鼠尾草酸调控miR-34a/SIRT1-p66shc信号通路抗酒精性肝损伤的分子机制研究

Uncontrolled formation of reactive oxygen species (ROS) resulted by mitochondrial oxidative stress is a major cause of alcoholic liver disease(ALD) with complex pathogenesis. Recent studies suggested that the modification of ROS levels was modulated by specific signal pathways. Carnosic acid (CA), which is found in the extracts of rosemary leaves, has antioxidant properties. We firstly showed that CA protected rats against chronic alcohol-induced liver injury. And we further found that the protection of CA was related with the inhibition of p66shc expression as a potential molecular mediator of hepatocellular mitochondrial oxidative stress and apoptosis by activating SIRT1 and was involved in downregulating miR-34a. On the basis of bioinformatics and researches, we firstly point out that the miR-34a/SIRT1-p66shc pathway maybe an important prevention strategy against alcohol-induced liver injury and the pathway plays a key role in the molecular mechanism that CA modulates. In this study a variety of molecular biology methods will be used for focusing on the significant role of miR-34a/SIRT1-p66shc pathway in the mechanism of chronic alcoholic liver injury and the regulation of CA. The study may provide new mechanisms for therapeutic intervention of ALD and represent an attractive pharmacological target for the development of CA to arrest ALD.

酒精性肝病发病机制复杂，线粒体氧化应激所致ROS失衡是其损伤的重要因素。近期研究显示，这种ROS失衡受某些信号通路的调控。鼠尾草酸为迷迭香中抗氧化成分最丰富的提取物, 我们前期研究首次发现其对大鼠慢性酒精性肝损伤具有明显的保护作用。深入研究发现，该保护作用与SIRT1的诱导进而抑制衔接蛋白p66shc调节的肝细胞线粒体氧化应激凋亡有关，与其对miRNA-34a的抑制作用有关。结合生物信息学结果和研究报道,首次提出：调控miR-34a/SIRT1-p66shc通路可能是酒精性肝损伤防治的重要策略，也是鼠尾草酸发挥作用的关键分子机制。本研究采用分子生物学技术和体内外实验模型，探讨miR-34a/SIRT1-p66shc通路在慢性酒精性肝损伤中的作用及机制，探讨鼠尾草酸对该通路的调控作用。本研究对揭示酒精性肝损伤发病新机制及为鼠尾草酸的开发利用提供重要的药理学依据。

线粒体氧化应激所致ROS失衡是酒精性肝病的重要因素。ROS失衡受某些信号通路的调控。鼠尾草酸为迷迭香中抗氧化成分最丰富的提取物, 其对大鼠酒精性及非酒精性肝损伤具有明显的保护作用。该保护作用与SIRT1的诱导进而抑制衔接蛋白p66shc调节的肝细胞线粒体氧化应激凋亡有关，与其对miRNA-34a的抑制作用有关。本研究利用对慢性酒精性肝损伤的动物模型以及L02细胞模型，采用Westernblot 、qRT-PCR、RNAi等分子生物学方法，明确鼠尾草酸在酒精液体饲料造模的大鼠体内抗酒精性肝损伤的药效评价；探讨大鼠酒精性肝损伤模型miR-34a变化与SIRT1、p66shc等表达的相关性及鼠尾草酸的干预作用；阐明miR-34a/SIRT1-p66shc在酒精性肝细胞损伤中的调控作用及鼠尾草酸对酒精性肝损伤的保护机制。.主要研究成果：鼠尾草酸在体内体外对慢性酒精性肝损伤具有保护作用；其保护作用与激活SIRT1有关，且具有时间与剂量的依赖性。通过RNA干扰技术，证明鼠尾草酸通过激活SIRT1，进而抑制ChREBP和p66shc的表达；首次证实鼠尾草酸通过对SIRT1/ChREBP和SIRT1/p66shc通路的调控作用减轻脂质堆积和氧化应激，从而减轻慢性酒精性肝损伤。首次揭示SIRT1/ChREBP和SIRT1/p66shc是慢性酒精性肝损伤发生、发展的关键环节，也是鼠尾草酸抗酒精性肝损伤的关键靶点。进一步研究中首次发现鼠尾草酸在体内外显著抑制miR-34a的表达，并可靶向调控SIRT1-p66shc信号。首次揭示鼠尾草酸通过抑制miR-34a，激活SIRT1/p66shc信号通路，进一步调节凋亡相关因子表达，发挥其肝保护作用。.本项目发现天然产物鼠尾草酸的抗酒精性肝损伤的作用，并通过体内外实验，应用分子生物学、生物信息学等技术，阐明鼠尾草酸抗酒精性及非酒精性肝损伤的分子机制。为鼠尾草酸在预防和治疗肝脏疾患的研究和开发提供重要的药理学依据。首次发现鼠尾草酸在体内外显著抑制miR-34a的表达。提示可通过高通量筛选找到更多miRNA的抑制剂，为miRNA在肝脏疾病治疗和药物开发方面提供新线索。揭示miR-34a/SIRT1-p66shc是鼠尾草酸抗慢性肝损伤的关键靶点，为酒精及非酒精性肝损伤的研究提供了新思路，为开发治疗该疾病的新药提供有希望的药理学靶点。