EPAC-1在脑出血后继发性脑损伤中的作用机制及雷公藤红素的调控作用研究

The prevention and treatment of secondary brain injury (SBI) after intracerebral hemorrhage (ICH) takes a critical part in improving the prognosis of patients with ICH. Mitochondrial injury is an important factor for brain injury after stroke. Our preliminary results showed that the mitochondria was injured, meanwhile, EPAC-1 was activated and translocated to mitochondria after ICH. And the novel small molecular, celastrol (Cel), could interact with EPAC-1 directly. Thus, we propose the hypothesis: EPAC-1 may translocate to mitochondrial membrane, which could induce the mitochondrial membrane potential collapse and damage of the neuronal cells, leading to the SBI induction. Furthermore, Cel may target to EPAC-1 to interfere with its phosphorylation and prevent the occurrence of SBI. To verify this hypothesis, we intend to analyze the changes of EPAC-1 after ICH from multiple levels such as animal, tissue, cell, organelle and molecular levels. We will use siRNA, overexpression and mutation technologies to evaluate the effects and mechanisms of EPAC-1 on mitochondrial injury. Then, molecular docking, cellular thermal shift assay (CETSA), immunofluorescence and co-immunoprecipitation will be used to investigate the potential effects of Cel on targeting EPAC-1 to protect mitochondria and prevent SBI. Through this study, we will provide a new therapeutic target and a small molecule candidate for the prevention and treatment of SBI after ICH in clinic.

脑出血（ICH）后继发性脑损伤（SBI）的科学防治对改善ICH患者的预后具有重要作用，线粒体损伤是脑卒中后脑损伤的关键因素。我们预实验结果表明：ICH后，神经元线粒体损伤，EPAC-1被激活并发生线粒体转位，雷公藤红素（Cel）可与EPAC-1发生直接结合作用。为此，我们提出假说：ICH后EPAC-1发生线粒体转位破坏线粒体外膜，损伤神经元线粒体，参与SBI的发生；Cel能够靶向EPAC-1，干预其磷酸化水平，逆转SBI的发生。为了验证该假说，我们拟从动物、组织、细胞、细胞器及分子水平等多层次分析ICH后EPAC-1的变化；利用siRNA、过表达及位点突变技术评价EPAC-1在线粒体损伤中的作用及机制；应用分子对接、热迁移、免疫荧光及免疫共沉淀技术探讨Cel靶向EPAC-1保护线粒体，改善SBI的可能性。通过本课题的深入研究，将为临床ICH后SBI的防治提供新的作用靶点及小分子候选药物 。

脑出血（ICH）后继发性脑损伤（SBI）的科学防治对改善ICH患者的预后具有重要作用，线粒体损伤是脑卒中后脑损伤的关键因素。在本研究中，我们主要揭示了ICH后EPAC-1损伤神经元线粒体的作用机制以及雷公藤红素的调控作用。首先，我们用氧合血红蛋白（OxyHb）刺激原代培养大鼠神经元构建体外ICH模型，以体重300 g的SD雄性大鼠作为实验动物，建立活体ICH模型，结合线粒体分离技术，分析了EPAC-1的活化水平、磷酸化水平以及线粒体定位情况；第二，我们运用磷酸化位点突变和质粒转染技术，调控神经元细胞EPAC-1的表达和磷酸化水平，观察了EPAC-1的磷酸化水平对其线粒体定位及神经元凋亡的影响；第三，我们分别在体内外ICH模型中，用雷公藤红素给予干预，结合Autodocking和体内热迁移实验，探讨了雷公藤红素对EPAC-1的调节作用，评价了其神经保护作用。我们的结果显示：ICH发生后，在神经元细胞中EPAC-1-Rap1被激活，EPAC-1 Ser108位磷酸化水平降低，促使其向线粒体的转位，损伤神经元线粒体，诱导神经元发生凋亡；ICH后通过调控EPAC-1 Ser108位的磷酸化能够改变EPAC-1的线粒体定位，减轻ICH诱导的线粒体损伤；雷公藤红素则能够直接与EPAC-1发生相互作用，改变其Ser108位的磷酸化水平，抑制EPAC-Rap1的激活，进而抑制EPAC-1的线粒体转位，发挥神经保护作用。因此，EPAC-1可作为ICH后线粒体保护的潜在靶点；雷公藤红素可作为EPAC-1的调控分子，有望成为临床ICH后SBI的防治的候选药物。