基于 CREB1/HtrA2/Hax-1 研究早期过度营养促进 NAFLD 发生发展的作用机制

Non-alcoholic fatty liver disease (NAFLD) has emerged as the most common chronic liver disease. The postnatal overfeeding induced by small rearing (SL) increases the risk of NAFLD, however, the mechanism remains elusive. Abnormal hepatic lipid metabolism is the major pathognomonic factor for NAFLD. The mitochondria dysfunction contributes to the dysregulation of lipid metabolism, while autophagy can improve the mitochondrial damage and alleviate hepatic lipid metabolism disorder. We observed that early overfeeding rats have clinical pathological changes of NAFLD at 3 weeks. Moreover, the mitochondrial function gene and ATP in the liver tissue is significantly decreased. The protein level of HtrA2, p-CREB1, LC3II / I and Beclin-1 is also reduced, while Hax-1 shows the the opposite result. Therefore, we hypothesis early overfeeding can regulate autophagy and then participate in mitochondrial dysfunction, which lead to the development of NAFLD. And CREB1/HtrA2/Hax-1 signaling pathway may be involved in this phenomenon. In this study, we will establish animal model by small litter rats during lactation and given different intervention. We will elucidate the clinical outcomes and mechanism of early overfeeding-indeced autophagy inhibition and mitochondria dysfunction in the promotion of NAFLD. Furthermore, with siRNA and overexpression plasmid, we will confirm that the biological function of FFA is mediated by CREB1/HtrA2/Hax-1 pathway. Finally, knockout mice will be used to verify the conclusion, as well as the molecular mechanism. These results obtained are benefit for us to clarifying the underlying mechanisms of postnatal overfeeding-induced NAFLD and may provide the molecular target and method for early nutritional programming intervention.

NAFLD的发病率逐年升高，生命早期过度营养是NAFLD发生的高危因素，但具体机制不明。肝脏脂质代谢异常是关键致病环节，线粒体损伤在脂质代谢方面具有重要地位，而自噬是减轻线粒体受损，缓解脂质代谢紊乱的关键途径。我们前期研究发现：早期过度喂养诱导CREB1/HtrA2/Hax-1信号通路改变，抑制自噬。本研究假设CREB1/HtrA2/Hax-1调控自噬，参与早期过度喂养引起的脂质代谢和线粒体损伤，进而影响NAFLD发生发展。本项目拟通过小窝组喂养建立哺乳期过度营养模型及断乳后不同干预，考察该通路、自噬及线粒体功能在早期过度喂养促进NAFLD中的变化情况；进一步通过肝细胞脂化模型，结合SiRNA和过表达质粒，在细胞水平分析CREB1/HtrA2/Hax-1在早期过度喂养促进NAFLD发生发展中的作用及机制；最后使用敲除鼠进行验证，研究结果为NAFLD的早期防治提供新策略、新靶点、新思路。

近年来，随着人们生活水平的提高，非酒精性脂肪肝（NAFLD）已经成为最常见的慢性肝脏代谢综合征，影响着全球数百万人。研究证明改善肝细胞线粒体功能和自噬是预防NAFLD的一种重要手段。高温需要蛋白A2（High-temperature requirement protein A2， HtrA2/Omi）能够维持肝细胞中的线粒体稳态和自噬。因此，我们探讨了HtrA2/Omi在NAFLD发生发展中的作用及HtrA2/Omi是否能改善线粒体功能和自噬。在本研究中，我们建立了高脂肪饮食（HFD）喂养的小鼠NAFLD模型和游离脂肪酸（FFA）处理的L02肝细胞模型，并使用腺相关病毒（AAV）恢复肝体内模型中HtrA2/Omi表达，在体外使用质粒恢复L02肝细胞模型中HtrA2/Omi的表达，分别在肝组织和L02肝细胞模型中评估了HtrA2/Omi对线粒体稳态和自噬能力的影响。研究结果显示，HFD诱导的小鼠NAFLD模型中的肝组织和经FFA处理的L02细胞中，HtrA2/Omi表达显著降低。恢复肝脏中HtrA2/Omi的表达可改善肝脏脂肪变性，同时，线粒体功能障碍和自噬阻断也被减弱。肝脏HtrA2/Omi过表达增加了线粒体DNA水平并提高了ATP的含量。TEM分析显示，与HFD小鼠相比，肝脏HtrA2/Omi过表达小鼠显著改善肝脏的线粒体结构和自噬体形成。肝脏HtrA2/Omi过表达也增强线粒体脂肪酸β-氧化基因表达，提高LC3II蛋白水平，降低SQSTM1/p62蛋白水平。此外，肝脏HtrA2/Omi过表达增加了小鼠的呼吸交换率和热量产生。在体外实验中，HtrA2/Omi过表达降低了L02肝细胞模型的线粒体功能障碍和自噬抑制。综上所述，我们首次证明HtrA2/Omi能够通过改善线粒体功能以及恢复自噬流量来治疗NAFLD。另一方面，研究中发现肥胖小鼠体内炎症因子明显升高，异于正常小鼠，我们发现aprepitant可以通过抑制JNK和p38的磷酸化和抑制NF-κB信号通路来抑制小鼠炎症因子，缓解由于炎症因子刺激神经末梢所引起的炎性疼痛。