HO-1-induced autophagy establishes a HO-1-p62-Nrf2 positive feedback loop to reduce gut permeability in cholestatic liver disease

The gut-liver axis disruption is a unified pathogenetic principle of cholestatic liver disease (CSLD). Increased gut permeability is the leading cause of gut-liver axis disruption. HO-1 is capable of protecting against gut-liver axis injury. However, it has rarely been reported whether autophagy is involved in HO-1 protecting gut-liver barrier integrity and the underlying mechanism. Mice underwent bile duct ligation (BDL) was established as CSLD model in vivo. Caco-2 cells with LPS treatment was established as in vitro cell model. Immunofluorescence, western blot and transepithelial electrical resistance (TER) assay were used to observe epithelial tight junction (TJ) and autophagy. Liver injury and fibrosis were evaluated as well through H&amp;E staining, masson staining, sirius red staining and ELISA. Our study demonstrated that the epithelial TJ and TER were notably reduced both in BDL mice and in LPS treated intestinal epithelial cells. Increased HO-1 expression could significantly induce intestinal epithelial cell autophagy. Additionally, this increased autophagy level reversed the reduction effects of BDL or LPS on epithelial TJ and TER <i>in vivo</i> and <i>in vitro</i>, therefore decreased transaminase level in serum and relieved liver fibrosis in BDL mice. Besides, increased autophagy level in turn upregulated the expression of HO-1 by p62 degradation of Keap1 and subsequent activation of Nrf2 pathway. Collectively, these results indicate that HO-1 reduces gut permeability by enhancing autophagy level in CSLD, the increased autophagy establishes a HO-1-p62-Nrf2 positive feedback loop to further improve gut-liver axis disruption. Therefore, our study confirms the critical role of autophagy in HO-1 ameliorating gut-liver axis injury during CSLD, highlighting HO-1 as a promising therapeutic target.

肠-肝轴紊乱是胆汁淤积性肝病（cholestatic liver disease, CSLD）的统一发病机制。肠道通透性升高是引发肠-肝轴紊乱的首要致病因素。血红素氧合酶-1（heme oxygenase-1, HO-1）可对肠-肝轴损伤发挥保护作用，但目前鲜有研究探讨自噬是否参与HO-1对肠-肝屏障完整性的保护作用及其潜在分子机制。本研究通过构建接受胆管结扎术（bile duct ligation, BDL）的小鼠作为体内CSLD模型，以脂多糖（lipopolysaccharide, LPS）处理Caco-2细胞构建体外细胞模型。采用免疫荧光、蛋白质印迹（western blot）及跨上皮电阻（transepithelial electrical resistance, TER）实验检测上皮紧密连接（epithelial tight junction, TJ）与自噬水平；同时通过苏木精-伊红染色（H&E staining）、马松三色染色（masson staining）、天狼星红染色（sirius red staining）及酶联免疫吸附测定（ELISA）评估肝损伤与肝纤维化程度。研究结果显示，BDL小鼠及LPS处理的肠上皮细胞的上皮紧密连接与跨上皮电阻均显著降低。HO-1表达升高可显著诱导肠上皮细胞自噬。此外，体内外实验中，自噬水平的升高可逆转BDL或LPS对上皮紧密连接与跨上皮电阻的抑制作用，从而降低BDL小鼠血清转氨酶水平并缓解其肝纤维化。进一步研究发现，升高的自噬水平可通过p62降解Keap1及后续核因子E2相关因子2（nuclear factor erythroid 2-related factor 2, Nrf2）通路激活，反过来上调HO-1的表达，形成HO-1-p62-Nrf2正反馈环路。综上，本研究证实，在CSLD进程中，HO-1可通过提升自噬水平降低肠道通透性；而升高的自噬可形成HO-1-p62-Nrf2正反馈环路，进一步改善肠-肝轴紊乱。本研究明确了自噬在HO-1缓解肠-肝轴损伤中的关键作用，提示HO-1是极具潜力的治疗靶点。