小窝蛋白1选择性自噬对肝窦内皮细胞去窗孔化和肝纤维化的作用机制

The defenestration of liver sinusoidal endothelial cell (LSEC) plays a key role in liver fibrogenesis. Caveolin 1 (Cav1), a structural protein around the fenestrae as well as on vesicles in LSECs, displays close links with autophagy and actin cytoskeletal reorganization. However, the mechanisms about how Cav1 regulates the defenestration of LSECs are unclear. Our previous study showed that LSEC underwent defenestration before liver fibrogenesis, along with enhanced autophagy and the reduction of Cav1 protein expression; autophagy activator promoted the defenestration of LSEC via inducing ubiquitin-autophagic degradation of Cav1 and cytoskeleton remodeling. Thus, we supposed that Cav1 ubiquitin-selective autophagy promotes the defenestration of LSEC and liver fibrosis. To investigate the molecular mechanisms about how the ubiquitin-autophagic degradation of Cav1 induces LSEC defenestration, we employed the CCl4-induced liver fibrosis rat models and cultured primary LSEC isolated from normal rats in vitro, administered with overexpressed or knockdown Cav1, autophagy regulators, as well as intervening ubiquitin and actin. The fenestrae, autophagosomes, and autolysosomes of LSECs were observed by electron microscopy; meanwhile, the MLCK/MLC signaling pathway, cytoskeleton protein, and autophagy related proteins were detected by western blot and co-immunoprecipitation. These results would clarify the significance of LSEC defenestration regulated by Cav1-selective autophagy on liver fibrosis.

肝窦内皮细胞(LSEC)去窗孔化对肝纤维化的启动有重要作用。小窝蛋白1(Cav1)是LSEC窗孔周围和胞浆内的结构蛋白，参与自噬和调节细胞骨架重组。Cav1调控LSEC去窗孔化的机制尚未明确。我们前期研究显示肝纤维化形成前LSEC发生去窗孔化，伴随LSEC自噬增加且Cav1表达下降；自噬促进剂可诱导Cav1发生泛素化自噬性降解，促进细胞骨架重构以加速细胞去窗孔化。本研究提出“Cav1泛素化选择性自噬促进LSEC去窗孔化和肝纤维化”这一假设。以过表达或敲低Cav1、自噬调节剂和干扰泛素化蛋白的方法干预大鼠原代LSEC和四氯化碳(CCl4)诱导的肝纤维化大鼠，电镜观察窗孔、自噬小体和自噬溶酶体，采用蛋白印迹、免疫共沉淀等方法检测MLCK/MLC通路、骨架蛋白和自噬相关蛋白，研究Cav1泛素化自噬性降解促进LSEC去窗孔化的分子机制，阐明Cav1选择性自噬调控LSEC去窗孔化在肝纤维化中的意义。

肝窦内皮细胞(LSECs)去窗孔化对肝纤维化的启动有重要作用。小窝蛋白1（Cav1）存在于LSECs窗孔周围和胞浆的结构蛋白，参与调节自噬和细胞骨架。此外，细胞应激性早衰参与肝纤维化发病和进程。但Cav1介导的自噬和细胞早衰对LSECs去窗孔化和肝纤维化的作用机制尚未明确。我们前期研究发现肝纤维化过程中LSECs发生去窗孔化，且自噬增加、Cav1表达下降，提示Cav1的选择性自噬可能调控LSECs表型和功能。本研究紧密围绕“Cav1泛素化选择性自噬促进LSECs去窗孔化和肝纤维化”这一假设，从体内、外层面探讨Cav1泛素化自噬性降解促进LSECs去窗孔化的分子机制。同时，研究中还发现另一有趣现象，氧化应激诱导细胞发生应激性早衰，通过p53-早衰蛋白（progerin）通路促进骨架蛋白重构和LSECs去窗孔化；促进progerin的核自噬降解可缓解这一过程。本研究深入剖析胞浆中Cav1的选择性自噬和核自噬对LSECs去窗孔化和肝纤维化的作用机制。为逆转早期肝纤维化寻找新的干预靶点提供实验依据。