Ghrelin改善非酒精性脂肪性肝炎（NASH）的作用和机制

Progression from simple steatosis to non-alcoholic steatohepatitis (NASH) is the hallmark of the deterioration of non-alcoholic fatty liver disease (NAFLD). Kupffer cells significantly contribute to the development of NASH. Our preliminary data shows that ghrelin, a gastric hormone, significantly improves the hepatic inflammation and fibrosis induced by low dose of lipopolysaccharides (LPS) in mice fed high fat diet. We thus hypothesize that ghrelin contributes to the improvement of NASH. The following experiments are designed to demonstrate this concept: (1) to examine the effect of global or macrophage specific ghrelin receptor deletion on the development of NASH; (2) to investigate the effects of ghrelin on the M2 polarization of Kupffer cells and its subsquent suppression of hepatic inflammation and fibrosis in LPS-induced NASH. Completion of this project will establish ghrelin as the new therapeutic strategy for intervention of NASH.

非酒精性脂肪性肝病（NAFLD）从单纯性脂肪肝（NAFL）进展到脂肪性肝炎（NASH）是NAFLD恶化、显著增加肝内危害和肝外代谢性疾病的关键节点。Kupffer细胞在NAFL发展为NASH过程中起重要作用。申请者前期实验观察到胃激素ghrelin明显改善低剂量LPS诱导高脂喂养小鼠肝脏的炎症状态，提示ghrelin可能具有改善NASH的作用。本项目将利用全身和巨噬细胞特异性ghrelin受体敲除小鼠在体实验和肝实质细胞与Kupffer细胞离体实验，研究ghrelin改善NASH的作用和机制，探讨ghrelin是否通过诱导Kupffer细胞M2极化分泌抗炎细胞因子进而改善肝脏炎症、纤维化和脂肪变，项目的实施将有助于揭示ghrelin在NAFLD发生发展中的作用，为临床防治NAFLD提供新的理论基础。

Ghrelin改善慢性炎症导致的非酒精性脂肪型肝炎，肝脏脂肪变、炎症和纤维化都减轻。机制是肝脏Kupffer细胞受LPS刺激后升高的TNFα、iNOS被ghrelin抑制，同时增加Arg-1。基础状态和LPS处理后的Kupffer细胞上清给予肝实质细胞后都增加脂质沉积，而ghrelin显著抑制这一现象。小鼠缺失PPARγ无论在肝脏还是肝细胞都显著减轻LPS引起的脂肪变。本研究说明胃分泌的激素ghrelin是胃-肝脏器官对话的重要媒介分子。Ghrelin可能是NASH潜在的治疗靶点。. Ghrelin能促进肝细胞增殖，利用Cell Counting Kit 8和EdU染色进行细胞计数都增加，处于S期的细胞比例上升。Ghrelin显著增加肝脏原代细胞PCNA和cyclin D1表达，降低增殖抑制因子p27表达。敲除受体GHSR1a之后完全消除了ghrelin对原代肝细胞的促增殖作用。Ghrelin激活GHSR1a受体刺激肝脏细胞增殖的作用可能是通过GSK3β/β-catenin信号通路实现的。. 肥胖小鼠埋置微量泵外源性给予ghrelin处理后能增加肝脏中Clock基因的表达，峰值向白天偏移，而Per2基因表达峰值向黑夜偏移，部分逆转了脂肪肝时恶化的节律。体外分离培养小鼠肝脏原代细胞，给予糖皮质激素以同步化细胞时间，检测到肥胖小鼠肝脏原代细胞节律基因表达紊乱并且能被ghrelin改善。小鼠敲除ghrelin受体GHSR1a后再喂养高脂饮食，肥胖引起的节律基因紊乱进一步恶化。肝脏中mTOR/S6信号表现出与节律基因一致的震荡变化，因而ghrelin可能通过mTOR修复肝脏脂肪变时紊乱的昼夜节律。