DDC通过MMP13/CXCL12轴调控巨噬细胞功能转化促进肝纤维化逆转的机制研究

Macrophages play the important role in liver fibrosis and resolution, and CXCL12 is involved in the process of macrophage transition. Our previous study showed that diethyldithiocarbamate (DDC) could modulate the transition of macrophage. However, the mechanism remains unclear. We found that DDC could up-regulate matrix metalloproteinase-13 (MMP-13), and overexpression of MMP-13 can result in decrease of active from of CXCL12, which is a substrate of MMP-13. Therefore, we hypothesized that DDC modulates the phenotype and function transition of macrophage through MMP-13/CXCL12 axis, and then influences the process of fibrolysis. In this study, we will first investigate the effect of DDC on the expression of MMP-13, the activity of CXCL12, the phenotype of macrophages and the stage of liver fibrosis in the animal model of liver fibrosis. Second, we plan to explore whether MMP-13/CXCL12 axis is involved in DDC induced transition of macrophage, by using AAV-8 recombinant virus and the conditional knockout mice to intervene the expression of MMP-13 and CXCL12. Finally, through intervening the expression of MMP-13 and CXCL12, we will further explore the change of macrophage subsets, and signaling pathways and transcriptional factor involved in the process of DDC induced transition of macrophage by using the in vitro tri-culture system. The study will provide evidence for the prevention of liver fibrosis by DDC through regulating the transition of macrophage.

巨噬细胞在肝纤维化形成及降解过程中发挥重要作用，CXCL12参与了巨噬细胞的转化过程。前期结果提示DDC对巨噬细胞的转化有调控作用，但其机制仍不清楚。我们发现DDC可上调MMP13，而过表达MMP13可导致其底物CXCL12的活化形式减少。据此，我们提出假设：DDC通过MMP13/CXCL12通路调控巨噬细胞功能转化，影响纤维降解过程。在本研究中，我们首先探讨DDC对肝纤维化小鼠MMP13表达、CXCL12活性、巨噬细胞表型及纤维降解的影响；接下来应用重组腺相关病毒及条件性敲除小鼠干预MMP13及CXCL12，明确MMP13/CXCL12通路在DDC调控巨噬细胞转化中的作用；最后通过体外实验干预MMP13、CXCL12，明确参与调控的信号通路及转录因子，阐明DDC通过上调MMP13调控巨噬细胞转化参与纤维降解的分子机制。旨在从巨噬细胞功能调控的角度为DDC抗肝纤维化治疗提供实验依据。

非酒精性脂肪性肝病（non-alcoholic fatty liver disease，NAFLD）的主要特征是肝脏脂肪的过度堆积，与胰岛素抵抗和遗传易感等因素密切相关。NAFLD包括两种预后不同的病理学形态，非酒精性脂肪肝（non-alcoholic fatty liver，NAFL）和非酒精性脂肪性肝炎（non-alcoholic steatohepatitis，NASH），后者包括纤维化、肝硬化和肝细胞癌（hepatocellular carcinoma，HCC）。随着肥胖与代谢综合征的流行，NAFLD已成为全球第一大肝病，但目前仍缺乏有效的治疗药物。在本研究中，我们首先建立了MCD及CDAA饮食诱导的NASH肝纤维化小鼠模型，给予二乙基二硫代氨基甲酸（diethyldithiocarbamate, DDC）治疗。结果显示，DDC对于两种NASH肝纤维化小鼠模型均有明显的治疗作用。接下来，我们通过转录组学、脂质组学等方法分析了DDC治疗NASH肝纤维化的可能机制。结果显示，DDC主要通过调控脂代谢及线粒体功能发挥治疗作用。最后，我们建立了脂变肝细胞与肝星状细胞的共培养体系，进一步验证了DDC抗NASH肝纤维化的分子机制。在本研究中，我们首次报道了二乙基二硫代氨基甲酸DDC可以显著改善小鼠非酒精性脂肪性肝纤维化。并且，与其他临床前期药物相比，DDC可能通过新的作用机制发挥治疗作用。DDC是戒酒药物双硫仑的主要代谢活性产物，作为一种已经在临床应用的药物，其药代动力学、药效动力学及毒理学已有全面深入的研究，如能用于治疗非酒精性脂肪性肝炎，将能够更快地向临床转化，应用于广大非酒精性脂肪性肝病患者。