以丁酸钠为探针研究组蛋白去乙酰化酶抑制剂促进胆固醇逆转运的分子作用机制

Serum high-density lipoprotein cholesterol (HDL-C) levels correlate inversely with cardiovascular disease (CVD) risk. The anti-atherogenic effects of HDL have been largely attributed to its ability in mediating cellular cholesterol efflux and thereby facilitating the removal of excess cholesterol from peripheral tissues, and delivering to the liver for elimination through reverse cholesterol transport (RCT) pathway. ATP-binding cassette transporter A1 (ABCA1) has been demonstrated to play a key role in RCT process. Enhancing ABCA1 expression could reduce susceptibility to atherosclerosis by promoting free cholesterol efflux from macrophage cells to extracellular lipid-poor apolipoprotein (Apo) A-I, forming nascent HDL. Therefore, the regulation of ABCA1 gene expression could provide a novel target in the prevention of atherosclerosis. Modification of histones by acetylation, regulated by the balance of histone acetyltransferases (HAT) and histone deacetylases (HDAC), is one of major epigenetic regulation mechanisms which regulate the process of transcription in target gene. Regulating disorders in the histone acetylation machinery contribute to the pathogenesis of atherosclerosis. Our previous work found sodium butyrate (NaB), a member of HDAC inhibitors, could upregulate the ABCA1 gene expression and cholesterol efflux in RAW 264.7 murine macrophage cells. Based on these results, this project intends to study the molecular mechanism of action for NaB on regulating ABCA1 gene expression. First, the cis-elements, transcription factors and signal pathway involved in transcriptional regulation will be clarified. Then, histone acetylation status and its resulting effect on ABCA1 expression by HDAC inhibition after NaB treatment will be elucidated at molecular level. Third, HDAC inhibition caused protein interaction and acetylation of non-histone proteins (transcription factors and kinases) will be further illustrated to reveal the relationship between different regulation pathways mediated by NaB. Finally, animal model will be exploited to verify the molecular mechanism of NaB on ABCA1 expression and anti- atherogenic activity. The results of this study will provide new insight in the HDL-based drug discovery, and lay the groundwork for the utility of HDAC inhibitors in CVD treatment.

胆固醇逆转运（RCT）是血浆高密度脂蛋白胆固醇（HDL-C）抗动脉粥样硬化（AS）的重要途径，巨噬细胞表面的膜转运蛋白ABCA1参与了RCT过程的关键步骤，通过生成有功能的HDL-C和增强RCT发挥抗AS作用，是AS治疗的新靶点。本项目的前期研究发现组蛋白去乙酰化酶（HDAC）抑制剂丁酸钠能显著上调巨噬细胞中ABCA1基因的表达和其介导的胆固醇外排功能。本项目拟以丁酸钠作为分子探针，研究与调控相关的顺式元件、转录因子和信号通路，初步阐明其作用机制；继而以组蛋白乙酰化修饰为研究对象，阐明丁酸钠作用后，组蛋白H3、H4乙酰化及目标HDAC亚型对ABCA1表达调控的影响；进一步考察丁酸钠介导的HDAC抑制途径对转录因子等非组蛋白的作用和修饰的影响，以期全面阐释丁酸钠调控ABCA1表达的分子作用机制；并将主要研究结果在动物模型中进行验证，从而为HDAC抑制剂在心血管疾病领域中的应用奠定研究基础。

胆固醇逆转运（RCT）是血浆高密度脂蛋白胆固醇（HDL-C）抗动脉粥样硬化（AS）的重要途径，巨噬细胞表面的膜转运蛋白ABCA1参与了RCT过程的关键步骤，是AS治疗的新靶点。本研究在细胞水平和体内水平，对丁酸钠上调ABCA1基因表达的活性及作用机制进行了研究。一方面，丁酸钠通过影响遍在转录因子Sp1来调控ABCA1基因的表达；另一方面，丁酸钠发挥组蛋白去乙酰化酶抑制剂的作用，影响胞内组蛋白乙酰化水平，并作用于相应的HDAC亚型（HDAC4和HDAC9），进而可能调控ABCA1基因的表达。在ApoE-/-动脉粥样硬化小鼠的研究中对体外主要研究结果进行了验证。丁酸钠作用后小鼠主动脉斑块处的脂质沉积有所减少，这可能反映了丁酸钠上调ABCA1在体内的影响。同时考虑到丁酸是肠道菌群代谢的主要产物之一，有可能是菌群发挥抗AS作用的效应分子，因此本研究进一步加入了丁酸钠对肠道菌群的影响以及对动脉粥样硬化作用的研究，为进一步明确肠道菌群与其代谢产物的相互作用、以及干预动脉粥样硬化的机制研究奠定基础。