促红细胞生成素诱导肝细胞自噬改善肝脏脂质代谢的机制研究

Erythropoietin (EPO) has been shown to have beneficial effects on glucose and lipid metabolism. We recently demonstrated that EPO activated hepatic phosphatidylinositol 3-kinase (PI3K)/AKT to improve insulin resistance in vivo and vitro. Importantly, we also found that EPO dramatically decreased hepatic lipid accumulation. However, the precise mechanisms still remain unclear. Autophagy is a major catabolic pathway involved in the degradation of intracellular organelles and macromolecules and was recently found to contribute to the breakdown of intracellular lipid droplets. Here, we found that EPO administration increased expression of autophagy markers Beclin1 and microtubule-associated protein light-chain 3-Ⅱ (LC3-Ⅱ) /LC3-Ⅰ, and inhibition of autophagy significantly attenuated the ability of EPO to decrease cellular lipid storage in palmitate-pretreated HepG2 cells, indicating that EPO reduced hepatic lipid accumulation through restoration of hepatic autophagic function. Based on these results, further studies will be performed to explore the molecular mechanism by which autophagy mediates EPO-induced reduction in hepatic lipid accumulation with gene knockdown mice and cell models. This study will provide a clue into the specific therapeutic target for EPO improving hepatic lipid over-accumulation and obesity.

促红细胞生成素（EPO）在改善肥胖及糖脂代谢紊乱方面有潜在作用，我们近期体内和体外实验发现EPO可激活磷酯酰肌醇3激酶/蛋白激酶B（PI3K/AKT）通路而改善肝脏胰岛素抵抗，同时也有助于改善肝脏脂质沉积，但其分子机制不明确。自噬是真核生物中高度保守的清除和降解细胞内受损细胞器以及多余生物大分子的主要代谢通路，近期研究发现其可促进肝细胞内脂滴降解而抑制脂质过度沉积。我们前期研究发现EPO可上调自噬相关蛋白酵母自噬基因Atg6同系物(Beclin1)和微管相关蛋白1轻链3-Ⅱ（LC3-Ⅱ）/LC3-Ⅰ的表达，而抑制自噬可拮抗EPO改善肝脏脂质沉积的作用，表明EPO可能通过诱导自噬而改善肝脏脂质沉积。本研究将通过建立基因敲除的小鼠模型和细胞模型等方法，进一步阐明自噬介导EPO改善肝脏脂质沉积的分子机制。研究结果将为EPO改善肝脏脂质代谢和肥胖的靶点研究奠定理论基础。

近年研究发现促红细胞生成素（EPO）可以降低肝细胞中甘油三酯的含量，但具体作用机制尚不明确。本研究通过动物及细胞实验，发现EPO干预能降低肥胖小鼠肝脏及棕榈酸处理的肝细胞系中的脂质含量，并通过多种监测手段证实EPO能激活细胞自噬，包括western blots技术检测自噬标记蛋白水平的上调，电镜观察自噬小体数量的增多，向肝细胞内转染mRFP-GFP-LC3自噬荧光双标腺病毒后通过共聚焦显微镜观察自噬小体与自噬溶酶体数量的增加。而免疫荧光染色观察到EPO干预后脂滴与自噬标记蛋白LC3共定位的增多，进一步通过小干扰RNA转染或抑制剂干预，证实抑制细胞自噬削弱了EPO降低肝细胞脂质含量的作用。同时，沉默信息调节因子（SIRT1）是自噬与细胞能量代谢中重要的调控者。本研究中观察到EPO能增加SIRT1的蛋白表达水平并上调烟酰胺腺嘌呤二核昔酸（NAD+）/NADH比值、增加其酶活性，从而降低LC3的乙酰化水平有助于自噬进程的启动，抑制SIRT1的表达则阻碍了EPO通过诱导自噬改善肝脏脂肪变的作用。通过构建肝脏特异性SIRT1敲除小鼠，进一步在体内模型中明确SIRT1在介导EPO诱导自噬改善肝脏脂代谢的重要作用。因此，本研究首次表明EPO可以通过激活SIRT1，降低LC3乙酰化水平，诱导细胞自噬，从而发挥改善肝脏脂肪变的重要作用, 为扩展EPO在代谢领域的应用提供有力的实验依据。