缺氧诱导HIF-2α上调加剧非酒精性脂肪肝病进展的机制研究

Emerging evidence indicated that chronic intermittent hypoxia (CIH) was an independent risk factor for the progress of nonalcoholic fatty liver disease (NAFLD). Heterotopic lipid accumulation and activation of immune inflammatory system in the liver are the initiating factors of NAFLD progression, and hypoxia inducible factors HIF-1α/2α play an important role in this process. HIF-1α mainly regulates glucose metabolism, and HIF-2α mainly regulates lipid metabolism. Our previous studies showed that hypoxia reprogramed 3T3-L1 differentiating adipocytes for enhanced triglyceride accumulation, glucose uptake and fatty acid β oxidation, but HIF-1α didn’t change accordingly. So we hypothesize that CIH aggravates liver lipid accumulation and immune inflammatory injury via upregulation of HIF-2α. In the present project, we will treat cell model (human LO2 hepatocyte and primary cultured mice hepatocyte) and mice model (high fat diet induced NAFLD model and liver specific HIF-2α gene knock mice) with high fat diet, CIH, lipopolysaccharide (LPS) or silencing HIF-2α intervention, then observe their effects on inflammatory cell infiltration, inflammatory cytokine secretion, glucose and lipid metabolism and HIF-1α /2α gene expression. If the hypothesis is confirmed, it would elucidate the mechanism of hypoxia-induced HIF-2α upregulation in the progress of NAFLD, provide an accurate and novel target for the prevention and treatment of NAFLD.

国内外研究表明：慢性间歇性缺氧(CIH)是非酒精性脂肪肝病（NAFLD）进展的独立危险因素，肝脏异位脂质聚集及免疫系统活化诱导的炎症因子增多是NAFLD进展的启动因素；缺氧主要通过HIF-1α/2α发挥作用，前者调控糖代谢，后者调控脂代谢。我们前期研究发现：缺氧可改编3T3-L1细胞成脂分化程序，增加细胞内甘油三酯聚集，促进葡萄糖摄取和脂肪酸β-氧化，但HIF-1α变化不明显。据此我们假设：CIH诱导HIF-2α上调加重肝脏脂质聚集和免疫性炎症损伤。拟在细胞（人LO2+小鼠原代肝细胞）和动物（高脂饮食诱导的NAFLD+肝脏特异性HIF-2α基因敲除小鼠）水平，予高脂、CIH、脂多糖或沉默HIF-2α等干预，观察对肝细胞糖脂代谢、炎症细胞浸润、炎症因子分泌及HIF-1α/2α基因表达的影响，阐明HIF-2α在NAFLD进展中的作用机制，为NAFLD的防治提供精准治疗的新靶点。

脂质沉积、巨噬细胞活化及肠道菌群失调是非酒精性脂肪肝病（NAFLD）进展的重要机制。我们在本项目的支持下，建立了FFA诱导的人LO2脂肪变性肝细胞和高脂饮食（HFD）诱导的小鼠NAFLD模型，给予慢性间歇性缺氧(CIH)干预，结果发现：CIH主要通过HIF-2α/PPARα通路促进肝脏脂肪变性，给予HIF-2α siRNA或PPARα激动剂，可逆转缺氧诱导的肝脏脂肪变性增加，给予PPARα抑制剂干预，肝脏脂肪变性加重，研究结果已发表于2019年Am J Physiol Endocrinol Metab. 317(4):E710-E722。同时采用Cre-LoxP技术，成功构建肝脏特异性HIF-2α敲除鼠，给予HFD干预制备NAFLD模型，结果发现：HIF-2α-KO小鼠能缓解HFD诱导的肥胖和NAFLD，KO小鼠内脏脂肪含量更低，肌肉含量增加，对葡萄糖反应更敏感。给予CIH和LPS干预后，M1型巨噬细胞极化的标志物促炎因子IFN-γ，IL-1β, IL-17降低，趋化因RANTES也降低，M2型巨噬细胞极化的标志物IL-10明显升高，说明HIF-2α可调节巨噬细胞极性。肝脏组织学检测发现：与WT小鼠相比， KO小鼠肝铁沉积减少， 在CIH干预后铁调素(HAMP）表达增加，铁转运蛋白（FPN)表达下降。对小鼠粪便进行肠道菌群检测分析发现：厚壁杆菌、拟杆菌在WT和KO存在明显差异，给予CIH干预后，差异减少或者消失，但肠道脱铁杆菌门在各组之间均有显著性差异，CIH干预后，差异进一步扩大。应用多因子检测试剂盒检测小鼠肠道激素，结果发现：HFD干预后，KO组小鼠具有较高的肠促胰素（GLP-1）和胃饥饿素（Ghrelin）水平，给予LPS刺激后，KO组具有较高的GLP-1, PYY和GIP水平。通过上述研究，我们发现：缺氧通过激活HIF-2α//PPARα通路促进肝脏脂肪变性, HIF-2α-KO小鼠能缓解HFD诱导的肥胖和NAFLD, 其机制与肝脏脂质合成减少，脂质过氧化增加、肝铁沉积减少以及肠道菌群改变有关。