PPARα/ACOT1信号通路在全氟辛酸暴露致大鼠脂肪酸代谢紊乱中的作用研究

Perfluorooctanoic acid (PFOA) is a perflourinated carboxylic acid, considered to be a persistent organic pollutant (POP). Environmental contamination and exposure to PFOA is considered dangerous yet poorly understood at present. Previous studies have found that rats exposed to PFOA can cause hepatomegaly, excessive oxidative stress in the liver and dyslipidemia among others; notably we observed activation of PPARα and downstream ACOT1 protein expression. Following lentivirus-mediated RNAi of PPARα, ACOT1 was found to be positively regulated by PPARα and further studies have confirmed that PPARα may act as a scavenger of reactive oxygen species (ROS). However, the regulation of the PPARα/ACOT1 signaling pathway play in fatty liver progression following PFOA exposure is poorly understood. We intend to use SD rats exposed to oral PFOA to identify proteins that interact with ACOT1 by immunoprecipitation in tandem with discovery based mass spectrometry, alongside traditional biochemical confirmatory techniques such as RNAi. We anticipate that this study will help in the elucidation of the biochemical mechanism underpinning lipid metabolism through the PPARα/ACOT1 in response to PFOA. This study will demonstrate that the regulating role of PPARα/ACOT1 pathway in the regulation of fatty acids metabolism disorders caused by PFOA exposure. It is the hope that this study will identify PPARα/ACOT1 pathway associated proteins which may be valuable potential drug targets for treatment of hepatic injury patients induced by PFOA exposure.

全氟辛酸（PFOA）是一种持久性有机污染物。我们前期研究发现，PFOA暴露可导致大鼠肝肿大、肝脏内产生过量的氧化压力、脂代谢紊乱等；并显著激活PPARα和下游ACOT1蛋白表达。通过慢病毒介导的RNAi实现PPARα干扰后，发现ACOT1受到PPARα正调控，并证实了PPARα在其中可起到清除活性氧（ROS）的作用。然而PPARα/ACOT1信号通路在PFOA致肝脏脂代谢紊乱中究竟起到怎样的调控作用？有待解决。本课题将以SD大鼠为研究对象，通过免疫沉淀和质谱技术鉴定与ACOT1相互作用的蛋白；RNAi等分子生物学方法进一步研究PFOA暴露后肝脏中PPARα/ACOT1通路相关蛋白的变化，评估这些变化与脂质代谢、生化指标之间的关联。以阐明PFOA暴露后PPARα/ACOT1通路在肝脏脂肪酸代谢紊乱中的调控作用；相关蛋白亦可能成为治疗PFOA暴露所致肝损伤患者的潜在药物靶点。

全氟辛酸（PFOA）是一种持久性有机污染物。PFOA暴露可激活PPARα并引起ACOT1显著升高，具体机制尚不清楚。本项目以大鼠、大鼠肝细胞和ACOT1为主要研究对象，通过PFOA暴露大鼠，代谢相关指标及血清生化检测，基于iTRAQ的定量蛋白质组学，qPCR和Western blot分析肝脏蛋白质组变化。包装PPARα干扰慢病毒，感染大鼠肝细胞，进行PPARα敲降，再进行暴露，测定活性氧簇（ROS）及ACOT1的变化。同时，为了研究PPARα/ACOT1信号通路中ACOT1蛋白在PFOA暴露致大鼠肝脏脂质代谢紊乱过程中的作用，我们通过细菌重组方法合成纯化大鼠ACOT1蛋白，并通过分子对接技术评估促进其与PFOA结合的结构特征。此外，通过使用圆二色光谱（CD spectrum）、等温滴定量热法（ITC）、限制性酶切等技术和方法，获得以下创新性结果：（1）PFOA暴露可致大鼠体重减轻和肝肿大，并对尿素代谢有显著影响。（2）PFOA暴露后，血清中有4项指标（ALT、GGT、BILD和UREA）显著变化。（3）发现肝脏中有8种蛋白质与脂肪酸的氧化有关，两种蛋白质与尿素循环障碍有关。PFOA暴露加速了大鼠肝脏中脂肪酸的β-氧化，同时抑制了肝脏中尿素的合成。（4）PPARα/ACOT1在大鼠肝细胞中可能起到清除ROS的作用。（5）通过与分子伴侣共表达成功实现了ACOT1蛋白的可溶性表达。（6）PFOA能够以中等亲和力与Acot1结合，可作为PFOA进入细胞核激活PPARα等核内受体途径中一条潜在途径。（7）PFOA按照1：1的摩尔比与WT-Acot1结合。二者之间主要的相互作用力为静电吸引和氢键。在结合过程中，ACOT1蛋白的N326和H373氨基酸残基起到关键作用。相关研究结果共发表论文9论文，其中SCI论文3篇，MEDLINE论文5篇，培养硕士研究生3人。