Nrf2/ARE/HO-1通路介导余甘子抗酒精性肝氧化损伤的机制研究

Alcoholic liver disease (ALD) caused by excessive ethanol consumption is a worldwide health problem. Thus, it is important to explore more effective and safety medicines to cope with ALD. Since oxidative stress plays an important role in the initiation and progression of ALD, the endogenous antioxidal system mediated and activated by exogenous antioxidant is crucial for the prophylaxis of alcoholic oxidative damage. Emblica has been used widely in many local traditional medicinal systems. It is reported to have significant hepatoprotective activities. However, the underlying mechanisms of these activities remain unclear. Polyphenols of emblica (PE) are main hepatoprotective compounds due to their highly antioxidant activity. Nrf2 is an important transcription factor for cell defense against oxidative stress reaction and PE can induce the express of Nrf2 protein. HO-1is an important downstream anti-oxidant and detoxication protein of Nrf2-ARE signaling pathway and can be induced by polyphenols. This research plans to confirm the antioxidation of PE against ethanol-induced heptocyte damage and further explore the expression of HO-1 protein in order to clarify the potential protective mechanism of the Nrf2-ARE pathway in ALD. The present study will hightlight the prospects of nutritional interventaion for ALD by occurring naturally phytochemicals.

酒精性肝损伤(ALD) 是一个世界性的医学难题，防治措施进展缓慢。氧化应激是ALD发病的重要原因，内源性抗氧化系统的激活是防治酒精性肝氧化损伤的关键。余甘子是我国传统保肝利胆的药食两用植物，但其抗肝损伤的机制研究仍是一个盲区。前期研究表明余甘子多酚具有显著的抗氧化活性，是起保肝作用的主要成分。余甘子多酚可以通过上调细胞防御氧化应激的调节因子Nrf2的表达保护细胞，而HO-1是 Nrf2-ARE信号通路下游重要的内源性抗氧化蛋白，且可以被多酚等抗氧化剂诱导激活。本项目拟以大鼠原代肝细胞为研究对象，以HO-1为靶点，评价余甘子多酚在酒精性肝损伤模型中的抗氧化作用和保护效应，探讨余甘子多酚通过Nrf2/ARE信号通路调控HO-1拮抗酒精性氧化应激肝损伤的分子机制，为酒精性肝损伤的营养防治提供理论与实验依据。

余甘子被用作治疗肝损伤，余甘子多酚具有显著的抗氧化活性，是起保肝作用的主要成分。本项目对余甘子原料筛选结果表明，四川凉山产地余甘子的多酚类物质含量及抗氧化活性相对较高，其总多酚含量为232.3mg/g，确定为实验用原料；确定了余甘子多酚的最佳提取方案：提取时间为135 min，浸提温度为70 ℃，溶于45%的乙醇，此条件下的多酚提取率为242.7mg/g，为大规模提取余甘子多酚提供了参考；采用反相-高效液相色谱法（RP-HPLC）对余甘子多酚提取物进行分析，结果表明GA、CO和EA是余甘子提取物中主要的成分（含量分别为4.5、3.8、1.6 mg/g），同时建立了三种多酚单体物质的快速检测方法；体外抗氧化实验表明，余甘子多酚的DPPH自由基清除率、羟自由基清除率、自发性肝脂质氧化均显著高于茶多酚；细胞实验结果表明，余甘子多酚通过增加内源性抗氧化酶超氧化物歧化酶（SOD）和谷胱甘肽（GSH）含量，降低丙二醛（MDA）、谷丙转氨酶（ALT）、谷草转氨酶（AST）含量，提高细胞存活率，从而抑制酒精性BRL-3A细胞氧化损伤，与余甘子中主要活性成分 GA、CO、EA含量正相关；通过Q-PCR实验结果表明，余甘子多酚可以降低Nrf2的mRNA水平和蛋白质含量，阻止Nrf2的过度表达；上调HO-1的mRNA水平和蛋白质含量，促进细胞抗氧化酶基因HO-1的表达，增强细胞自身抗氧化能力，抑制酒精性肝细胞氧化损伤。证实了余甘子多酚可通过调控Nrf2/HO-1信号通路抑制酒精性肝细胞损伤的分子机制，为酒精性肝损伤的营养防治提供了重要的理论与实验依据。