c-FLIPL在I/R诱导神经元程序性坏死/凋亡中的作用及调控机制

The death mode of retinal ganglion cells (RGCs) following ischemia-reperfusion injury is mainly necrosis at early stage and apoptosis at advanced stage. Our previous study shows partial RGCs necrosis can be intervened (necroptosis). However, whether there is a molecular switch between apoptosis and necroptosis is still unclear. According to genechip and literatures we screened cellular FLICE-like protein isoform L (c-FLIPL), which may have a concentration-related regulation of apoptosis and necroptosis. Our previous study suggested that the expression of c-FLIPL in RGCs following ischemia-reperfusion injury is different at different stages, caused by different c-FLIPL generating amount. RNA-binding proteins (RBPs), which regulate the alternative splicing of cflar pre-mRNA, play an important role in c-FLIPL generating. Heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1) has been proved to be the main RBP regulating c-FLIPL generating while not the same in our previous study, suggesting that there are other RBPs regulating this procedure. Therefore, our proposal plans to study roles of different concentration of c-FLIPL on apoptosis and necroptosis of RGCs following ischemia-reperfusion injury at different stages, investigate the distinct RBP regulating c-FLIPL generation, and convinced by animal models in vivo. Our research will further elucidate the roles of c-FLIPL in apoptosis and necroptosis of RGCs, find out the distinct RBP regulating c-FLIPL generation, look for the key intervention targets on RGCs, which will afford the fundamental theory basis in treatment of neurons injury related diseases following ischemia-reperfusion.

RGCs在I/R早期以坏死，中晚期以凋亡为主。研究显示部分坏死RGCs可被调控（程序性坏死），但能同时调控程序性坏死和凋亡的“开关分子”有待阐明。前期基因芯片结合文献筛选出c-FLIPL对细胞凋亡和程序性坏死均有调控，其作用可能与浓度有关。预实验显示c-FLIPL在RGCs I/R后不同时期浓度不同，该差异主要由c-FLIPL产生不同所致。RBP可调控cflar pre-mRNA选择性剪接，对c-FLIPL产生起重要作用。前期工作表明hnRNP A2/B1可能不是调控c-FLIPL产生主要RBP，有存在其他RBP调控可能。故本项目拟用形态学、细胞生物和分子生物学等多方法研究c-FLIPL浓度在I/R后不同时期对RGCs程序性坏死和凋亡影响；探索调控c-FLIPL产生的特异性RBP，并用动物实验验证。本研究将寻找干预RGCs程序性坏死和凋亡关键靶点，为神经元损伤疾病治疗提供理论依据。

细胞FLICE抑制蛋白（c-FLIP）是一种抗凋亡调节剂，与caspase-8具有显著相似性，而caspase-8在GSDMD的剪切中起关键作用。据报道，氧糖剥夺/再恢复（OGD / R）模型和脂多糖（LPS）/三磷酸腺苷（ATP）处理可引起炎症和细胞焦亡。但是，尚不清楚c-FLIP在视网膜神经元的细胞焦亡中的调节作用。因此，在这项研究中，我们假设c-FLIP可能通过GSDMD剪切来调节视网膜神经元焦亡。为了研究该假设，我们在体外（OGD / R损伤和LPS / ATP处理）和体内（急性高眼压[aHIOP]）诱导了视网膜神经元损伤。我们的研究结果表明，这三种损伤诱导了焦亡相关蛋白表达的上调，并且c-FLIP可能剪切GSDMD从而生成GSDMD的功能性N端（NT）结构域，从而引起视网膜神经元焦亡。此外，体内外c-FLIP抑制可改善由急性IOP升高介导的已建立的视力障碍。综上所述，这些发现表明，c-FLIP表达的降低可能通过抑制GSDMD-NT的生成来保护视网膜神经元免于发生焦亡。因此，c-FLIP可能提供了有关焦亡相关疾病发病机理的新见解，并有助于阐明新的治疗靶点和潜在的治疗策略。