基于AMPK/SIRT1/UCP2通路探讨参苓白术散对NAFLD肝细胞线粒体功能障碍的调控机制

Studies have shown that mtochondrial dysfunction is one of the critical steps in the pathogenesis of nonalcoholic fatty liver disease (NAFLD),and the AMPK/SIRT1/UCP2 pathway is closely related to mitochondrial dysfunction and disturbance of lipid metabolism. Our previous study found that shenling baizhu powder could up-regulate AMPK protein expression and lower UCP2 mRNA and protein expression in liver tissues，and achieve the purpose of lipid-lowering，but the specific regulatory mechanism is unknown. Research purposes:In vitro and vivo,To observe the effect of related metabolites (such as ATP、ROS、MDA and so on )and the molecular mechanism of shenling baizhu powder，which are activated by AMPK/SIRT1/UCP2 pathway in the liver cells of NAFLD rats. Expected Results:①It is identificated that the AMPK/SIRT1/UCP2 pathway play an important role in regulatory mechanism of NAFLD between mitochondrial dysfunction and disturbance of lipid metabolism.②TO clarify the preliminary regulation mechanism of shenling baizhu powder in AMPK/SIRT1/UCP2 pathway of the NAFLD liver cells. ③To find the potential drug targets for shenling baizhu powder. ④TO reveal the relationship between "Dampness 、turbidity、Phlegm、dyspepsia"and the products of oxidative stress and lipid peroxidation，which deepen the understanding of Chinese medicine for the etiology and pathogenesis in NAFLD.

研究表明，线粒体功能障碍是非酒精性脂肪性肝病（NAFLD）发病的关键环节之一，AMPK/SIRT1/UCP2通路与线粒体功能障碍、脂质代谢紊乱关系密切，我们前期研究发现参苓白术散可以上调NAFLD大鼠磷酸化AMPK蛋白的表达和下调UCP2 mRNA及其蛋白的表达，实现降脂的目的，其作用与影响线粒体代谢有关，但具体机制不明。目的：体内、外观察NAFLD大鼠肝细胞AMPK/SIRT1/UCP2通路对线粒体ATP及ROS、MDA等代谢产物的调控作用和参苓白术散干预的分子机制。预期：①明确AMPK/SIRT1/UCP2通路在NAFLD线粒体功能障碍与脂质代谢紊乱之间的调控机理。②初步阐明参苓白术散对NAFLD肝细胞AMPK/SIRT1/UCP2通路的作用机制。③发现参苓白术散治疗NAFLD的潜在药物靶点。④揭示湿浊痰积与氧化应激产物、脂质过氧化产物之间的内在关系，深化中医对NAFLD病因病机认识。

背景：线粒体功能障碍是非酒精性脂肪性肝病（NAFLD）发病的关键环节之一，AMPK/SIRT1/UCP2通路与线粒体功能障碍、脂质代谢紊乱关系密切，我们前期研究发现参苓白术散可以上调NAFLD大鼠磷酸化AMPK蛋白的表达和下调UCP2 mRNA及其蛋白的表达，实现降脂的目的，其作用与影响线粒体能量代谢有关，但具体机制不明。目的 观察参苓白术散对高脂饲料建立的非酒精性脂肪性肝病（NAFLD）大鼠脂质代谢及AMPK/SIRT1/UCP2通路的调控机制。方法 选用SPF级SD大鼠32只，随机分为4组：正常组、模型组、参苓白术散高剂量组（ig，30.0g•kg-1•d-1），参苓白术散低剂量组（ig，10.0g•kg-1•d-1），每组8只。采用高脂饲料喂养大鼠16周建立NAFLD大鼠模型，各药物干预组大鼠分别灌服参苓白术散的高、低剂量进行干预，16周后取血和肝组织样本，全自动生化仪检测血清和肝匀浆甘油三酯（TG）、总胆固醇（TC）含量；HE染色观察肝组织病理学变化，油红O染色观察肝组织脂质蓄积情况；透射电镜观察肝细胞的超微结构改变；Western blotting检测肝组织AMPK、pAMPK、SIRT1和UCP2蛋白表达水平。结果 与正常组比较，模型组大鼠病理证实肝组织脂质蓄积严重，肝组织匀浆和血清TG、TC含量显著升高（P＜0.01，P＜0.05），肝组织pAMPK、SIRT1蛋白表达均显著降低（P＜0.05），UCP2蛋白表达显著升高（P＜0.05），与模型组比较，参苓白术散高、低剂量组肝组织脂质蓄积明显改善，肝组织和血清TG、TC含量明显下降（P＜0.05，P＜0.01），肝组织pAMPK、SIRT1蛋白表达均显著上调（P＜0.05，P＜0.01），UCP2蛋白表达显著降低（P＜0.01），其中参苓白术散高剂量组较低剂量组效果较好；但二者比较差异无统计学意义。结论 参苓白术散能够改善16周高脂饮食诱导的NAFLD大鼠脂肪代谢紊乱、减轻肝脏脂质蓄积，其作用机制可能与肝细胞内AMPK/SIRT1/UCP2通路的激活有关。科学意义: ①初步明确AMPK/SIRT1/UCP2通路在NAFLD线粒体功能障碍与脂质代谢紊乱之间的调控机理。②初步阐明参苓白术散对NAFLD肝细胞AMPK/SIRT1/UCP2通路的作用机制。③揭示湿浊痰积与氧化应激产物之间的内在关系。