FXR调控自噬在肝脏缺血再灌注损伤中的作用及机制研究

Autophagy protects against ischemia reperfusion (I/R) injury. However, the autophagy flux is impaired during reperfusion, the mechanism of which is unclear. We have identified the TRPML1, a key lysosome protein, is transcript down regulated during reperfusion, which attenuates the autophagosome and lysosome fusion. Farnesoid X receptor (FXR), a bile acid receptor, negatively regulates autophagy flux by suppressing transcription factor EB (TFEB), a key transcriptional activator of lysosome genes. Hence we hypothesize that transcription of TRPML1 genes by TFEB is suppressed by FXR activation during reperfusion, which exacerbates liver I/R injury. Our preliminary experiments found high FXR expressions in human allografts associate with poor transplant outcomes. FXR suppression protects against I/R injury. TRPML1 knock-down abolished this effect. In current study, we aim to investigate role of FXR in regulating hepatocyte survival in in vitro I/R model, demonstrate autophagy is regulated by FXR, verify whether FXR suppresses autophagy by inhibiting the transcriptional activity of TFEB, and identify whether TRPML1 is transcript regulated by TFEB. Finally, we confirm the regulatory role of FXR on autophagy and I/R injury in liver I/R model. This study aims to uncover the mechanism of autophagy flux impairment at reperfusion and may provide a novel therapeutic target for the prevention and treatment of liver I/R injury in clinics.

自噬在缺血再灌注（I/R）损伤中起重要保护作用，然而再灌注时自噬流却受阻，原因不明。我们前期发现肝I/R后溶酶体蛋白TRPML1转录下调，引起自噬体积聚而无法降解。已知胆汁酸受体FXR可通过抑制转录因子EB（TFEB）对溶酶体基因的转录而抑制自噬流。因此我们假设FXR可能调控肝I/R损伤：再灌注时大量胆汁酸入肝激活FXR，抑制了TFEB对TRPML1的转录，导致自噬流受阻，加重了I/R损伤。我们预实验发现供肝FXR高表达与移植预后不佳有关。抑制FXR可促进自噬流，保护I/R损伤，敲减TRPML1则该保护作用丧失。本课题拟在体外I/R中证实FXR通过调控自噬流影响细胞生存，阐明FXR通过抑制TFEB调控自噬流，并确证TFEB可转录调控TRPML1；在体内干预FXR观察对自噬的调控作用及对I/R损伤的影响。本研究旨在揭示I/R后自噬流受阻的原因，进而提出了肝I/R损伤治疗的新靶点。

在肝脏缺血/再灌注(I/R)损伤中，FXR如何调节炎症反应的确切机制尚不清楚，这限制了FXR激动剂在炎症相关肝病中的临床应用。本研究旨在探讨FXR在肝脏缺血/再灌注(I/R)损伤中的确切作用机制。在本研究中，我们发现FXR在人肝脏I/R后表达下调。在肝脏FXR KO的小鼠中，肝脏坏死加重，炎症相关通路上调。FXR KO原代肝细胞中P65DNA结合活性增强，而FXR可通过与P65的TAD结合而抑制P65的DNA结合活性，从而减弱P65与P300之间的相互作用。FXR激动剂或转染可显著减少细胞凋亡，而转染p65则可减弱这一作用。相反，FXR抑制剂增加了凋亡细胞，并可被NEO2734挽救。总而言之，本研究发现了FXR抑制P65和P300之间的相互作用，从而保护肝脏免受I/R损伤。