基于BAMBI在Smads信号转导中的作用探讨下瘀血汤抗肝纤维化机制

With the promoting blood circulation for removing blood stasis of Xia-yu-xue decoction (XYXD), is an effective classical prescriptions in liver fibrosis treatment. BMP and activin membrane bound inhibitor (BAMBI) plays an important role in inflammation and fibrogenesis. We previously proved that BAMBI translocated from cytoplasm to nucleus, and the combination of BAMBI and Smad7 reduced significantly but the binding of Smad2/3 and BAMBI increased notably in HSC activation in response to LPS stimulation, and XYXD could regulate remarkably these pathological changes. Based on these new findings, we hypothesized that XYXD inhibit liver fibrosis by regulating on BAMBI/Smads signal transduction. For these aims, we would detect the inhibitory effects of XYXD on BAMBI nuclear translocation and Smads signal transduction and their relationships using immunofluorescence microscopy and nuclear/membrane separation in CCl4-indcued liver fibrosis in mice. Next, we would analyze the function of BAMBI/Smads signal transduction in HSC activation and fibrogenesis, meanwhile; we would explore the regulation of XYXD on these changes using primary human stellate cells and fibrotic model by chromatin immunoprecipitation (ChIP) sequence and co-immunoprecipitation. The project will help us to elucidate the pathological biological mechanism of XYXD on inhibiting liver fibrosis by promoting blood circulation for removing blood stasis principle, and laying a foundation for its further development and clinical application.

活血祛瘀的下瘀血汤是治疗肝纤维化有效经典方剂，BMP激活素膜结合抑制因子(BMP and activin membrane bound inhibitor，BAMBI)是炎症和纤维化的重要介体；本课题前期发现肝星状细胞（HSC）活化过程中有BAMBI核转位及BAMBI与Smad7结合减少而与Smad2/3结合增多，下瘀血汤对此有显著调控作用；提出“下瘀血汤可能通过调控BAMBI/Smads信号转导抗肝纤维化”的假说。为此，采用免疫荧光和核浆蛋白分离技术，通过CCl4肝纤维化模型观测BAMBI核转位/Smads信号转导的关联性及下瘀血汤干预前后的变化；结合hHSC培养，通过染色质免疫共沉淀测序、蛋白质免疫共沉淀等技术，分析BAMBI/Smads信号转导在HSC活化、肝纤维化病理机制中的作用以及下瘀血汤的干预效应，阐释下瘀血汤“活血祛瘀”抑制肝纤维化的病理生物学机制，为其发展应用奠定基础。

肝纤维化是中医治疗学的优势病种，下瘀血汤是抗肝纤维化基本方剂，负责人在项目研究中发现下瘀血汤调控BAMBI/Smads信号抑制HSC活化机制。通过临床肝活检石蜡切片、冻存组织及血清样本，检测BAMBI的核转位、蛋白和mRNA表达及血清含量；运用CCl4和BDL肝纤维化模型检测下瘀血汤抗肝纤维化效应；采用人原代HSC培养，核蛋白分离结合蛋白质免疫共沉淀联合质谱分析等技术，从临床-小鼠-细胞-分子多平层面揭示：1）肝纤维化患者血清BAMBI显著升高；BAMBI阳性面积和核转位率在肝纤维化患者显著升高；人肝纤维化进展中BAMBI mRNA表达下调、Smad2/3显著上调；2）采用腺病毒过表达BAMBI显著抑制肝纤维化形成，Smad2/3显著下调，Smad7上调；采用AddnBAMBI敲减BAMBI结果与此相反。3）BAMBI在正常HSC表达细胞膜，LPS处理BAMBI核转位，过表达BAMBI显著抑制Smad2/3核转位、促进Smad7表达；ChIP实验发现LPS和TGF刺激后NF-κB及Smads结合BAMBI DNA上的结合位点。4）下瘀血汤显著抑制小鼠肝纤维化，在CCl4模型，BAMBI细胞浆蛋白表达下调而核蛋白表达上调，下瘀血汤显著抑制BAMBI细胞核转位；从而证实下瘀血汤通过调控BAMBI/Smads抑制HSC活化的科学假说。在完成上述工作之外：我们还新增：1）蛋白质质谱分析发现并证实细胞核BAMBI与Smad2结合，下瘀血汤促进BAMBI和Smad7结合；2）BAMBI转录后调控机制：miR-942特异与BAMBI3-UTR结合进而诱导HSC活化。3）采用均匀设计法U63筛选下瘀血汤抑制胶原沉积的最佳配伍，发现桃仁（5g）和地鳖虫（3g）显著抑制肝组织胶原沉积。项目解析下瘀血汤调控BAMB/Smads信号抗肝纤维化机制，为临床应用提供理论基础。