杨梅酮基于miR-146b/TRβ调控肝脏脂代谢防治NAFLD的机制研究

Because of the high incidence and serious disease progression of non-alcoholic fatty liver disease (NAFLD), seeking the effective strategies for the prevention and treatment of NAFLD and the possible mechanism is critical. Thyroid hormones regulate hepatic lipid metabolism, in which thyroid hormone receptor β (TRβ) plays an important role in modulating expressions of type 1 deiodinase (D1) and T3 responsive genes. We previously found that myricetin was effective in attenuating hepatic steatosis through altering signaling pathways, including PPAR signaling pathway, in which the differentially expressed genes were targeted by T3, but the exact mechanism is largely unknown. We used next-generation sequencing technology to screen the hepatic differentially expressed microRNAs between diet-induced obesity and diet-induced obesity resistant mice and then used bioinformatics algorithms to find that the differentially expressed miR-146b target on TRβ. Quantitative PCR analysis further verified that expression of miR-146b was up-regulated in fatty liver samples, while TRβ and D1 was down-regulated, which could be normalized by myricetin treatment. Based on these findings, with microRNA mimics and inhibitor, RNA interference, as well as miR agomir to induce miR-146b overexpression in mice, current project will further study the detailed mechanism of myricetin in regulating hepatic lipid metabolism to attenuate hepatic steatosis via miR-146b/TRβ, which will provide the evidence for the further development of myricetin, as well as the new strategy to prevent and treat NAFLD.

基于非酒精性脂肪性肝病（NAFLD）的高发病率及严重疾病进程，探寻有效的防治手段及潜在机制至关重要。甲状腺激素调节肝脏脂代谢过程中，其受体β（TRβ）通过调控1型脱碘酶(D1)和T3靶基因转录发挥关键作用。前期研究发现杨梅酮通过诱导肝脏PPAR信号通路（差异表达基因为T3靶基因）等多条通路的显著变化，改善肝脏脂肪变性，但其具体机制尚未阐明。申请人以高通量测序结合生物信息学预测法筛选出脂肪肝差异表达且靶作用于TRβ的miR-146b，而杨梅酮干预使脂肪肝小鼠肝脏miR-146b的高表达水平恢复正常且上调TRβ及D1表达。本项目拟在细胞水平上以microRNA的模拟物和抑制物、RNA干扰，结合miR agomir诱导小鼠体内miR-146b过表达等手段，研究miR-146b/TRβ在杨梅酮调节肝脏脂代谢，改善脂肪变性中的机制，预期成果将为杨梅酮的后续开发提供理论支持，为脂肪肝的防治提供新策略。

前期研究发现杨梅酮改善非酒精性脂肪肝，可能通过调节PPAR信号通路，且涉及的差异表达基因均为T3靶基因。考虑到microRNA可通过调节TRb和DIO1的转录和翻译，调控肝脏甲状腺激素作用的发挥。因此本项目基于microRNA开展杨梅酮改善非酒精性脂肪肝的潜在机制研究。.高脂诱导小鼠肝脏脂肪变性，杨梅酮干预，Illumina高通量测序分析肝脏差异表达microRNA，发现高脂诱导12个microRNAs显著变化，高脂基础上杨梅酮干预诱导14个miRNAs显著变化。结合定量PCR验证发现miR-205和miR-146b可被高脂显著上调，被杨梅酮恢复至对照组水平。TargetScan在线预测显示miR-205和miR-146b分别靶向作用至DIO1和TRb。原代肝实质细胞转染miR-205 mimics和inhibitor，均未能改变DIO1表达水平，排除miR-205与DIO1靶向结合的可能性。miR-146b mimics转染诱导 miR-146b过表达，TRb的mRNA和蛋白水平显著下降；miR-146b inhibitor转染诱导miR-146b下调，TRb的mRNA和蛋白水平显著上调。荧光素酶报告基因检测显示miR-146b与TRb的3’UTR区结合。在游离脂肪酸诱导的原代肝实质细胞脂肪变性模型中转染miR-146b inhibitor，与阴性对照组相比，甘油三酯显著下降，TRb显著升高，脂质生成基因、脂肪酸氧化及转运基因、能量代谢基因均显著上调。在转染miR-146b mimics的原代肝实质细胞中采用杨梅酮干预，有效抑制miR-146b的水平，显著上调DIO1和TRb的水平，调节脂代谢基因的表达。在转染scrambled siRNA和TRb siRNA的细胞中，分别以FFA诱导脂肪变性模型，杨梅酮进行干预。在对照组中，杨梅酮有效提高TRb的蛋白水平，降低细胞内TG含量，调节脂代谢基因的表达；在TRb siRNA转染的细胞中，各组细胞的TRb蛋白水平无显著差异，脂代谢基因相对表达量无显著差异，TG水平显著升高，表明杨梅酮调节肝实质细胞脂代谢的作用依赖于TRb。.本项目明确杨梅酮通过调节miR-146b/TRb信号通路，进而调节肝脏脂肪变性，将为杨梅酮的后续开发提供理论支持，为脂肪肝的防治提供新策略。