心脏APJ下调的分子机制研究：肝X受体的调控

Apelin exibits significant cardioprotective effect via interacting with APJ receptor, which is located on cell membrane. However, the fact that APJ was down-regulated in heart failure or diabetic cardiomyopathy implicates that the protective effect would be attenuated. So far, the mechanism is unclear. This project is aimed to study how APJ expression and location pattern is regulated. Based on our preliminary data that Liver X receptor (LXR) activation down-regulated APJ level in cardiac cells, we hypothesize that LXR is responsible for APJ down-regulation in heart. To prove it, we will use GW3965 and T0901317 to active LXR in cardiac cells in cell culture level and also animal level. APJ mRNA and protein expression will be measured, and cell membrance fraction will be isolated to quantify APJ amount versus total amount. Meanwhile, adenoviral system for APJ-GFP expression will be constructed to monitor APJ movement in living cells. Autophagy level, which is responsible for membrane protein recycle, will be studied by western blotting and electromicroscopy. Meanwhile, RNAi technique will be utilized to silence LXRalpha or LXRbeta to study the role of different LXR isoform in this process. Rab4/7 and beta-arrestin will be measured to evaluate APJ internalization. Through this study, we will reveal the mechanism that how APJ is down-regulated, which will be beneficial for developing apelin/APJ as a therapeutical target in cardiovascular diseases.

脂肪因子apelin具有心肌保护效应，但其膜受体APJ表达量下调则预示着其疗效将减弱。因此，研究APJ下调的分子机制具有重要意义。本课题组前期工作发现肝X受体（liver X receptor, LXR)的激活与APJ下调密切相关，但具体机制尚未阐明。本项目拟从细胞和动物水平研究LXR调控APJ表达分布及分子机制。采用LXR激动剂T0901317处理细胞和动物后，检测APJ mRNA、蛋白总量以及细胞膜上含量变化。沉默LXRalpha或LXRbeta以研究LXR不同亚型对APJ-GFP动态变化的影响。检测beclin1、LC3等蛋白表达及细胞自噬体数量变化，并用bafilomycin A1阻断以研究自噬的作用。还将探讨Rab4和Rab7是否参与LXR的调控。通过本项目的研究，将证实LXR激活可下调心肌APJ，并揭示Rab4/7和自噬的作用，为进一步探索如何增强apelin疗效提供新的思路。