神经酰胺诱导神经细胞Parthanatos的分子机制研究

Ceramide plays an important role in the pathogenesis of cerebral microvascular disease, and the molecular mechanisms underlying ceramide induced nerve cell death have not been elucidated. Parthanatos is an important matter causing neural cell death in stroke, mainly through the pathway of PARP-1/PAR/AIF, and MIF may be a downstream molecule of AIF. In previous study, we found that ceramide induced nuclear translocation of MIF, leading to neural cell death, which could not be prevented by the caspase-3 inhibitor of z-DEVD-FMK. Therefore, we propose a hypothesis that ceramide can induce Parthanatos through the pathway of PARP-1/PAR/AIF/MIF in nerve cells. To evaluate this hypothesis, we treat nerve cells with ceramide, transfect cells with lentivirus expressing AIF shRNA and MIF shRNA, deeply study the effect of ceramide on the activation of PARP-1, PAR polymer formation, the interaction of AIF and MIF, and nuclear translocation of AIF and MIF, clarify the molecular mechanisms underlying ceramide induced Parthanatos and whether MIF is the downstream molecule of AIF. This investigation will shed new light on the molecular mechanisms of ceramide induced neuronal death, and also provide experimental and theoretical basis for preventing Parthanatos in neurons.

神经酰胺在脑小血管病中发挥着重要作用，其诱导神经细胞死亡的分子机制尚未阐明。Parthanatos主要通过PARP-1/PAR/AIF导致细胞死亡，是神经细胞死亡的一种方式。前期工作中，我们发现神经酰胺诱导MIF核转位，导致神经细胞死亡，且不能被caspase-3的抑制剂z-DEVD-FMK阻止。我们推测神经酰胺通过PARP-1/PAR/AIF/MIF诱导神经细胞Parthanatos。为了验证这一假说，我们运用神经酰胺处理细胞，构建AIF shRNA和MIF shRNA慢病毒载体转染细胞，深入研究神经酰胺对PARP-1激活、PAR聚集、AIF与MIF相互作用以及AIF、MIF核转位的影响，探讨神经酰胺能否诱导Parthanatos以及MIF是否为AIF的下游分子。本研究将进一步阐明神经酰胺诱导神经细胞死亡的分子机制，为阻断神经细胞发生Parthanatos提供实验基础和理论依据。

神经酰胺在许多中枢神经系统疾病中发挥着重要作用，它能诱导神经元凋亡和坏死，但是其诱导神经元发生parthanatos的作用机制尚未完全阐明。在这项研究中，我们研究了神经酰胺诱导神经元发生parthanatos的分子途径以及巨噬细胞迁移抑制因子（macrophage migration inhibitory factor, MIF）在其中的作用。研究发现神经酰胺显著降低了神经元的活力并导致神经元死亡，这种现象不能被泛半胱天冬酶抑制剂Z-VAD-FMK所抑制，相反，却能被多聚ADP核糖聚合酶-1（poly (ADP-ribose) polymerase-1, PARP-1）的抑制剂 ABT-888 所阻止。进一步研究发现神经酰胺诱导PARP-1过度激活，增加多聚ADP核糖（poly (ADP-ribose), PAR）聚合物水平，并促使凋亡诱导因子（apoptosis-inducing factor, AIF）和MIF的核转位，导致神经元DNA损伤和死亡。通过腺病毒介导的shRNA敲低AIF的表达，抑制了神经酰胺诱导的MIF核转位、DNA损伤和神经元死亡，而敲低MIF的表达能抑制神经酰胺诱导的DNA损伤和神经元细胞，但是AIF的核转位不受影响，表明MIF可能是AIF的下游。同时我们发现神经酰胺增加神经元的活性氧（reactive oxygen species, ROS）水平，而N-乙酰半胱氨酸（N-acetyl cysteine, NAC)能显著抑制PAR的产生、DNA损伤和神经元死亡。这些结果表明，神经酰胺通过增加ROS水平、诱导PARP-1过度激活、PAR聚合物水平增加、AIF和MIF核转位，引起神经元发生parthanatos，导致神经元死亡。总之，敲低MIF的表达可能是神经系统疾病的潜在治疗策略。