膜联蛋白A1在抗肝纤维化中的作用机制

Liver fibrosis remains a significant clinical problem throughout the world that needs to be solved urgently, it is of great practical significance to study the treatment of liver fibrosis in China. There was no effective pharmacological agent have been developed that directly prevent or reverse the fibrotic process, we have previously reported that expression of AnxA1 was significantly increased in mice liver and AnxA1-/-mice developed more server hepatic fibrosis after CCl4 treatment. These findsings suggest that AnxA1 can play a central role in the treatment of hepatic fibrosis, which can be an ideal therapeutic target for hepatic fibrosis. We confirmed that β-catenin is a downstream target gene of AnxA1 by bioinformatics analysis and experimental verification, which may promotes activation of hepatic stellate cells by regulating the function of macrophages, but the specific mechanism remains to be further studied. Based on the previous studies, we aim to study the effects of Wnt/β-catenin signaling pathway in the regulation of macrophage function and modulates hepatic stellate cell activation through gene knockout mice and cell transfection, and to explore the effect of AnxA1 on anti-hepatic fibrosis in vitro and in vivo. This study is highly significant which elucidate the pathogenesis of hepatic fibrosis and ultimately may have clinic application.

抗肝纤维化治疗是慢性肝病治疗的棘手问题，也是目前急需解决的迫切问题，研究抗肝纤维化治疗在我国具有重要现实意义。目前全世界针对抗肝纤维化治疗没有很好的手段，课题组前期研究发现膜联蛋白A1(Annexin A1，AnxA1)在CCL4诱导的肝纤维化小鼠肝组织中表达显著增高，且CCL4诱导AnxA1-/-小鼠较普通小鼠的肝纤维化严重，说明AnxA1在抗肝纤维化治疗能起到很好的作用，可作为理想的抗肝纤维化治疗靶标。我们通过生物信息学分析及靶标验证证实β-catenin是AnxA1的下游靶基因，发现其可能通过调节巨噬细胞功能进而调控肝星状细胞活化，但具体机制尚待研究。课题组拟在前期研究基础上通过AnxA1基因敲除小鼠和细胞转染技术，研究分析Wnt/β-catenin信号通路对巨噬细胞功能的影响及肝星状细胞活化的作用，探明AnxA1在体内体外抑制肝纤维化的能力与机制，为肝纤维化防治提供新思路。

肝纤维化是各种病因所引起的慢性肝脏疾病的共同病理过程，可进展为肝硬化，但其潜在机制尚未完全清楚。课题组通过腹腔注射20%CCL4诱导野生小鼠/Anxa1敲除小鼠肝纤维化，同时造模组分别腹腔注射Ac2-26、Boc2干预CCL4诱导小鼠肝纤维化。检测炎症因子、胶原沉积的表达，wnt/β-catenin通路在肝纤维化中的作用。我们研究结果表明：与对照组比较，CCL4诱导肝纤维化野鼠肝脏Annexin-1、TGF-β1、IL-1β、IL-6的表达明显升高，促进胶原沉积及a-SMA、collageI、CTGF的表达增加，并随时间进行性增加。CCL4诱导Anxa1基因敲除小鼠肝组织TGF-β1和IL-1β、IL-6的表达升高更明显，肝脏炎症、肝纤维化程度及a-SMA、collageI、CTGF的表达均比野鼠组明显增加。Ac2-26治疗后肝纤维化小鼠肝脏炎症因子表达、胶原沉积的程度及a-SMA、collageI、CTGF的表达均较治疗前下降，Boc2抑制Ac2-26的抗炎及抗纤维化的作用。此外，AnxA1还可下调ccl4诱导的肝纤维化wnt/β-catenin通路的表达。体外细胞实验，LPS诱导HC及HSC表达Anxa1，Ac26-6能够抑制LPS诱导的RAW264.7细胞活化及HSC细胞增殖,降低HSC细胞α-SMA和胶原I和CTX4的表达，同时抑制HSC活化后wnt/β-catenin通路的表达，而这一治疗作用可被Boc2所抑制。综上，AnxA1具有抑制小鼠肝纤维化的作用，其作用机制可能与AnxA1通过靶向作用于甲酰肽受体发挥调节巨细细胞功能来抑制HSCwnt/β-catenin通路的活化有关，最终抑制肝脏炎症及胶原沉积。这些变化过程有可能成为我们治疗肝纤维化的重要靶点，为我们治疗肝纤维化提供理论基础。