LOXL-1/Fibulin-5通过调控弹性蛋白交联及降解影响肝纤维化可逆性的机理

Liver fibrosis is a wound-healing process of the liver in response to repeated and chronic liver injury with quantitative deposition extracellular matrix. Now liver fibrosis is considered as a dynamic and reversible process when the underlying causative etiologies are eradicated, yet we found nearly half patients do not achieve histological liver regression even if the HBV virus is undetectable. Our preliminary data showed that the deposition of mature elastin in extracellular matrix could stabilize collagen fibrils and is responsible for the irreversibility of liver fibrosis. However it is still unclear how elastin crosslinks together, how it deposits into the extracellular matrix in liver fibrosis and how mature elastin is degraded during regression. It still needs further exploration to find therapeutic strategies based on elastin crosslinking and degradation for expediting the regression of liver fibrosis.. In this study, we will reveal the dynamic expression and histological localization of elastin, lysyl oxidase like-1(LOXL-1) and Fibulin-5 at different stages of carbon tetrachloride liver fibrosis mice and spontaneous regression mice by immunohistochemistry and second harmonic generation/two-photon excitation fluorescence. Secondly, we are going to isolate the primary hepatic stellate cells and Kupffer cells from different stages of carbon tetrachloride induced liver fibrosis mice and spontaneous regression mice by indirectly co-cultured with elastin to reveal the molecular pathway of elastin crosslink and degradation. Thirdly, we would like to conditionally delete LOXL-1 in hepatic stellate cells by GFAPCreER-ΔLOXL1 mice in order to elucidate the regulatory effects of LOXL-1 on elastin crosslinking and degradation and on the reversibility of liver fibrosis. Finally, we are going to screen the receptor and interaction proteins of Fibulin-5 in hepatic stellate cells by co-immunoprecipitation and mass spectrometric analysis to explore the positive feedback mechanism of Fibulin-5 in activating hepatic stellate cells and further confirm the anti-fibrotic effects by LOXL-1/Fibulin-5 intervention. . This study could provide evidence for understanding the role of elastin in regulating reversibility of liver fibrosis and finding potential searching for novel therapeutic target to promoting resolution of liver fibrosis.

临床研究发现有效控制肝纤维化病因后，仍有近一半病人难以逆转。我们前期工作提示弹性蛋白具有稳定胶原纤维致其拮抗降解的作用,但弹性蛋白交联沉积与降解的分子机制尚不清楚,通过干预弹性蛋白逆转纤维化的途径仍待阐明。本研究拟首先利用双光子二次谐波观察弹性蛋白及其交联调控分子—LOXL-1和Fibulin-5在纤维形成和逆转不同阶段的动态变化和定位特点；其次分离纤维形成和逆转期的原代肝星状细胞和枯否细胞与弹性蛋白间接共培养,并干预星状细胞中LOXL-1/Fibulin-5表达以阐明弹性蛋白交联与降解的细胞和分子机制；第三，利用GFAP Cre ER -ΔLOXL-1小鼠选择性敲除星状细胞中LOXL-1基因,明确阻断LOXL-1进而抑制弹性蛋白交联的逆转纤维化作用；最后应用免疫共沉淀结合质谱分析明确Fibulin-5的受体并揭示其活化星状细胞的分子机制。为通过干预弹性蛋白逆转肝纤维化的途径提供实验依据。

细胞外基质（ECM）过度沉积是纤维化形成的关键因素，其中赖氨酰氧化酶样蛋白1（LOXL1）参与的ECM交联形成尤其是特异性参与弹性蛋白的交联形成是维持ECM稳定不易被降解的关键环节，但LOXL1及其参与的ECM蛋白交联对纤维化形成/逆转的作用及机制尚不清楚。因此本课题的目的是明确纤维化形成/消退过程中ECM蛋白（胶原和弹性蛋白）的动态变化特点，探讨参与ECM交联形成的关键蛋白（LOXL1/FBLN5）在肝纤维化进程中的作用。首先我们利用四氯化碳（CCl4）诱导的小鼠肝纤维化形成/逆转模型动态观察了胶原和弹性蛋白表达、沉积和降解以及LOX家族的变化特点。结果发现在肝纤维化形成/逆转阶段胶原和弹性蛋白的表达模式不同：胶原的变化与纤维化进展和严重程度显著相关。与胶原不同，成熟的弹性蛋白具有"缓升慢降"的特征，特别在肝纤维化晚期显著升高，主要在纤维间隔和门脉区与胶原交织在一起沉积。LOX家族成员的变化同样证实，仅有LOXL1表达水平与交联/不溶性弹性蛋白一致，即在纤维化晚期显著升高，且活化的肝星状细胞（HSCs）是LOXL1的主要来源。提示弹性蛋白和LOXL1在纤维化进展特别是晚期阶段发挥重要作用。其次建立四氯化碳诱导的肝纤维化小鼠模型，通过尾静脉注射重组腺相关载体（rAAV）2/8 shRNA靶向抑制肝脏LOXL1的表达，结果发现抑制肝脏LOXL1表达能显著抑制HSCs活化、减少ECM蛋白（胶原和弹性蛋白）的沉积，具有治疗纤维化的作用。最后，在肝硬化患者中，我们发现弹性蛋白及参与弹性蛋白组装蛋白Fibulin5（FBLN5）显著升高。小鼠体内研究发现，通过rAAV2/8-Fibulin-5-shRNA靶向抑制肝脏FBLN5的表达，延缓CCl4诱导的肝纤维化进展，表现为抑制HSCs活化、减少胶原沉积和纤维化相关指标的表达。综上所述，弹性蛋白沉积和LOXL1在肝纤维化发展过程中发挥重要作用，靶向抑制LOXL1/FBLN5可以减少弹性蛋白交联进而阻止CCl4诱导的肝纤维化进展，可能成为新的潜在的抗纤维化治疗的靶点。