血红素加氧酶-1在大鼠慢性间歇性缺氧性心功能不全中的保护作用及分子机制

Obstructive sleep apnea syndrome (OSAS) is characterized by chronic intermittent hyoxia (CIH). Accumulating evidence has reported that OSAS is associated with cardiac dysfunction independent of other risk factors，yet the underlying mechanisms are not clear. The induction of heme oxygenase-1(HO-1) in cardiac tissue was proposed to inhibit cell apoptosis and thereby protecting against ischemia-reperfusion-induced heart damage, yet its role in OSAS- related cardiac dysfunction has not been illustrated. We have previously demonstrated that the induction of HO-1 could inhibit the IH-stimulated apoptosis of vascular endothelial cells in vitro. Moreover, CIH for 4 weeks could increase cholesterol accumulation within cells and facilitate cardiomyocyte apoptosis，leading to the cardiac dysfunction in Sprague-Dawley (SD) rat model. Based on our results as well as reports that cholesterol accumulation could accelerate cell apoptosis and that HO-1 plays an important role in the inhibition of cholesterol accumulation within cells, we hypothesize that the induction of HO-1 could suppress cellular cholesterol accumulation by CIH, therefore rescuing the cardiomyocyte apoptosis and improving heart function. SD rat models will be established for different exposure periods. Cholesterol metabolism within cardiomyocytes and cell apoptosis will be examined by a series of molecular biological techniques. Parameters of heart function will be measured with echocardiography and cardiac pressure-volume loop (PV-LOOP) catheter. On the other hand, HO-1 is induced by hemin or inhibited by cobalt protoporplyrin Ⅸ(ZnPP Ⅸ) treatments, and their effects on these parameters will be evaluated. This project will demonstrate the role of HO-1 in CIH-induced heart injury and involved mechanisms，therefore providing a novel target for the prevention of OSAS-related cardiac dysfunction.

阻塞性睡眠呼吸暂停综合征（OSAS）主要病理生理学特征为慢性间歇性缺氧（CIH），是心功能不全的独立危险因素，血红素加氧酶-1 (HO-1) 可减轻缺血-再灌注所致的心功能损害，但在CIH致心功能不全中作用尚不明确。前期体外实验发现药物诱导及基因敲除方法高表达HO-1可抑制血管内皮细胞凋亡，提示HO-1保护心功能的可能机制；而4周CIH可致大鼠心肌细胞内胆固醇积聚及细胞凋亡，进而诱导心功能损害。基于文献中HO-1对细胞胆固醇代谢影响及胆固醇积聚与细胞凋亡相关性的报道，我们进一步假设诱导HO-1高表达可抑制CIH致心肌细胞内胆固醇积聚，从而减轻细胞凋亡并改善心功能。本课题拟构建大鼠CIH模型，使用分子生物学、组织荧光染色、线粒体功能测定、超声心动图及心脏容积压力导管等方法，观察HO-1表达对胆固醇代谢、细胞凋亡和心功能的影响。本项目期望阐明HO-1在CIH致心功能不全中的保护作用及分子机制。

阻塞性睡眠呼吸暂停综合征（OSAS）主要病理生理学特征为慢性间歇性缺氧（CIH），是心功能不全的独立危险因素，血红素加氧酶-1 (HO-1)具有抗炎、抗凋亡的作用， 可减轻缺血-再灌注所致的心功能损害，但在CIH致心功能不全中作用尚不明确。前期体外实验发现药物诱导及基因敲除方法高表达HO-1可抑制血管内皮细胞凋亡，提示HO-1保护心功能的可能机制；而4周CIH可致大鼠心肌细胞凋亡，进而诱导心功能损害。本项目通过构建细胞及动物CIH模型，观察CIH对炎症反应、细胞凋亡、线粒体动态变化和心功能的影响，以及HO-1表达在CIH致心功能不全中的保护作用及分子机制。18只Sprague-Dawley (SD)大鼠分为3组：对照组、CIH处理组 (PBS,i.p.)和 CIH+hemin 组 (hemin,4 mg/kg,i.p.)；新生小鼠心肌细胞分为4组：对照组、CIH处理组CIH+HO-1siRNA组和 CIH+hemin 组 (10µM)。使用ELISA方法测定血浆及心脏组织炎症因子（CINC-1, MCP-1, adiponectin, TNF-a, IL-6）水平；western blotting方法测定线粒体形态（Mfn2, Drp1）及凋亡相关蛋白（cleaved caspase3, caspase3, Bcl2, Bax）水平；线粒体染色观察线粒体形态；TUNEL染色观察细胞凋亡；超声心动图及心脏容积压力导管测定大鼠心功能。CIH增加大鼠血浆和心肌组织中炎症因子的表达水平；无论在体外或体内模型中，CIH降低心肌细胞Mfn2而增加Drp1表达水平,导致线粒体断裂以及细胞凋亡，而通过hemin处理诱导HO-1高表达则减少线粒体断裂和细胞凋亡的发生；相反，在新生小鼠心肌细胞中敲除HO-1表达进一步加剧线粒体断裂和细胞凋亡。另外，CIH导致大鼠左心室肥大、收缩功能下降以及心肌间质纤维化，而hemin诱导HO-1高表达有效缓解心功能不全和心肌间质纤维化的程度。该项目着重探讨了CIH导致心功能不全的分子学机制；炎症反应、线粒体形态损伤和细胞凋亡等有可能作为OSAS相关性心功能不全发生发展的重要的病理生理学机制。而通过药物诱导HO-1高表达则可能作为改善OSAS患者心功能不全的新的治疗靶点。