非酒精性脂肪肝（nonalcoholic fatty liver disease, NAFLD）是以肝内脂质大量堆积为特点、当前发病人数及发病率最高的肝脏脂代谢紊乱疾病，极大威胁人们生命健康，对社会经济造成严重负担。饮食疗法是目前公认最为经济、有效的NAFLD治疗方法。以往研究中多关注于饮食中脂质以及简单糖对NAFLD的影响，而对淀粉这种日常生活中主食的主要成分却鲜有研究，尽管高淀粉饮食的负面影响已在人群研究中初步显露，但由于人群试验的多变量性，难以明确淀粉在其中的作用影响。本研究旨在系统性的阐明高淀粉饮食对NAFLD的影响及调控机制，为进一步理解淀粉这一重要营养物质的代谢特点、探寻靶向高淀粉饮食所介导NAFLD的治疗方法提供新的思路。本论文中，我们通过设计多角度实验，发现并证实高淀粉饮食诱导NAFLD发生，并较高脂饮食进一步介导NAFLD恶化。肝脏代谢组学研究表明，在高淀粉饮食干预下，脂肪酸内流的增加是导致肝内脂质进一步沉积的原因，肝脏脂质代谢并不是介导此过程的作用途径。随后，通过肝脏转录组学测序分析，发现高淀粉饮食显著提升小鼠肝脏内NADPH氧化酶2（NADPH oxidase 2, NOX2）的表达及生理功能的发挥。敲低NOX2可特异性的抵抗高淀粉饮食导致的肝内脂质堆积、减少肝脏脂肪酸内流和炎症浸润，在细胞水平以及动物水平均表现出对高淀粉饮食介导的NAFLD的缓解。进一步的，利用Seahorse实验，证明NOX2催化产生的活性氧是调控脂肪酸内流的关键因子。此外，通过追溯脂肪酸来源，我们发现高淀粉饮食通过增加肾周脂肪组织脂解，为脂肪酸内流提供原料，胰岛素抵抗是介导脂解增加的重要原因。最后，我们通过抑制剂实验，证明高淀粉饮食通过抑制AMPK活性介导NOX2的高表达，并且，利用co-IP实验，发现AMPKα1与NOX2亚基p47phox的相互作用是高淀粉饮食增加NOX2表达的依赖途径。综上，本研究独创性的设计了无脂质无游离糖的高淀粉饮食，阐明了高淀粉饮食通过增加特异性调控因子NOX2表达、以促进脂肪酸内流入肝的方式导致NAFLD进一步恶化，发现其不同于简单糖的独特代谢特点；并进一步的证明了NOX2催化产生的ROS在调控脂肪酸内流中的作用，探究了该过程中脂肪酸的供给来源；最后，探明AMPKα1/p47phox是高淀粉饮食介导NOX2高表达的依赖途径。从而系统论证了淀粉这一营养物质对NAFLD的影响及作用机制，为NAFLD的治疗和可靠药物靶点的探寻提供了新的思路和方向。
Nonalcoholic fatty liver disease （NAFLD） is a lipid metabolism disorder disease in the liver with high incidence and morbidity at present, which greatly threaten the health of people and impose a serious burden on the social economy. Diet is currently the most economical and effective treatment for NAFLD. Previous studies focused more on the effects of lipids and simple sugars on NAFLD, while starch, as the main component of the staple food in daily life, has rarely been studied. Although the negative effects of the high-starch diet on lipid metabolism have been gradually revealed in population studies, due to the multivariate of population trials, it is difficult to focuse on starch and track its function. This study aims to systematically elucidate the effects and regulatory mechanisms of high-starch diet on NAFLD, and provides further understanding on the characteristics of starch and new ideas for exploring therapeutic targets for NAFLD induced by the high-starch diet.In this study, we designed experiments to find and confirm in multiple perspectives that a high-starch diet can induce NAFLD, and aggravate NAFLD even worsen than a high-fat diet. Liver metabolomics studies showed that increased fatty acids influx is the cause for further lipid deposition in the liver under a high-starch diet, while lipid metabolism in the liver is not the mediating way. Subsequently, liver transcriptome studies showed that the expression and assembly of NADPH oxidase 2 （NOX2） in the liver of mice were increased under a high-starch diet. Knocking-down NOX2 can specifically resist NAFLD deterioration caused by a high-starch diet, reducing liver fatty acid influx and inflammatory infiltration. The phenomenon that knocking down NOX2 alleviates NAFLD was confirmed at both the cellular level and animal level. Furthermore, we found that ROS produced by NOX2 is a key factor in regulating the influx of fatty acids by the Seahorse experiment. In addition, by tracing the source of fatty acids, we found that perirenal white adipose tissue is the primary source of fatty acids for triglyceride synthesis, and insulin resistance is an important reason in this process. Finally, we demonstrated that AMPK activity was inhibited, which led to the overexpression of NOX2 in the liver of mice, and co-IP assay showed that the high expression of NOX2 mediated by high starch diet depends on the interaction between AMPKα1 and p47phox, a subunit of NOX2.In conclusion, this study creatively designed a high-starch diet without lipids and free sugars, demonstrating that the high-starch diet aggravates NAFLD by NOX2-mediated increased fatty acids influx, illustrating a unique metabolic characteristic that is different from simple sugars. The role of ROS catalyzed by NOX2 in regulating the influx of fatty acids was proposed, and the supply sources of fatty acids in this process were explored. Finally, AMPKα1/p47phox was identified as a dependent pathway of high-expressed NOX2 mediated by the high-starch diet. Therefore, this study systematically elaborated the effects and mechanism of the high-starch diet on liver metabolism, providing new ideas and directions for the treatment of NAFLD and the exploration of reliable drug targets.
