非经典Wnt信号通路介导肝星状细胞活化促进肝纤维化的作用研究

How to treat liver fibrosis is the key to prevent the progression of chronic liver diseases.Wnt signaling pathway is involved in mutiple hepatic physiological processes.Many studies about liver fibrosis focus on Wnt/β-catenin canonical pathway,whereas little is known on noncanonical Wnt pathway.Our previous studies have shown that Wnt5a was upregulated in activated HSC and experimental liver fibrosis model. Wnt5a could promote the proliferation, contraction and extracellular matrix secretion of HSC. With cDNA microarray and bioinformatics analysis we found Wnt5a noncanonical signaling pathway mediated the activation of HSC. According to previous reports and our results we speculate: Wnt5a noncanonical signaling pathway may: 1) promote the proliferation and releasing of proinflammatory factors of HSC by activating the JNK pathway;2) promote HSC contraction through the activation myosin light chain kinase ( MLCK) causing myosin light chain (MLC) phosphorylation. In order to confirm this hypothesis,we study the pathway activation status and mechanisms, as well as the effects of regulating this pathway on HSC activation and liver fibrosis with gene transfection and RNA interference in vivo and vitro. The results of this research may reveal a new signaling mechanism in HSC activation, thus providing a new strategy for liver fibrosis treatment.

如何有效治疗肝纤维化是阻止慢性肝病进展的关键，Wnt信号通路调控肝脏多种病理生理过程，已有肝纤维化研究集中在Wnt/β-catenin经典信号通路，而对非经典Wnt通路鲜有研究。我们前期研究表明HSC活化后及肝纤维化模型Wnt5a表达水平显著上升，Wnt5a可促进HSC增殖、收缩及ECM分泌，利用生物信息学分析发现Wnt5a介导的非经典信号通路参与HSC活化。结合文献推测：Wnt5a通路可能：1）通过激活JNK促进HSC增殖及炎症因子分泌；2）通过激活HSC肌球蛋白轻链激酶（MLCK），使肌球蛋白轻链（MLC）磷酸化，促进HSC收缩。为此，本课题拟采用基因转染、RNA干扰等技术从体内、体外实验两个层面探讨该通路激活状态、对经典Wnt通路的影响以及调控该通路对HSC活化及肝纤维化的阻断作用。从Wnt5a信号通路促进HSC活化机制全新角度探讨其在肝纤维化中的作用，为肝纤维化治疗提供新策略。

有效治疗肝纤维化是阻止慢性肝病进展的关键。有关非经典Wnt通路在肝纤维化进程中作用的研究较少，本课题主要研究了Wnt5a介导肝星状细胞活化和肝纤维化中的作用和机制。. 本课题首次发现：1）炎症刺激因子LPS和TNF-α能促进肝星状细胞(HSC) Wnt5a表达；2）稳定过表达Wnt5a可促进炎症因子（IL-1β、IL-6）在HSC中的表达和分泌，促进肝脏炎症反应。Wnt5a基因沉默可抑制炎症因子（IL-1β、IL-6）在HSC中的表达和分泌；3) 稳定过表达Wnt5a可诱导HSC分泌胶原蛋白（COL1、COL3），促进肝脏细胞外基质的沉积。Wnt5a基因沉默则减弱这一效应；4）Wnt5a过表达促进HSC的增殖，Wnt5a基因沉默可抑制HSC增殖；5）Wnt5a过表达促进β-catenin和JNK的磷酸化，Wnt5a基因沉默则抑制这一效应。在HSC中，Wnt5a非经典信号通路能抑制经典Wnt信号通路；JNK的磷酸化可能与HSC分泌炎症因子、胶原蛋白和促进HSC增殖的现象有关；6）Wnt5a过表达可提高HSC细胞内Ca2+浓度和MLCK的表达，减少MLCK的磷酸化；Wnt5a基因沉默抑制此效应。. 上述实验结果表明，Wnt5a促进HSC增殖及炎症因子分泌可能与JNK磷酸化的激活有关；Wnt5a通过激活HSC肌球蛋白轻链激酶（MLCK），使肌球蛋白轻链（MLC）磷酸化，从而促进HSC收缩，Wnt5a信号通路为肝纤维化治疗提供新策略。