蛋白激酶Cα的转位与活化在脓毒症致心肌细胞凋亡中的作用

Sepsis has high morbidity and mortality rates in intensive care units. Septic cardiomyopathy is a severe complication of sepsis. Sepsis induced apoptosis signals activation is involved in septic myocardial dysfunction. PKCα can promote apoptosis and its translocation to plasma membrane is closely related to sepsis. RACK1 plays an important role in facilitating the translocation of PKCα to plasma membrane. The translocation of BAX to mitochondria can activate apoptotic pathways. We have proved in our previous studies that hypoxia/reoxygenation can activate apoptotic pathways through promoting the disassociation and translocation of PKCα and BAX. While blocking the disassociation of PKCα and BAX inhibits cell apoptosis. Based on our previous work, we hypothesize that sepsis can activate cytosolic PKCα and disassociate it from BAX. BAX translocates to mitochondria and activates apoptosis pathways with enhanced cytochrome c release and caspase-3 activation. RACK1 mediates the translocation of PKCα to caveolae. The translocated PKCα is activated through p38 MAPK signaling. Inhibiting the activity of PKCα or using peptide βC2-4, a competitive inhibitor of PKCα, which inhibits the translocation of PKCα will block the above processes. To test the above hypothesis, we will use cell and molecular biology techniques.

脓毒症在重症监护病房中有较高的发病率和死亡率，脓毒症性心肌病是脓毒症的严重并发症。脓毒症可以激活细胞凋亡信号，参与心肌损伤发生。PKCα有促凋亡作用，其向胞膜的转位与脓毒症密切相关。RACK1在PKCα向胞膜转位的过程中起重要介导作用。BAX向线粒体的转位可激活凋亡途径。我们既往研究证实，缺氧/复氧损伤可促进PKCα与BAX的解离与转位,激活凋亡途径，而抑制二者解离则阻断细胞凋亡。本课题在此工作基础上提出假设：脓毒症可以激活心肌细胞胞浆中的PKCα，使其与BAX解离，BAX转位到线粒体激活凋亡途径，促进细胞色素c释放和caspase-3活化，而PKCα在RACK1的介导下转移至caveolae，转位后的PKCα通过p38 MAPK信号而活化，抑制PKCα的活性或应用PKCα的竞争性抑制剂βC2-4肽抑制PKCα的转位，可以阻断上述过程。我们将通过细胞和分子生物学技术验证上述假说。

脓毒症在重症监护病房中有较高的发病率和死亡率，脓毒症性心肌病是脓毒症的严重并发症之一。脓毒症可以激活细胞凋亡信号，参与心肌损伤的发生。PKCα有促进凋亡的作用，其向细胞膜的转位与脓毒症密切相关。RACK1在PKCα向胞膜转位的过程中起重要的介导作用。BAX向线粒体的转位可激活凋亡途径。我们既往的研究证实，缺氧/复氧损伤可促进胞浆内的PKCα与BAX的解离与转位，激活凋亡途径，而抑制二者解离则阻断细胞凋亡。本课题在此工作基础上提出假设：脓毒症可以激活心肌细胞胞浆中的PKCα，使其与BAX解离，BAX转位到线粒体激活凋亡途径，促进细胞色素c释放和caspase-3活化，而PKCα在RACK1的介导下转移至细胞膜并活化，抑制PKCα的活性或应用PKCα的竞争性抑制剂βC2-4肽抑制PKCα的转位，可以阻断上述过程。在本研究中，我们分离培养了原代新生SD大鼠心肌细胞，将βC2-4肽或打乱顺序的βC2-4s肽导入心肌细胞，用LPS刺激心肌细胞，提取了细胞的胞浆蛋白、胞膜蛋白、线粒体蛋白和全细胞蛋白，通过Western blot实验检测了细胞各组分的PKCα、BAX、RACK1、细胞色素c和caspase-3的表达情况。我们观察到，LPS刺激可以促进BAX从胞浆向线粒体的转位与活化，而这种作用可以被βC2-4肽抑制，LPS刺激还增加了RACK1在细胞膜的表达。我们的结果提示LPS促进BAX向线粒体的转位与活化参与凋亡途径的激活，而这一作用可能与PKCα与BAX的解离及转位相关。本研究的其他相关结果正在进一步验证中。