PPARα基因V227A多态性参与肝细胞氧化应激损伤和凋亡的机制研究

Nonalcoholic fatty liver disease (NAFLD) has emerged as one of the most common chronic liver diseases around the world. Unfortunately, the mechanism is still not very clear. It is generally considered that the progression of NAFLD to non-alcoholic steatohepatitis and cirrhosis is governed by interplay of oxidative stress injury and proinflammatory pathways. PPARα is considered as an important regulator of fatty acid oxidation and inflammatory response. Our previous study in vitro showed that the L02 cell transfected with PPARα 227A has higher cell viability and lower apoptosis rate compared with the PPARα V227 group.And the expression of NF-κB-p65 in PPARα 227A liver cells is decreased. Nuclear factor κB (NF-κB) is known as an important cellular transcription factor in regulating inflammation and apoptosis. The results suggested that PPARα 227A may have higher activity and may be involved in the apoptosis. To further explore the mechanism of PPAR α V227A polymorphism in lipid metabolism and apoptosis, surface plasmon resonance biosensor and molecular docking and molecular dynamics simulations were used to investigate the change of protein structure and the interactions between PPAR α V227A and ligand,RXR and target genes.And then hepatocellular steatosis was induced with palmitic acid in the study. And PPAR alpha agonist WY14643 and antagonist GW6471 were used to treat the liver cells. Liver cell proliferation and apoptosis, cell ultra-microstructure and the change of inflammatory cells, fat acid metabolism and the level of ATP, oxidative stress, mitochondrial beta oxidative damage were observed. And the expression of fatty acid metabolism related enzymes, cytokines, NF-κB and apoptosis linked genes were investigated. This study will provide insight into the mechanism of PPAR α V227A on the oxidative stress and apoptosis.

非酒精性脂肪性肝病（NAFLD）已成为全球患病率最高的肝病，其机制不甚明确，目前认为氧应激和炎症反应是NAFLD进展的关键，而PPARα被认为是调节脂肪酸氧化和炎症反应的交汇点。前期对PPARα V227A多态性研究发现：突变型较野生型细胞存活率高而凋亡率低，且NF-κBp65表达明显降低，为进一步探索其机制，本研究拟用表面等离子共振生物传感器实验、分子对接和分子动力学模拟分析PPARαV227A蛋白构象的改变及与配体受体的相互作用，并用软脂酸诱导肝细胞脂变，并用PPARα激动剂WY14643和拮抗剂GW6471进行干预，分别观察细胞的增殖与凋亡、细胞器超微结构、脂肪酸代谢与能量水平、氧应激水平、线粒体β氧化损伤等情况，并检测脂肪酸代谢相关酶、NF-κB及凋亡调控因子的表达，探索PPARαV227A影响氧化应激和凋亡的分子机理。该研究为明确PPARαV227A影响机体功能的机理提供了依据。

非酒精性脂肪性肝病（NAFLD）已成为全球患病率最高的肝病，其机制不甚明确，目前认为氧应激和脂肪酸氧化是NAFLD进展的关键，而PPARα被认为是调节脂肪酸氧化的关键。前期分子流行病学研究显示PPARα 227A携带者体重指数、腰臀比及腹壁脂肪厚度等指标较非227A携带者明显降低，因此考虑PPARα V227A可能是一有益的突变。本研究旨在分析PPARαV227A蛋白构象的改变，构建PPARα 227V和PPARα 227A稳定转染肝细胞L02模型，并用脂肪酸（油酸和软脂酸）诱导肝细胞脂变，并用PPARα激动剂WY14643进行干预，分别观察细胞的增殖与凋亡、氧应激水平、线粒体β氧化损伤等情况，并检测脂肪酸代谢相关酶CPT-1、CPT-2、 HADHA、CEBP-Β、 SREBP1、 LPL和PPARγ等的表达，探索PPARαV227A影响氧化应激的分子机理。结果发现PPARα 227A明显降低了肝细胞的甘油三酯水平，而增加了ROS、catalase、MDA等水平，进一步研究发现PPARα 227A上调了CPT-2的表达。该研究结果表明PPARαV227A是一个功能性的变异，PPARα227A通过增加CPT-2的表达有效地降低了甘油三酯水平却增加了脂质过氧化损伤。本项目基本完成了研究目标和内容，在中华肝脏病杂志发表了一篇论文，该研究内容还入选了2014年亚太消化年会的会议口头报告，赢得了参会者的一致好评。课题承担者陈韶华还赴澳大利亚国立大学肝病研究所与国际著名的肝病学教授Geoffery Farrell进行合作研究。