胞外微纤维相关糖蛋白1(MAGP1)介导弹性蛋白交联沉积在肝纤维化逆转中的作用及机制

Crosslinked elastin is excessively deposited in the extracellular matrix of fibrotic livers, which is hardly degradable and considered as a vital factor responsible for the irreversibility of liver fibrosis. Microfibril-associated glycoprotein 1 (MAGP1) constitutes a key component of microfibril scaffold where elastin globules accumulate. Our preliminary study reveals that, during advanced liver fibrosis, MAGP1 is closely associated with the formation of liver elastic fibers; the expression and localization of MAGP1 is highly conserved with elastin and lysyl oxidase-like 1 (LOXL1). In addition, there also exists a direct interaction between MAGP1 and LOXL1 in mouse primary hepatic stellate cells. However, the specific role and its underlying mechanism of MAGP1 in fibrotic liver elastin crosslink and deposition, and in the reversion of liver fibrosis remain unclear. In order to provide direct evidence to illuminate the relationships among MAGP1, elastin and LOXL1, our present study will first clarify the dynamic changes of the expression and localization of the three members using colloidal gold immune electron microscopy in mice models of mild or advanced liver fibrosis. Afterwards, the MAGP1 knockout mice and AAV2/8 vector-induced MAGP1 overexpression mice will be utilized to, in vivo, illuminate the specific role of MAGP1 in liver elastin crosslinking and deposition, and the effect of MAGP1 on the reversibility of liver fibrosis. The primary hepatic stellate cells lacking or overexpressing MAGP1 are then isolated and cultured. Also, the cells conditionally expressing MAGP1 and continuously expressing LOXL1 are established. The two in vitro models are used to further understand the role of MAGP1 in LOXL1-mediated liver elastin crosslinking and deposition. Our study aims to provide potential targets and experimental evidence for optimized treatment of liver fibrosis.

细胞外基质弹性蛋白交联沉积不被降解是肝纤维化不易逆转的重要因素。微纤维相关糖蛋白1（MAGP1）是弹性蛋白交联沉积所需微纤维骨架的主要结构蛋白。我们前期研究发现MAGP1与晚期肝纤维化弹性蛋白交联沉积相关，与赖氨酸氧化酶同系物1（LOXL1）直接互作且表达模式一致。然而MAGP1在弹性蛋白交联沉积及肝纤维化逆转中的具体作用和机制尚不明确。本研究拟在肝纤维化进展/逆转模型中，利用胶体金免疫电镜观察MAGP1/弹性蛋白/LOXL1表达与定位的动态变化；应用基因敲除及AAV2/8载体过表达MAGP1小鼠，阐明MAGP1及其介导的弹性蛋白交联沉积在肝纤维化进展/逆转中的作用；原代分离培养MAGP1敲除/过表达肝星状细胞，同时构建条件表达MAGP1并持续表达LOXL1的稳转细胞系，探讨MAGP1在LOXL1调控纤维化肝脏弹性蛋白交联沉积中的作用。本研究旨在为肝纤维化临床治疗提供潜在靶点和实验依据。

细胞外基质弹性蛋白交联沉积不被降解是肝纤维化不易逆转的重要因素。微纤维相关糖蛋 白1（MAGP1）是弹性蛋白交联沉积所需微纤维骨架的主要组分。然而MAGP1在弹性蛋白交联沉积及肝纤维化逆转中的具体作用和机制尚不明确。本研究观察了肝纤维化进展和自发逆转小鼠模型中MAGP1蛋白表达与定位的动态变化；干预MAGP1对CCL4诱导的肝纤维化进展的影响和对弹性蛋白交联沉积的作用；弹性蛋白在肝纤维化动态变化过程中的病理特征；LOXL1调控LOX介导胶原、弹性蛋白沉积及肝星状细胞促纤维化行为的作用等。研究发现MAGP1蛋白表达仅在肝纤维化后期显著上调，肝纤维化逆转期表达下调；肝星状细胞是MAGP1的主要细胞来源；体内敲除MAGP1对早期肝纤维化无显著影响，但可显著减轻CCL4长期刺激诱导的肝纤维化；MAGP1对肝纤维化的影响存在性别差异，敲除MAGP1仅能减轻雄性小鼠肝纤维化进展；无论是雄性还是雌性，无论是早期还是后期肝纤维化，MAGP1敲除均可以显著影响肝脏弹性蛋白的沉积；弹性蛋白在肝纤维化进展及逆转过程中具有“缓升慢降”的特征；阐明了LOXL1可通过调控LOX的表达，进而影响肝星状细胞的促纤维化行为的作用。本研究可在为肝纤维化治疗提供潜在靶点和实验依据。