茶黄素TF3通过血浆激肽释放酶（PK）调控AMPK信号通路改善非酒精性脂肪肝病的作用机制研究

The incidence of nonalcoholic fatty liver disease (NAFLD) is increasing and the patients tend to be younger. It has become the leading cause of chronic liver diseases in the worldwide recently. There is evidence revealing that the pathogenesis of NAFLD is mostly related to lipid metabolism disorders. Others and our previous studies confirmed that black tea and its active ingredient, theaflavins significantly regulate lipid metabolism disorders and reduce fat accumulation in liver. But the mechanism is still unclear. Our preliminary experiments show that theaflavin monomer TF3 has a significant effect on reducing lipid droplets in hepatic cells. TF3 directly binds to plasma kallikrein (PK) and inhibits its activities, further activates AMPK, thus regulating its downstream proteins which relative to fat oxidation and fat synthesis. Therefore, we speculate that “TF3 alleviates NAFLD through activating AMPK signaling pathway by directly interacting with PK”. In this project, by using NAFLD mouse and HepG2 cells as models, we intend to elucidate the regulation of theaflavin TF3 on AMPK signal pathway, to clarify the critical intermediate role of PK, and to reveal the molecular mechanism of TF3 on relieving NAFLD. Our project provides clues for the relationship of black tea and human health as well as new thoughts and targets for the development of highly effective and safe therapeutic natural products of NAFLD.

非酒精性脂肪肝（NAFLD）发病率不断上升且低龄化，近年来已成为全球范围内慢性肝脏疾病的首要病因。研究显示，脂质代谢异常在NAFLD形成中起关键作用。他人和我们的前期研究表明，红茶及其活性成分茶黄素可改善脂质代谢紊乱，减少肝脏中脂肪积累，但具体机制尚不清楚。我们预实验发现，茶黄素单体TF3显著减少肝脏细胞脂滴的生成，与血浆激肽释放酶（PK）直接结合并抑制其活性，进一步激活腺苷酸活化蛋白激酶（AMPK），并调控其下游脂肪合成与脂肪分解相关蛋白的表达水平。由此推测“TF3通过与PK直接相互作用激活AMPK信号通路，进一步改善NAFLD”。本研究拟利用NAFLD小鼠和肝细胞HepG2为模型，阐明TF3对AMPK信号通路的调控作用，明确靶点PK的关键介导作用，揭示TF3缓解NAFLD的机制，为红茶与健康的关系研究提供科学依据，也为高效、安全的NAFLD治疗性天然药物开发提供新靶点与新思路。

非酒精性脂肪肝（NAFLD）已成为一个全球关注的公共健康问题，发病率逐年升高。肝脏器官脂肪的长期过度堆积，使NAFLD伴随着脂肪性肝炎，甚至进一步恶化为肝硬化或肝癌。目前还没有专门针对NAFLD治疗的药物。天然植物及其活性成分自古以来就是药物发现的主要来源之一，本项目以茶叶单体成分茶黄素-3, 3'-没食子酸酯（TF3）为材料，分别在体内外模型上明确了TF3缓解NAFLD的作用及分子机制。在ob/ob肥胖小鼠脂肪肝模型上，与模型对照相比，TF3显著减轻了小鼠体重、腰围、肝脏重量和其它内脏器官脂肪重量；明显逆转了弥漫性肝脂肪变性伴核浓缩、细胞质疏松、脂肪空泡增多等症状。TF3还显著地降低了肥胖小鼠体内血清和肝脏组织中异常高水平的脂质类总胆固醇、甘油三酯、游离脂肪酸、低密度脂蛋白胆固醇和肝功能酶谷丙转氨酶和谷草转氨酶。通过肝脏组织转录组测序和RT-qPCR验证分析进一步显示，脂质代谢过程包括不饱和脂肪酸的生物合成（Fads1、Tecr、Scd1 和 Elovl1）、花生四烯酸和亚油酸代谢（Cyp4f14、Cyp1a2 和 Cyp2c70）以及类固醇生物合成（Fdft1、Tm7sf2、Ebp、Dhcr7）在TF3干预组明显上调，而PPAR信号通路基因Fabp4、Plin4、Lpl和Acadm的表达减少。肠道菌群16S rRNA测序及关联分析显示，TF3治疗NAFLD小鼠增加了菌群的多样性和丰富度，主要通过上调增加短链脂肪酸如丁酸的肠道菌群Prevotellaceae_UCG -001、norank_f__Ruminococcaceae和GCA-900066575有关。进一步地，通过靶点预测和细胞水平上基因过表达等技术确认TF3缓解NAFLD的直接作用靶点，分析获得TF3通过靶向抑制血浆激肽缓释酶（PK）活性，进而激活腺苷酸活化蛋白激酶（AMPK）及调控其下游脂质代谢相关蛋白的分子机制。本项目首次在NAFLD中提出TF3-PK-AMPK作用信号轴，并在体内实验证实了TF3是一种安全有效的缓解NAFLD的活性成分。本研究不仅为揭示红茶健康功效提供了依据，也为NAFLD药物的研发提供了研究基础，并为临床上以PK为作用靶点防治NAFLD提供了新的可能。