脯氨酰内肽酶介导肝细胞脂肪变及中性粒细胞诱导的肝内炎症微环境在小鼠NASH发病中的作用

Non-alcoholic fatty liver disease (NAFLD) has been a health problem in China and around the world. Non-alcoholic steatohepatitis (NASH) is its severe type, and the pathogenesis is complex, which still needs to be further elucidated. Hepatic steatosis and intrahepatic inflammatory microenvironment are the two basic factors of NASH initiation and progression. Knowing that infiltrated neutrophils induce intrahepatic inflammatory microenvironment in NASH and that prolyl oligopeptidase (PREP) promotes the infiltration and activation of neutrophils, and based on our newly discovered mechanisms that hepatocyte steatosis induces upgegulation of PREP expression and inhibition of PREP ameliorates hepatocyte steatosis, we hypothesize that “PREP participates in the onset and development of NASH by promoting hepatocyte steatosis and neutrophil-induced intrahepatic inflammatory microenvironment.” Using PREP gene knock-out mice and hepatocytes and neutrophils cultured in vitro, together with high-fat diet- or methionine/choline deficient diet-induced NAFLD/NASH model, PREP inhibitors, and PREP over-expression technology, etc, the impact of PREP on the onset and development of NASH, and the potential cellular and molecular mechanisms will be explored. The aim of this project is to illustrate the critical role of PREP in NASH, and to provide new thought for the development of highly effective and selective PREP inhibitors and possible application for the prevention and treatment of NASH.

非酒精性脂肪性肝病（NAFLD）已是中国和世界性的健康问题，非酒精性脂肪性肝炎（NASH）为其严重类型，发病机制复杂仍亟待深入阐明。肝细胞脂肪变和肝内炎症微环境是NASH发生和进展的两个基本要素。本项目结合中性粒细胞诱导NASH时的肝内炎症微环境、脯氨酰内肽酶（PREP）促进中性粒细胞组织内浸润激活等背景，在我们新近发现脂肪变肝细胞表达PREP上调和抑制PREP缓解其脂肪变基础上，提出“PREP促进肝细胞脂肪变及中性粒细胞诱导的肝内炎症微环境参与NASH发生与进展”的假说，并以Prep基因敲除小鼠、体外培养肝细胞和中性粒细胞等为对象，利用高脂或蛋氨酸/胆碱缺乏饲料诱导NASH模型、PREP过表达和化学抑制剂等手段，观察PREP对NASH发生与进展的影响，探讨潜在细胞分子机制。目的是阐明PREP在NASH发病中的关键作用，为开发高效特异性PREP抑制剂及应用于NASH防治提供新思路。

本项目研究脯氨酰内肽酶（PREP）调控非酒精性脂肪性肝炎（NASH）发病与进展的作用及机制。高脂饮食诱导的单纯性脂肪肝（NAFL）/NASH模型中，发现疾病进展过程中PREP基因表达及活性增加，伴随着PREP-脯胺酰甘氨酰脯氨酸（PGP）/乙酰化PGP（AcPGP)/AcSDKP系统向促炎方向偏移，导致NASH持续进展。PREP基因敲除鼠高脂饲料所诱导NAFL/NASH模型中发现，PREP基因敲除可激活AMPK-SIRT1通路减轻高脂饮食诱导肝脏脂代谢紊乱，血清学脂代谢指标（TG、TC）、肝脏损伤（NAS评分）、脂代谢基因（ACC、FASN、SREBP1c、SCD1和CD36）均较野生型高脂饮食组显著改善；抑制PREP-MMP8/9-PGP通路，减少中性粒细胞以及巨噬细胞肝脏浸润、肝脏炎症反应（IL-1β、TNF-α、IL-6）、炎症信号分子（P-ERK、P-p65）以及血清生化指标（AST、ALT）；改善高脂饮食诱导肠道菌群紊乱并保护肠粘膜屏障，ZO-1和Occludin表达增高。利用蛋氨酸缺乏饮食（MCD）诱导NASH模型中，进一步证实了PREP基因敲除对NASH相关炎症的减轻作用，并结合棕榈酸诱导的HepG2细胞体外实验，发现PREP基因敲除或抑制剂可通过激活P53及其下游抗氧化应激通路（SESN2/NRF2/HO-1）减轻肝脏氧化损伤（ROS、MDA），增强抗氧化指标（CAT、GSH-PX）；PREP天然抑制剂迷迭香酸干预高脂饮食诱导小鼠NASH模型，显著抑制肝内源性PREP活性，降低 ALT 水平；PREP化学合成抑制剂KYP-2047干预高脂饮食小鼠模型，减轻小鼠体重、睾脂指数及肝指数，并改善生化及糖脂代谢指标，降低小鼠炎症细胞浸润和炎症基因（MCP-1、CCR5、TNF-α、LCN-2、IL-1β）、脂质代谢基因（SREBP-1c、CYP7A1、ACACA）表达。高脂加小剂量四氯化碳的NASH相关肝纤维化模型发现PREP基因敲除长期将会加重肝纤维化。本项目揭示PREP基因抑制可减轻NAFL/NASH模型中脂肪变性、炎症以及氧化应激，延缓NASH的进展，是NASH治疗的潜在靶点，但长期抑制的安全性需要慎重评估。PREP基因与NAFLD等慢性肝病关系密切，但其作用在不同肝病类型、疾病病程不同阶段中存在差异，值得进一步研究和临床转化。