-PCR和Western blot等技术，观察三甲散对肝纤维化模型大鼠LSECs和体外培养的LSECs窗孔的影响，进一步探索三甲散治疗肝纤维化的分子机制，为阐释基于玄府理论的开通玄府法的现代科学内涵提供部分实证依据，拓展中医药防治肝纤维化的理法方药。

有效治疗肝纤维化对于改善慢性肝病预后和降低病死率至关重要。课题组前期研究发现，开通玄府法经典名方三甲散在临床应用和实验研究中均显示出良好的抗肝纤维化疗效，但其抗肝纤维化的现代医学效应机制有待阐释。研究表明，肝窦内皮细胞(LSECs)表型分化与肝纤维化发展密切相关，LSECs失窗孔化与玄府郁闭似有一定契合度，受此启发，课题组提出研究假说：三甲散可能通过调控LSECs表面窗孔，维持LSECs正常分化表型，减少血管新生，阻断肝纤维化。我们通过动物实验和细胞实验，采用扫描电镜、免疫荧光染色、qRT-PCR和Western blot等技术，观察三甲散对肝纤维化模型大鼠LSECs表型分化、血管新生和肝HSCs活化的影响。研究结果显示，三甲散通过调控miR29b-3p/VEGF信号轴及下游的促血管生成因子VEGFR2、PDGF等蛋白表达和抑制促纤维化因子TGFβ、Smads蛋白及HSCs自噬相关蛋白Beclin-1、P62、LC3表达，维持LSECs正常表型，减少肝窦毛细血管化和病理性血管新生，减少肝窦内皮下基膜形成，促进ECM降解，抑制HSCs自噬，减少HSCs激活，阻断肝纤维化进展。研究结论为揭示开通玄府法代表方三甲散治疗肝纤维化的现代科学机制提供了部分实证依据，拓展了中医药防治肝纤维化的临床应用思路。