Bezafibrate促进肉碱棕榈酰基转移酶Ⅱ缺陷症能量代谢的作用机制研究

Carnitine palmitoyltransferase Ⅱ (CPT Ⅱ) deficiency is one of the most common inherited metabolic diseases characterized by myalgia, exercise intolerance and rhabdomyolysis, which is a rare autosomal recessive inheritance. It results in mitochondrial long-chain fatty acid β-oxidation (FAO) disorder and energy crisis. Although numerous studies had been carried out on these dieases, but the precise molecular mechanisms of CPT Ⅱ deficiency were not completely understood, especially the studies of the clinical treatment on CPT Ⅱ deficiency patients had no further progress. In our previous study, we reported the variants CPT Ⅱ performed reduced residue enzyme activity, thermal instability, short half-life, decreased fatty acid β-oxidation, low ATP production and loss of mitochondrial membrane potential in transfected COS-7 cells. Recently, we cultured CPT Ⅱ deficiency patients fibroblast and analyzed their genomics DNA sequence, treated fibroblasts with fibrate group of hypolipidemic drugs. The data indicated that the Bezafibrate significantly improve the CPT Ⅱ residue enzyme activity and the fatty acid β-oxidation related genes expression. In this study, we intends to further analyze the energy crisis of treatment with Bezafibrate in CPT Ⅱ deficiency cells and CPT Ⅱ point mutant mice. Clarify fatty acid β-oxidation and ATP production levels, and further elucidate the molecular mechanisms of Bezafibrate up-regulated energy crisis in CPT Ⅱ deficiency cells and animal models. The study will further reveals the pathogenesis of CPT Ⅱ deficiency patients and provides the basic data for clinical research of drug therapy.

CPT Ⅱ缺陷症是一种常见的遗传性代谢病，其缺陷直接导致线粒体脂肪酸β-氧化紊乱，能量代谢障碍，但其发病机理至今未完全明确，尤其是对CPT Ⅱ缺陷症病人的临床药物治疗研究未有深入。我们通过转染COS-7细胞发现变异的CPT Ⅱ表现为酶活性降低和热不稳定性、蛋白半衰期缩短、脂肪酸β-氧化和ATP生成不足。我们对CPT Ⅱ缺陷症进行基因组序列分析并获得了CPT Ⅱ缺陷症细胞系，用Fibrate类降血脂药处理CPT Ⅱ缺陷症细胞，研究发现Bezafibrate能显著提高CPT Ⅱ残基酶活性及脂肪酸代谢相关基因的表达。我们拟进一步用Bezafibrate处理CPT Ⅱ缺陷症细胞和CPT Ⅱ点突变小鼠，分析脂肪酸β-氧化和ATP生成变化，从细胞水平和动物整体水平阐明其促进CPT Ⅱ缺陷症能量代谢的分子机制。该研究的深入有望进一步揭示CPT Ⅱ缺陷症病人的发病机理，并为临床药物治疗研究提供基本数据。

本项目采用CPT Ⅱ缺陷症病人的成纤维细胞系：F352C (Homozygote)，V368I (Homozygote)，F352C (Heterozygote) + V368I (Homozygote)进行研究，分析了CPT Ⅱ缺陷与CPT Ⅱ酶活性、线粒体脂肪酸β-氧化和ATP生成水平之间的关系，揭示生物体内由于CPT Ⅱ缺陷导致机体能量代谢障碍的分子机制。探索了临床用于CPT Ⅱ缺陷症治疗有效药物。研究结果显示：CPT Ⅱ缺陷症病人的CPTⅡ残基酶活性明显降低并呈热不稳定性；CPT 2基因表达无明显差异而F352C (Heterozygote) + V368I (Homozygote) 的CPT Ⅱ蛋白表达显著降低，在酶动力学特征上表现为显性负效应；变异的CPT Ⅱ蛋白表达不稳定，半衰期显著缩短；细胞膜电位显著降低并对温度敏感；CPT Ⅱ缺陷导致细胞凋亡；脂肪酸β-氧化能力和ATP的生成水平显著降低。Bezafibrate不仅能显著提高CPT Ⅱ酶活性及脂肪酸代谢相关基因的表达，而且能显著提高脂肪酸β-氧化和ATP生成水平，显著提高细胞膜电位。.该研究中我们采用CPT Ⅱ缺陷症病人的成纤维细胞系：F352C (Homozygote)，V368I (Homozygote)，F352C (Heterozygote) + V368I (Homozygote)等变异体，研究CPT Ⅱ缺陷症引起能量代谢障碍的分子病理机制，并用Bezafibrate处理CPT Ⅱ缺陷症病人的成纤维细胞，分析药物处理后正常的和CPT Ⅱ缺陷的细胞线粒体脂肪酸β-氧化、ATP生成水平的变化；研究分析Bezafibrate处理CPT Ⅱ缺陷的细胞后ATP供应能否克服能量代谢的障碍，维持自身所需的ATP水平；进一步深入探究Bezafibrate促进CPT Ⅱ缺陷症能量代谢的分子机制，探索治疗CPT Ⅱ缺陷症病人的临床药物。该研究揭示了CPT Ⅱ缺陷症病人的能量代谢障碍的分子机制，提出了临床治疗CPT Ⅱ缺陷症病人可能药物，为今后进一步研究CPT Ⅱ缺陷症及神经系统并发症的关系提供了基本数据和理论依据。