肝脏生物钟通过USP2-45/PPARα信号通路调控肝脏甘油三酯含量的机制研究

肝脏脂肪代谢稳态与人类健康密切相关。研究证明人体生物钟调控功能紊乱可导致脂肪肝的产生，但其具体分子机制还不清楚。我们研究发现，钟基因Per1/2/3敲除小鼠肝脏甘油三酯含量显著降低，去泛素化酶Usp2-45表达升高，PPARα蛋白及其下游信号通路上升。本研究以“肝脏生物钟在转录水平调控Usp2-45的表达，USP2-45通过去泛素化调节PPARα蛋白的降解，从而影响肝脏脂肪代谢”为假说，通过Per全身性敲除、肝细胞特异性Bmal1敲除、Usp2敲除小鼠模型，利用油红O染色、转录组测序、RT-qPCR、WB、IHC、启动子活性分析、实时生物荧光测定、ChIP、CoIP等技术，研究肝脏生物钟对肝脏脂肪代谢的影响，并揭示肝脏生物钟通过USP2-45/PPARα信号通路调控甘油三酯含量的分子机制。为全面认识肝脏脂肪代谢调控网络，从生物钟角度寻找治疗脂肪肝等代谢性疾病的靶位点提供理论基础和实验依据。

The homeostasis of hepatic lipid metabolism is closely related to human health. It has been shown that circadian clock dysfunction of human body can cause fatty liver disease. However, the underlying molecular mechanism remains largely unknown. Our preliminary results showed lower hepatic triglyceride content, higher mRNA expression of deubiquitinase Usp2-45, higher PPARα protein level and its downstream signaling pathways in circadian clock gene Per1/2/3 triple knock out mouse. We hypothesize that hepatic circadian clock regulates the transcription of Usp2-45 mRNA expression, USP2-45 controls the degradation and stability of PPARα by deubiquitination, PPARα thus affects hepatic lipid metabolism. The Per triple knock out mouse, clock gene Bmal1 hepatocyte specific knock out mouse, Usp2 knock mouse are used in this study. We will investigate the hepatic circadian clock control of hepatic lipid metabolism, and uncover molecular mechanism of hepatic circadian clock control of hepatic triglyceride content via USP2-45/PPARα signaling pathway, using Oil Red O staining, RNA-sequencing, Real time-quantitative PCR, Western blot, Immunohistochemistry, Promoter activity analysis, Real time bioluminescence monitoring system, Chromatin immunoprecipitation, and Co-immunoprecipitation techniques. Therefore, this study will provide us theoretical and experimental basis for a comprehensive understanding on regulatory networks of hepatic lipid metabolism, treatment of metabolic diseases such as fatty liver disease by identifying its molecular targets from the point of view of circadian clock.