NE在非酒精性脂肪性肝炎中性粒细胞介导Kupffer细胞活化中的作用和机制

The activation of kupffer cells (KCs) was confirmed to play a central role in the development of NASH. Neutrophils infiltration in liver is one of the typical histological characteristics of NASH in both animal models and human subjects, nevertheless, the interactions and relationships between neutrohpils and KCs remains elusive. Results from our studies demonstrated that neutrophils may play an initiating role in NASH by helping to recruit and activate KCs via neutrophil elastase (NE), and we hypothesize that NE play its mechanistic role by acting on the Toll-like receptor 4 (TLR4) of KCs.To confirm this hypothsis, firstly, we will investigate the role the NE during the process of KCs activation both on ApoE-/-NE-/- and bone marrow macphage/neutrophil specific ApoE-/-NEfl/flLysM-Cre+ mice combining with the applying of recombinant mouse NE. Then, we will explore its mechanistic role with the ApoE-/-TLR4-/- mice both in vivo and vitro. Finally, we will isolate KCs from liver and co-cultrue them with neutrophils, observing the levels of TLR4 –TRIF-IFNβpathway of KCs to explore its reaction to neutrophils. In summary, we will clarify the role of NE in NASH via the interaction between KCs and neutrophils.

Kupffer细胞（KCs）活化在非酒精性脂肪性肝炎(NASH）发生发展中起着重要作用。我们前期研究发现中性粒细胞（PMNs）在肝内募集后通过分泌弹性蛋白酶（NE）介导NASH早期炎症启动，并可能通过TLR4通路活化KCs导致炎症进一步放大，但具体分子机制尚不清楚。本研究首先应用ApoE-/-NE-/-小鼠和肝脏特异性ApoE-/-NEfl/flLysM-Cre+小鼠，以及NE过表达等技术，证实NE在PMNs介导KCs活化中的关键作用；其次采用ApoE-/-TLR4-/-小鼠及其分离的KCs，通过体内外实验来阐明NE介导KCs活化中的TLR4依赖机制；最后通过分离NASH小鼠KCs，并与小鼠PMNs共培养等方法，探讨NE介导的TLR4-TRIF-IFNβ负反馈通路在KCs调控PMNs凋亡的机制及其在促/抑炎稳态调控中的作用。以此阐明PMNs和KCs在NASH发生发展中的相互作用和调控机制。

非酒精性脂肪性肝炎 (Nonalcoholic steatohepatitis, NASH) 是由营养过剩导致的肝脏脂肪变性与炎症的肝脏疾病。天然免疫失衡是非酒精性脂肪性肝炎发病的关键环节。中性粒细胞弹性蛋白酶 (Neutrophil elastase, NE) 是中性粒细胞所分泌的一种蛋白酶，有研究显示NE是肥胖的潜在致病因子，但NE是否参与NASH的发生和发展以及具体的机制尚不清楚。本研究首先在APOE-/-及C57/B6小鼠中敲除NE基因，并以高脂高胆固醇饮食 (High fat and high cholesterol diet, HFHC) 进行诱导建立NASH小鼠模型，结果发现NE基因敲除的APOE-/-NE-/-以及NE-/-小鼠肥胖、脂代谢异常明显改善及库普弗细胞（Kupffer cell，KC）活化受到抑制 ；随后，我们利用ApoE-/-TLR4mut及ApoE-/-TLR4-WT小鼠，通过HFHC进行诱导建模，并予以rNE进行干预，结果表明rNE可加重HFHC诱导所致的肝脂肪变性，血脂紊乱与KC的活化，这些作用依赖TLR4信号通路；然而体外实验中并未能证实rNE促进KC的活化；进一步的rNE与原代肝细胞共培养后，发现rNE可显著上调肝细胞表达炎症趋化因子TNF-α及MCP-1，并且这种增加炎性趋化因子的表达是通过rNE上调肝细胞神经酰胺来实现的；本研究在人体中进一步证实了NASH患者组织学的损伤与血浆及肝内NE水平相关，血浆NE水平有助于NASH及NASH纤维化的鉴别诊断。本研究揭示了NE具有可促进高质高胆固醇饮食诱导NASH的发生和进展，具体作用机制为NE通过上调肝内神经酰胺代谢，促进肝内毒性脂质神经酰胺蓄积，导致肝细胞炎症及释放炎症趋化因子，最终导致KC的募集与活化，促进NASH的进展。