基于靶向垂钓策略探讨核桃楸皮保肝药效物质基础及作用机制

Peroxisome proliferator-activated receptor alpha (PPARα) is closely related to alcoholic liver disease (ALD), and the search for PPARα agonists is of great significance for ALD therapy. Cortex Juglandis Mandshuricae (CJM) has the effect of clearing heat and dampness, relieving liver and eyesight, and is very consistent with the pathogenesis of alcoholic liver disease. Previous studies have found that flavonoids of CJM can improve the serum and liver biochemical indexes of mice with alcoholic liver injury and increase the expression level of PPARα. Therefore, we speculate that flavonoids of CJM have therapeutic effects on ALD and can stimulate PPARα target to produce pharmacodynamics. However, the specific effective form and mechanism of action have not been elucidated. In the present study, the flavonoids of CJM are as the research object: Rat model of alcoholic liver injury was established to clarifies the therapeutic effect of flavonoids of CJM on ALD; Using the in vitro hepatocyte degeneration model to clarify the activity of PPARα in the flavonoids of CJM; PPARα target molecule affinity-ultrafiltration-mass spectrometry platform was used to screen the flavonoids of CJM; The structure-activity relationship between the capture and PPARα agonistic activity was studied by molecular docking technique, and the mechanism of action was further explored. The present study will not only provide an experimental basis for the treatment of ALD by Pueraria flos, but also make beneficial exploration in seeking for the leading compounds for the treatment of ALD fromnatural resources.

过氧化物酶体增殖物激活受体α（PPARα）与酒精性肝病（ALD）密切相关，探寻PPARα激动剂对ALD治疗具有重要意义。核桃楸皮具有清热燥湿、泄肝明目之功效，与酒精性肝病湿热蕴结的病机相吻合。前期研究发现核桃楸皮黄酮可改善酒精性肝损伤小鼠血清及肝脏生化指标，升高 PPARα 表达水平，由此推测核桃楸皮黄酮可激动PPARα靶点对ALD产生药效作用，然而其具体显效物质基础和作用机制尚不明确。本课题拟以核桃楸皮为研究对象：建立酒精性肝损伤大鼠模型，明确核桃楸皮黄酮对ALD的治疗作用；采用肝细胞变性模型，证明核桃楸皮黄酮激动PPARα的活性；运用“PPARα靶分子亲和-超滤质谱”平台，靶向筛选核桃楸皮黄酮显效成分；利用分子对接技术研究捕集成分与 PPARα 激动活性的构效关系，进一步探讨作用机制。本研究为核桃楸皮治疗ALD提供实验依据，是从天然药物资源中寻找治疗ALD先导化合物的有益探索。

随着人民生活水平的提高，长期大量饮酒所致的酒精性肝病（ALD）在临床上发病率逐年上升，已成为仅次于病毒性肝炎的第二大肝脏疾病。核桃楸皮具有清热燥湿、泄肝明目之功效，与酒精性肝病湿热蕴结的病机非常吻合。本项目以“阐明核桃楸皮黄酮（TFC）保肝药效物质基础及作用机制”为指导方针，运用体内外药效学评价及分子生物学验证、“肠-肝轴”研究、亲和超滤靶向筛选、分子对接等多学科相关技术，开展如下研究：（1）建立酒精性肝损伤小鼠模型，通过检测血清生化指标、肝脏酶学指标、肝组织病理结构变化、对肠道菌群的影响、测定相关蛋白表达，从整体动物水平证明TFC通过调节AMPK/PPARα通路、TLR4/NF-κB通路、肠-肝轴来预防酒精诱导的肝损伤。（2）建立酒精诱导L02细胞损伤模型，检测TFC对肝细胞株培养液中肝脏酶学指标、油红O水平、ROS改变及相关蛋白表达的影响，从体外细胞水平验证了TFC对酒精肝的保护作用及激活PPARa表达的分子机制。（3）建立“PPARα靶分子亲和-超滤质谱”平台，垂钓出TFC中与PPARα受体结合的化合物11个，为TFC保肝活性的作用机制研究奠定物质基础。（4）利用分子对接技术研究TFC中靶向显效成分与 PPARα 的结合位点、结合模式，发现氢键、强疏水键发挥重要作用。本项目阐明了TFC防治酒精肝的药效物质基础及作用机制，为保障核桃楸皮资源的合理开发和临床安全用药提供实验依据，并为筛选治疗ALD的先导化合物做了有益的探索。