DHA 调控Kupffer细胞极化途径缓解NASH肝细胞炎性损伤

Nonalocoholic steatohepatitis (NASH) is increasingly recorgnized as a major health burden, which is a poorly understood severe liver disease. Docosahexaenoic acid (DHA), a long-chain omega-3 polyunsaturated fatty acid (PUFA), is known to have important beneficial effects on inflammation in NASH. However, it is not clear about the mechanism underlying DHA-induced protection in NASH. The imbalance of proinflammatory M1 and anti-inflammatory M2 polarization in Kuffer cells plays an important role in NASH. Our pilot study indicates that DHA increases anti-inflammatory M2, but decreases proinflammatory M1 polarization in Kuffer cells. These findings led to a hypothesis that M2 activation/M1 inhibition–induced by DHA inhibits liver inflammation in NASH. The following 2 specific aims will be pursued to test our hypothesis. 1) NASH rat model will be engaged to determine whether DHA protects againist liver inflammation via the effect on the ratio of M2/M1 polarization in Kuffer cells; 2) Taking advantage of in-vivo cell culture of liver microphages, flow cytometry, immunohistochemistry, PCR and pharmacology tools will be served to determine whether DHA activates M2 polarization via GPR120/40, PPARs activation and the underlying pathways, including NLRP3, STAT6, RXR; Also, whether DHA inhibits M1 via TLRs—NF-κB pathway will be investigated. Findings from our proposal may not only reveal the fundamental mechanisms underlying DHA-induced anti-inflammation, but also may provide a rationale for the development of preventions and therapies for patients with NASH.

NASH是以炎性损伤为主要特征的严重脂肪肝疾病，广受关注。DHA是一种多不饱和脂肪酸类营养素，可改善NASH肝炎性损伤，但机制不清。肝驻守Kupffer细胞（KC）是局部组织免疫应激调控的中心，可激活形成促炎的M1和抗炎的M2两种极化亚型。前期实验提示， DHA可选择性促进M2极化，抑制M1极化。故提出假说：DHA通过激活M2/抑制M1而缓解NASH肝炎性损伤。对此展开研究：动物实验研究DHA是否激活M2和/或抑制M1，改善NASH肝炎性损伤；细胞实验，利用免疫组化，流式细胞术及信号通路的激动或阻断等方法，研究DHA是否通过GPR120、PPARs等受体介导，调控NLRP3、STAT6、RXR等信号途径，激活M2极化途径，并通过活化的转录因子与分泌产物抑制TLRs—NF-κB等M1极化途径，缓解NASH肝炎性损伤。研究将揭示DHA独特的抗炎作用及机制，为NASH防治提供理论依据。

非酒精性脂肪性肝病（NAFLD）是一个世界范围内广泛流行的营养相关的代谢性疾病，其主要特征是胰岛素抵抗及慢性系统低度炎症，其疾病谱包括从单纯性非酒精性脂肪肝（NAFL）、非酒精性脂肪性肝炎（NASH）以及逐渐恶化为肝纤维化和肝硬化。NASH阶段如果能得到有效的干预，可有效的避免疾病往肝纤维化、肝硬化、肝癌等不可逆的病程发展。已经有越来越多的研究证明，DHA可显著改善肝脏脂代谢的紊乱，降低促炎细胞因子的分泌和炎性细胞浸润，从而降低肝细胞的炎性损伤程度，缓解NASH发展。Kupffer细胞作为肝脏驻守的巨噬细胞，是肝组织局部免疫调节的中心，存在两条相反的极化途径，即经典活化型（M1型，促炎）和替代活化型（M2 型，抗炎）。因此，我们提出假设：DHA通过调节kupffer细胞的极化方向，即激活M2/抑制M1，从而缓解NASH肝炎性损伤。项目组从动物水平及细胞水平进行了系统的研究：1).通过在饲料中添加不同的油脂，检测不同时间段实验大鼠的肝组织中脂代谢、炎性损伤、kupffer细胞极性状态的各项指标，研究kupffer细胞极性与NASH病程发展的关系及DHA的干预效果；2).用软质酸（PA）及DHA对大鼠巨噬细胞RAW264.7进行不同的干预方式，观察PA及DHA对RAW264.7细胞极性转变的影响，明确DHA诱导Kupffer细胞M2极化对NASH肝细胞炎性损伤的缓解作用；3).在用不同的脂肪酸对细胞进行干预的基础上，同时利用内质网应激的兴奋剂及抑制剂对RAW264.7进行干预，探索DHA影响kupffer细胞极化的分子机制。实验的数据显示，长期的高脂饮食，促使kupffer细胞向促炎的M1方向转变，加重炎症损伤，而DHA可以有效促使kupffer细胞向抗炎的M2方向活化，缓解炎症损伤。细胞实验通过不同的脂肪酸诱导kupffer细胞的极化变化，有力地印证了动物实验的结果。我们还发现，饱和脂肪酸可能通过影响kupffer细胞的内质网应激促使其向M1极化，而DHA可显著地缓解其内质网应激促使其向M2转化。本项目的研究结果明确了kupffer细胞的极化是DHA缓解NASH炎性损伤的重要机制之一，为DHA干预NASH奠定一定的理论基础。