SS-20调控心磷脂过氧化对脓毒症性心肌抑制的影响

Cardiolipin plays an important role in mitochondrial injury and the activation of mitochondrial apoptotic pathway, which are the pathological basis of septic myocardial depression. We previously found that the mitochondrial penetrating peptide, SS-20, was able to inhibit the activation of mitochondrial apoptotic pathway induced by myocardial ischemia and reperfusion, but the underlying mechanisms are currently unknown. Thus, we hypothesized that SS-20 could prevent cardiolipin oxidation through its binding with cardiolipin, which in turn inhibit the release of cytochrome c from mitochondria, ensuring the production of ATP and reducing cellular oxidative stress. More importantly, the prevention of cardiolipin oxidation could inhibit the activation of mitochondrial apoptotic pathway through the prevention of mitochondrial permeability transition pore opening, cytochrome c release and the activation of proapoptotic factor Bid. We intend to use animal model of sepsis to investigate whether SS-20 is able to intimately binding with cardiolipin on the inner mitochondrial membrane by laser confocal microscopy; whether SS-20 has any effects on sepsis induced reduction of ATP, oxidative stress, the opening of mitochondrial permeability transition pore and cardiomyocyte apoptosis; whether SS-20 is able to reduce the extent of septic myocardial depression; and whether the incubation of mitochondria with exogenous oxidized cardiolipin is capable to mimic sepsis to induce mitochondrial injury and activate mitochondrial apoptotic pathway, all of which are totally blocked by the addition of SS-20. This study is expected to elucidate the mechanisms of SS-20's protective effects on sepsis induced mitochondrial injury and septic myocardial depression, provide new targets to improve the clinical efficacy of sepsis treatment.

心磷脂(CL)在脓毒症等所致线粒体(Mt)损伤和Mt凋亡途径激活中扮演重要角色，我们发现Mt穿膜肽SS-20能抑制心肌缺血再灌注诱导的Mt凋亡途径激活，但机制不明。据此提出如下假说：SS-20与CL结合→阻止CL过氧化→抑制Cyt c外流，MPTP开放和Bid活化→阻抑Mt损伤及Mt凋亡途径激活。我们拟在脓毒症动物模型观察SS-20能否在线粒体内膜与CL紧密结合；能否降低Mt过氧化酶活性，CL过氧化物含量，氧自由基和Cyt c浓度，MPTP通透性，Cyt c和tBid基因表达水平；能否影响Mt凋亡途径调控蛋白的表达和心肌细胞凋亡；能否对脓毒症性心肌抑制具有保护效应。并在体外验证外源性CL过氧化物能否模拟脓毒症诱导Mt损伤，激活Mt凋亡途径，上述效应可否被SS-20逆转。本研究旨在评价SS-20能否阻抑脓毒症所致线粒体损伤和心肌抑制，初步阐明潜在的机制，为提高脓毒症的临床疗效提供新靶标。

脓毒症是因感染而诱发的全身炎症反应综合征，且常合并或发展为多脏器功能障碍综合征。近年来的研究表明，约40~50％的脓毒症患者合并不同程度的脓毒症性心肌抑制，而且一旦发生死亡率将从20%上升到70%以上。Szeto-Schiller多肽是一类具有良好细胞膜穿透性的线粒体靶向抗氧化剂，SS-20虽在体外并不具备抗氧化活性，但在心肌损伤模型中表现出了对心肌的保护作用。本研究旨在以脓毒症性心肌抑制大鼠模型来验证SS-20对脓毒症性心肌抑制是否具有保护作用及其可能的机制。.SD大鼠随机分成Control组（腹腔注射8ml/kg的生理盐水）、LPS组（腹腔注射5mg/kg的LPS+4ml/kg的生理盐水）以及LPS+SS-20组（腹腔注射5mg/kg的LPS+5mg/kg的SS-20）。24小时后进行左心室插管，获取心功能数据后取心肌进行心肌HE染色、Tunel染色、使用透射电镜观察线粒体超微结构；检测心肌ATP含量、心肌和心肌线粒体MDA含量、MPTP开放情况；检测心肌凋亡相关基因和蛋白表达情况。取血清检测心肌酶含量、抗氧化能力、炎症因子、DNA损伤。.与Control组相比，LPS组大鼠心肌心功能下降、血清心肌酶含上升且HE染色显示存在心肌损伤；血清抗氧化能力降低、心肌和心肌线粒体内脂质过氧化水平较高且DNA损伤程度较重；MPTP开放程度较大、凋亡相关蛋白（Bax、caspase-3，8，9、tBid）表达上升Bcl-2表达下降而且Tunel染色显示凋亡细胞数量增多；炎症因子（TNF-α、IL-1β、IL-6）含量较高。与LPS组相比LPS+SS-20组大鼠心肌心功能提高、血清心肌酶含下降且HE染色显示存在心肌损伤较轻；血清抗氧化能力升高、心肌和心肌线粒体内脂质过氧化水平降低且DNA损伤程度降低；MPTP开放程度减小、凋亡相关基因（Bax、caspase-3，9）表达均下降、凋亡相关蛋白（Bax、caspase-3，9、tBid）表达下降Bcl-2表达上升而caspase-8表达无明显变化而且Tunel染色显示凋亡细胞数量减少；炎症因子（TNF-α、IL-1β、IL-6）含量减少。.本研究发现SS-20可以减轻氧化应激状态、减轻线粒体氧化应激损伤、减少心肌细胞凋亡以及减少心肌抑制因子产生从而降低心肌酶水平、减轻心功能下降的程度，对脓毒症性大鼠心肌发挥保护作用。