bFGF对缺血再灌注损伤心肌中能量代谢关键酶硝化损伤调控机制的研究

Oxidative and nitrosative stress plays a critical role in the occurrence of myocardial ischemia reperfusion (MI/R) injury. Previous report described that endogenous bFGF plays a significant role in protection of MI/R injury as well as improve myocardial function. However, the underlying mechanism of how bFGF protects MI/R injury is remain elusive. Our preliminary proteomic studies on the MI/R rat with bFGF indicated that application of bFGF significantly inhibited the nitration levels of ATP synthase subunit α (ATPSA) and Adenine nucleotide translocator 1 (ANT1), two important metabolic enzymes. Therefore, it suggests that bFGF may improve myocardial energy supply by modulating the nitrosative damages of ATPSA and ANT1 to prevent the apoptosis of myocardial cells. The purpose of this project is to understand how bFGF regulates the 3-NT modifications of ATPSA and ANT1 in the MI/R rat myocardium. Site-directed mutagenesis and in vitro recombinant protein expression systems will be introduced to examine the function of each nitrated tyrosine residue in regulation of ATPSA and ANT1 activities, which will be expected to further explore the relationship between the protection mechanism of bFGF on MI/R and nitrosative damages. This study will not only provide a theoretical basis for clinical therapy but also exploit a new method for the diagnosis of MI/R.

氧化硝化损伤在心肌缺血再灌注（MI/R）损伤过程中扮演重要的角色。外源性生长因子bFGF可显著增加心肌对MI/R损伤的抵抗，改善心肌功能。但bFGF对MI/R的作用机制尚不清楚。前期通过蛋白质组学技术对MI/R大鼠研究发现bFGF可显著抑制心肌中ATPSA和ANT1的硝化水平，而它们又是体内能量代谢的关键酶，是维持心肌正常功能的必要因素之一，提示bFGF可能通过调控ATPSA和ANT1的硝化水平，改善心肌的能量供应，从而减少心肌细胞的凋亡，发挥其对MI/R损伤的保护作用。本课题旨在前期实验基础上进一步研究bFGF对MI/R损伤心肌中ATPSA和ANT1硝基化修饰酪氨酸位点的调控，并通过体外表达、定点突变和体内外转染检测各修饰位点对ATPSA和ANT1活性的影响，以期深入探索bFGF对MI/R保护机制与硝化损伤的关系。本项研究结果不仅能在理论上指导临床，而且有望开辟MI/R诊治方法的新篇章。

随着人类生活水平的提高和生活方式的巨大转变，心肌梗死患者的数量急剧增多，心肌梗死严重威胁着人类的生活与生存。目前，临床已成功将外源性生长因子bFGF应用于预防心肌梗死损伤，并取得显著疗效。bFGF促心肌梗死修复的主要机制之一是减少心肌细胞的凋亡，但其信号传递途径和网络调控作用尚不明确。在前人报道研究中发现心梗能使心肌细胞中氧化应激水平上调，而本研究突出的成果是bFGF能通过下调氧化应激水平对其具有保护作用，并对其保护机制进行较深入的研究。通过研究发现，在心肌梗死情况下，bFGF作用于心肌细胞后，通过FGFR激活AKT磷酸化，抑制GSK3β的活性，抑制Fyn的入核，促进Nrf2的入核转录，抗氧化基因表达上升，降低细胞内的ROS水平，缓解氧化应激，抑制细胞凋亡，进而保护心肌梗死。该研究为临床更广泛的应用bFGF提供了一定的理论基础。