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。

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.