载脂蛋白O在巨噬细胞炎症反应中的调控作用及机制探讨

Apolipoprotein O (apoO) is a very recently discovered apolipoprotein in 2006. The biologic role of apoO remains incompletely unknown. Our data suggested an association between apoO and the inflammation of atherosclerosis. However, the mechanism involved remains to be fully elucidated. We observed that the expression of several inflammatory cytokines, such as TNF-α, IL-6, IL-1β and MCP-1, was significantly increased in apoO-silencing macrophages, indicating that apoO exerts an inhibitory effect on macrophage inflammation. Moreover, endogenous apoO positive influenced the level of UCP2, which could reduce the secretion of cytokines by suppressing mROS/NF-κB signaling. Notably, apoO interacted with FKBP8 in vitro in our preliminary experiments. In view of the fact that FKBP8 was involved in the degradation of UCP2 via ubiquitin-proteasome pathway, we propose the hypothesis that apoO may impair the activation of UCP2/mROS/NF-κB pathway in macrophage by its interaction with FKBP8 which could subsequently suppress of the degradation of UCP2. In this study, we will focus to examine the effect of apoO on the expression of inflammatory cytokines and the UCP2/mROS/NF-κB pathway and identify the functional region of apoO, in order to verify the mechanism by which apoO regulates macrophage inflammation. This study is expected to provide a new strategy for the prevention and treatment of atherosclerotic cardiovascular disease.

载脂蛋白O（apoO）是2006年新发现的一种载脂蛋白，其生理功能目前尚不清楚。根据前期研究结果，本课题组首次提出“apoO与动脉粥样硬化炎症反应有关”的观点，并观察到巨噬细胞内apoO通过上调线粒体质子载体UCP2蛋白水平而减轻细胞炎症反应。同时发现，apoO定位于巨噬细胞线粒体并可与线粒体外膜蛋白FKBP8相结合。鉴于FKBP8参与UCP2的泛素化降解过程，推测apoO与26S蛋白酶体竞争性结合FKBP8是其调控UCP2蛋白稳定性及下游炎症通路活性的关键机制。本研究拟在前期工作基础上，试图从分子、细胞和动物水平阐明apoO对巨噬细胞炎症因子表达及UCP2/mROS/NF-κB炎症通路的影响，明确apoO与FKBP8可逆性结合在该过程中的作用。本研究将首次阐明apoO调节巨噬细胞内炎症反应的分子机制，并为动脉粥样硬化性心血管疾病的防治提供新的干预靶点。

载脂蛋白O（apolipoprotein O，apoO）是新近发现的载脂蛋白家族新成员，由肝细胞、单核巨噬细胞等合成，其生理功能目前尚不清楚。我们首先探讨了apoO在巨噬细胞内的功能。巨噬细胞介导的炎症反应是动脉粥样硬化性心血管疾病重要的病理生理基础。线粒体质子载体解偶联蛋白2（uncoupling protein 2，UCP2）可抑制巨噬细胞炎症激活，减轻动脉粥样硬化血管的结构与功能损害。结合前期研究结果及apoO分子结构特性我们推测：巨噬细胞内apoO可能通过与26S蛋白酶体竞争性结合线粒体膜蛋白FKBP8，减少UCP2经泛素化降解，进而抑制UCP2/mROS/NF-κB炎症通路活性，减轻巨噬细胞炎症反应。本研究项目发现：过表达apoO确实能抑制巨噬细胞炎性分泌，但抑制UCP不能影响apoO对炎症反应的调控作用。分析后发现apoO主要存在于细胞线粒体内膜中，而调控UCP2蛋白代谢的FKBP8主要存在于线粒体外膜中，因此，apoO在体内难以大量与FKBP8结合。为了明确apoO的生理功能，我们通过尾静脉注射的方法构建apoO过表达C57小鼠后观察到，小鼠肝脏表达h-apoO升高最为明显。申请人前期研究结果已明确apoO参与调控肝细胞内炎症反应，因此我们调整了研究内容测定了过表达apoO对小鼠肝脏炎症反应的影响并发现：肝细胞内apoO可能通过激活TLR4/NF-κB炎症信号通路促进肝细胞炎症反应。这与巨噬细胞内的抑炎作用不同，这一差异可能与细胞类型不同有关。同时，apoO可通过影响线粒体功能及脂肪酸代谢导致肝脏脂质沉积及纤维化。这些发现提示apoO极有可能参与非酒精性脂肪肝（non-alcoholic fatty liver disease, NAFLD）这一与脂质代谢及炎症反应密切相关的疾病的发生发展。本研究为深入探讨apoO在细胞内的生理功能提供了重要依据，并为NAFLD及相关疾病的防治提供了新的思路。